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Volume 15 - Number 3 March 2011


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Scope

The Atlas of Genetics and Cytogenetics in Oncology and Haematology is a peer reviewed on-line journal in open

access, devoted to genes, cytogenetics, and clinical entities in cancer, and cancer-prone diseases.

It presents structured review articles ("cards") on genes, leukaemias, solid tumours, cancer-prone diseases, more

traditional review articles on these and also on surrounding topics ("deep insights"), case reports in hematology, and

educational items in the various related topics for students in Medicine and in Sciences.

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Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3)







Editor

Jean-Loup Huret

(Poitiers, France)

Editorial Board

Sreeparna Banerjee (Ankara, Turkey) Solid Tumours Section

Alessandro Beghini (Milan, Italy) Genes Section

Anne von Bergh (Rotterdam, The Netherlands) Genes / Leukaemia Sections

Judith Bovée (Leiden, The Netherlands) Solid Tumours Section

Vasantha Brito-Babapulle (London, UK) Leukaemia Section

Charles Buys (Groningen, The Netherlands) Deep Insights Section

Anne Marie Capodano (Marseille, France) Solid Tumours Section

Fei Chen (Morgantown, West Virginia) Genes / Deep Insights Sections

Antonio Cuneo (Ferrara, Italy) Leukaemia Section

Paola Dal Cin (Boston, Massachussetts) Genes / Solid Tumours Section

Louis Dallaire (Montreal, Canada) Education Section

Brigitte Debuire (Villejuif, France) Deep Insights Section

François Desangles (Paris, France) Leukaemia / Solid Tumours Sections

Enric Domingo-Villanueva (London, UK) Solid Tumours Section

Ayse Erson (Ankara, Turkey) Solid Tumours Section

Richard Gatti (Los Angeles, California) Cancer-Prone Diseases / Deep Insights Sections

Ad Geurts van Kessel (Nijmegen, The Netherlands) Cancer-Prone Diseases Section

Oskar Haas (Vienna, Austria) Genes / Leukaemia Sections

Anne Hagemeijer (Leuven, Belgium) Deep Insights Section

Nyla Heerema (Colombus, Ohio) Leukaemia Section

Jim Heighway (Liverpool, UK) Genes / Deep Insights Sections

Sakari Knuutila (Helsinki, Finland) Deep Insights Section

Lidia Larizza (Milano, Italy) Solid Tumours Section

Lisa Lee-Jones (Newcastle, UK) Solid Tumours Section

Edmond Ma (Hong Kong, China) Leukaemia Section

Roderick McLeod (Braunschweig, Germany) Deep Insights / Education Sections

Cristina Mecucci (Perugia, Italy) Genes / Leukaemia Sections

Yasmin Mehraein (Homburg, Germany) Cancer-Prone Diseases Section

Fredrik Mertens (Lund, Sweden) Solid Tumours Section

Konstantin Miller (Hannover, Germany) Education Section

Felix Mitelman (Lund, Sweden) Deep Insights Section

Hossain Mossafa (Cergy Pontoise, France) Leukaemia Section

Stefan Nagel (Braunschweig, Germany) Deep Insights / Education Sections

Florence Pedeutour (Nice, France) Genes / Solid Tumours Sections

Elizabeth Petty (Ann Harbor, Michigan) Deep Insights Section

Susana Raimondi (Memphis, Tennesse) Genes / Leukaemia Section

Mariano Rocchi (Bari, Italy) Genes Section

Alain Sarasin (Villejuif, France) Cancer-Prone Diseases Section

Albert Schinzel (Schwerzenbach, Switzerland) Education Section

Clelia Storlazzi (Bari, Italy) Genes Section

Sabine Strehl (Vienna, Austria) Genes / Leukaemia Sections

Nancy Uhrhammer (Clermont Ferrand, France) Genes / Cancer-Prone Diseases Sections

Dan Van Dyke (Rochester, Minnesota) Education Section

Roberta Vanni (Montserrato, Italy) Solid Tumours Section

Franck Viguié (Paris, France) Leukaemia Section

José Luis Vizmanos (Pamplona, Spain) Leukaemia Section

Thomas Wan (Hong Kong, China) Genes / Leukaemia Sections




Volume 15, Number 3, March 2011

Table of contents

Gene Section

AGER (advanced glycosylation end product-specific receptor) 239 Geetha Srikrishna, Barry Hudson

ANG (angiogenin, ribonuclease, RNase A family, 5) 244 Shouji Shimoyama

ATF5 (activating transcription factor 5) 252 Arthur KK Ching, Nathalie Wong

BRE (brain and reproductive organ-expressed (TNFRSF1A modulator)) 255 Yiu-Loon Chui, Kenneth Ka-Ho Lee, John Yeuk-Hon Chan

DDX1 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 1) 259 Takahiko Hara, Kiyoko Tanaka

DIO2 (deiodinase, iodothyronine, type II) 262 Ana Luiza Maia, Simone Magagnin Wajner, Leonardo B Leiria

GFI1B (growth factor independent 1B transcription repressor) 266 Lothar Vassen, Tarik Möröy

LRP5 (low density lipoprotein receptor-related protein 5) 270 Zhendong Alex Zhong, Bart O Williams

MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)) 276 Jan-Philipp Bach, Michael Bacher, Richard Dodel

NEU3 (sialidase 3 (membrane sialidase)) 280 Kazunori Yamaguchi, Taeko Miyagi

NPY1R (neuropeptide Y receptor Y1) 283 Massimiliano Ruscica, Elena Dozio, Luca Passafaro, Paolo Magni

REPS2 (RALBP1 associated Eps domain containing 2) 288 Salvatore Corallino, Luisa Castagnoli

XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) 293 Sabina Pucci, Maria Josè Zonetti

Leukaemia Section

t(6;22)(p21;q11) 297 Jean-Loup Huret

Solid Tumour Section

t(11;22)(q24;q12) in giant cell tumour of bone 299 Jean-Loup Huret

t(11;14)(q13;q32) in multiple myeloma Huret JL, Laï JL




t(11;22)(q24;q12) in rhabdomyosarcomas (RMS) 300 Jean-Loup Huret

t(11;22)(q24;q12) in solid pseudopapillary tumour of the pancreas 302 Jean-Loup Huret

Deep Insight Section

MTA1 of the MTA (metastasis-associated) gene family and its encoded proteins: molecular and regulatory functions and role in human cancer progression 303 Yasushi Toh, Garth L Nicolson

Role of p38α in apoptosis: implication in cancer development and therapy 316 Almudena Porras, Carmen Guerrero

Gene Section Review

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3) 239



AGER (advanced glycosylation end product-specific receptor) Geetha Srikrishna, Barry Hudson

Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037,

USA (GS), Columbia University Medical Center, 630 West 168th St. New York, NY 10032, USA (BH)

Published in Atlas Database: June 2010

Online updated version : http://AtlasGeneticsOncology.org/Genes/AGERID594ch6p21.html DOI: 10.4267/2042/44975

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

Other names: MGC22357; RAGE

HGNC (Hugo): AGER

Location: 6p21.32

Figure 1. Schematic of human chromosome 6.

DNA/RNA

Description

The human AGER (RAGE) gene lies within the major

histocompatibility complex class III region on

chromosome 6, which contains genes involved in

immune responses, such as TNFalpha, lymphotoxin,

complement components and homeobox gene HOX12.

It comprises 11 exons and 10 introns, and a 5' flanking

region that regulates its transcription. The resulting

transcribed mRNA of ~1.4 kb with a short 3'UTR is

alternatively spliced, and nearly twenty isoforms have

been identified in different tissues such as lung, liver,

kidney, smooth muscle, endothelial cells and brain. The

different RAGE gene splice variants have been named

RAGE, RAGE_v1 to RAGE_v19 according to the

Human Gene Nomenclature Committee. RAGE is

composed of a number of distinct protein domains; an

extracellular region (aa 1-342) composed of a

signal peptide (aa 1-22), followed by three

immunoglobulin-like domains, a V-type domain, (aa

23-116) and two C type domains (C1: aa 124-221 and

C2: 227-317), a single transmembrane domain (aa 343-

363), and a short cytoplasmic domain (aa 364-404)

necessary for signaling. The prevalent isoforms of

RAGE are full length RAGE, RAGE_v1 or endogenous

secretory (es RAGE) which lacks the cytosolic and

transmembrane domains and therefore can be secreted

into the extracellular space, and N-terminal truncated

RAGE (RAGE_v2) which lacks N-terminal V domain

and therefore cannot bind ligands. RAGE_v2 does not

form mature protein. Through its ability to scavenge

RAGE ligands, soluble RAGE isoforms (sRAGE) are

believed to act a decoy receptor by regulating signaling

mediated by activation of full length RAGE.

Expression of isoforms is tissue specific, suggesting

tight tissue-specific regulation of expression. sRAGE

can also be formed by ectodomain cleavage by

ADAM10/MMP9.

A number of NF-kappaB sites have been identified in

the RAGE 5' regulatory region. In addition,

transcription is also controlled by other pro-

inflammatory transcription factors such as SP-1 and

AP-2.

At least 30 polymorphisms are known, most of which

are single nucleotide polymorphisms (SNP). A Gly to

Ser change at an N-glycosylation sequon at position 82,

and two 5' flanking polymorphisms at position -374

and -429 lead to altered function and expression of

RAGE.

AGER (advanced glycosylation end product-specific receptor) Srikrishna G, Hudson B


Protein

Description

The V and C1 domains in the extracellular region of

RAGE form an integrated structural unit, while C2 is

fully independent, attached to VC1 through a flexible

linker. RAGE was originally identified as a receptor for

advanced glycation end products, but it also interacts

with other structurally unrelated ligands including

HMGB1, several members of the S100 family,

amyloid-beta peptide, transthyretin and beta2 integrin

Mac-1. By virtue of its multi-domain structure and

ability to recognize different classes of ligands, RAGE

behaves as a pattern recognition receptor (PRR) akin to

innate immune receptors such as Toll-like receptors

(TLRs) in orchestrating immune responses. However,

unlike other PRRs that predominantly bind exogenous

ligands, RAGE binds endogenous ligands, especially

those considered to be damage associated molecular

pattern molecules (DAMPs). AGEs, HMGB1, Abeta

peptides, S100B, S100A1, S100A2 and S100A5 bind to

the V domain, S100A12 binds to V-C1 domains, and

S100A6 interacts with V-C2 domain. Studies on S100

protein-RAGE interactions also suggest that

multimerization of ligand and receptor occurs and that

formation of these higher ordered complexes may be

essential for signal transduction. In addition to

contribution by protein interaction domains, post-

translational modifications such as glycosylation of the

receptors, or acetylation or phosphorylation of ligands

could also play important roles in defining specificity

of interactions, multimerization and downstream

signaling. RAGE has two N-glycosylation sites on the

V-domain and both sites are occupied by complex and

hybrid or high mannose N-glycans. A subpopulation is

modified by carboxylated glycans, which promote

interaction with HMGB1, S100A8/S100A9 and

S100A12. In addition, the quaternary structure of

RAGE might also account for the diversity of ligand

recognition. Though the cytoplasmic domain lacks

endogenous kinase activity or any other known

signaling motif, studies indicate that the cytoplasmic

domain is essential for intracellular signaling.

Expression

RAGE is highly expressed during embryonic

development, especially in the brain, but levels

decrease in adult tissues. RAGE is found at low levels

in neurons, endothelial cells, mononuclear phagocytes,

smooth muscle cells, and constitutively expressed at

high levels in the lung.

Localisation

- Full length: membrane: single pass type I membrane

protein.

- Isoforms: secreted.

Figure 2. Schematic of RAGE protein and its domains. RAGE is a multi-ligand receptor consisting of three Ig-domains (V, C1 and C2), a transmembrane domain and a cytosolic tail required for RAGE-mediated intracellular signaling. The V and C1 domains in the extracellular region of RAGE form an integrated structural unit, while C2 is fully independent, attached to VC1 through a flexible linker. Many ligands bind to the V domain, while some also interact with the V-C1 or V-C2 domains. The V domain has N-glycosylation sites both of which are modified. Ligand binding activates multiple signaling pathways and regulates gene expression through the transcription factors NF-kappaB, CREB and SP1 (From Rauvala H, Rouhiainen A. Biochim Biophys Acta. 2010 Jan-Feb;1799(1-2):164-70. Reproduced with permission from publishers).

Function

Normal physiological functions of RAGE include

embryonal neuronal growth, myogenesis, mobilization

of dendritic cells, activation and differentiation of T

cells, stem cell migration and osteoclast maturation.

HMGB1 interaction of RAGE results in stimulation of

myogenesis. RAGE mediates trophic and toxic effects

of S100B on embryonal neurons, and promotes neurite

outgrowth and neuronal regeneration promoted by

HMGB1. RAGE also plays an important role in the

regulation of osteoclast maturation and function, and

bone remodeling.

Ligand interaction promotes activation of intracellular

signaling pathways including the MAPK pathway,

RAC-1 and CDC42, NADPH oxidase, PI3 kinase and

AGER (advanced glycosylation end product-specific receptor)Srikrishna G, Hudson B


JAK/STAT pathway, and activation of NF-kappaB.

RAGE expression is induced in inflammatory settings,

since its transcription is controlled by several

transcription factors as mentioned above. Thus a

positive feed-forward loop evolves in ligand rich

inflammatory settings, perpetuating the pathology.

sRAGE is believed to regulate signaling mediated by

activation of full length RAGE. Binding of RAGE to

HMGB1 induces RAGE shedding by ADAM10

metalloprotease, thus possibly representing another

pathway for negatively regulating RAGE mediated

cellular activation.

Implicated in

Gastric cancer

Note

RAGE is constitutively expressed in human gastric

carcinoma cell lines, and poorly differentiated human

gastric carcinomas preferentially express RAGE.

Strong RAGE expression is seen in cells at the invasive

edge of tumors and correlates with invasion and lymph

node metastasis. Studies in Chinese population show

that Gly82Ser polymorphism on RAGE is associated

with increased risk for gastric cancer.

Colon cancer

Note

RAGE expression is increased in advanced colon

tumors. Co-expression of RAGE and its ligands

HMGB1 and S100P is strongly associated with

invasion and metastasis of human colorectal cancer.

RAGE appears to be at the interface of inflammation

and colon cancer, since RAGE deficient mice are

resistant to the onset of colitis associated colon cancer.

Pancreatic cancer

Note

Expression of RAGE is strongest in pancreatic cancer

cells with high metastatic ability, and RAGE may play

an important role in the viability of pancreatic tumor

cells against stress-induced apoptosis. RAGE ligand

S100P is overexpressed in pancreatic cancer.

Prostate cancer

Note

RAGE and ligands are highly expressed on prostate

cancer cell lines, untreated prostate cancer tissue and

hormone-refractory prostate cancer tissue, and RAGE

promotes growth and invasion of prostate cancer cells

in response to ligand activation.

Oral squamous cell cancer

Note

RAGE expression closely associates with histologic

differentiation, invasiveness, angiogenesis and

recurrence of oral squamous cell carcinoma.

Common bile duct cancer

Note

RAGE is expressed on human biliary cancer cells, and

expression correlates with their invasive ability.

Glioma

Note

HMGB1/RAGE signaling pathways promote the

growth and migration of human glioblastoma cells.

Inhibition of RAGE-HMGB1 interactions decreases

growth and metastases of gliomas in mice.

Skin cancer

Note

RAGE is expressed in human melanoma cells and

promotes ligand-dependent growth and invasion.

RAGE null mice are resistant to the onset of

inflammation mediated skin tumors in mice.

Lung cancer

Note

RAGE, as well as its ligands, is highly expressed in

normal lung, but unlike other cancers, RAGE is

markedly reduced in human lung carcinomas. Down-

regulation correlates with advanced tumor stages,

suggesting that RAGE may have tumor suppressive

functions in lung cancer.

Tumor microenvironment

Note

Many RAGE ligands are expressed and secreted not

only by cancer cells but also by cells within the tumor

microenvironment, including myeloid derived cells and

vascular cells. These ligands interact with the receptor

in both autocrine and paracrine manners, promoting

tumor growth, invasion, angiogenesis and metastasis.

Inflammation and immune responses

Note

RAGE and its ligands are highly enriched in immune

and inflammatory foci and their interaction promotes

upregulation of inflammatory cytokines, adhesion

molecules and matrix metalloproteinases. They are

therefore implicated in many inflammatory conditions

including colitis and arthritis. RAGE is upregulated in

synovial tissue macrophages and its ligands are

abundant in inflamed synovial tissue. Activation leads

to increased stimulation of chondrocytes and

synoviocytes, promoting ongoing inflammation and

autoimmunity in arthritis. RAGE mediates HMGB1

activation of dendritic cells in response to DNA

containing immune complexes, contributing to

autoimmune pathogenesis. Blockade of RAGE

interactions suppresses myelin basic protein induced

experimental autoimmune encephalomyelitis.

Inhibition of RAGE-ligand interactions or RAGE

deletion protects mice from septic shock induced by

AGER (advanced glycosylation end product-specific receptor) Srikrishna G, Hudson B


caecal ligation and puncture. RAGE null mice are also

resistant to skin and colon inflammation and

inflammation-based tumorigenesis.

Diabetes

Note

RAGE, as a receptor for advanced glycation end

products and other pro-inflammatory ligands,

contributes to micro and macrovascular changes in

diabetes. RAGE over-expression in transgenic mice is

associated with increased vascular injury, diabetic

nephropathy and neuropathy, while RAGE deletion

confers partial protection from these diabetes-

associated changes.

Atherosclerosis and ischemia

Note

Increased RAGE expression is found in endothelial

cells in non-diabetic patients with peripheral occlusive

vascular disease. sRAGE reduces atherosclerotic

lesions and inflammation in normoglycemic Apo E null

mice, and reduced neointima expansion in wild type

mice following femoral artery injury. Studies on

ischemia-reperfusion injury of the heart in wild type

and RAGE null mice show that infarct size and severity

of tissue damage is dependent on HMGB1-RAGE

interactions following necrotic cell death.

Neuronal degeneration

Note

RAGE is expressed on neurons, microglia and

endothelial cells in the brain, and binds the multimeric

form of amyloid-beta peptide. Binding leads to

activation of NADPH oxidase, generation of reactive

oxygen species, activation of NF-kappaB and CREB,

and upregulation of cytokines and chemokines, thus

promoting neuroinflammation. The associated up-

regulation and release of other RAGE ligands such as

HMGB1 and S100 proteins further amplifies this

cascade, leading to neuronal degeneration. sRAGE has

been shown to be beneficial in animal models of

Alzheimer's disease. RAGE null mice are also partially

protected from diabetes-induced loss of neuronal

function.

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Ding Q, Keller JN. Splice variants of the receptor for advanced glycosylation end products (RAGE) in human brain. Neurosci Lett. 2005 Jan 3;373(1):67-72

Andrassy M, Igwe J, Autschbach F, Volz C, Remppis A, Neurath MF, Schleicher E, Humpert PM, Wendt T, Liliensiek B, Morcos M, Schiekofer S, Thiele K, Chen J, Kientsch-Engel R, Schmidt AM, Stremmel W, Stern DM, Katus HA, Nawroth PP,

Bierhaus A. Posttranslationally modified proteins as mediators of sustained intestinal inflammation. Am J Pathol. 2006 Oct;169(4):1223-37

Bierhaus A, Stern DM, Nawroth PP. RAGE in inflammation: a new therapeutic target? Curr Opin Investig Drugs. 2006 Nov;7(11):985-91

Zhou Z, Immel D, Xi CX, Bierhaus A, Feng X, Mei L, Nawroth P, Stern DM, Xiong WC. Regulation of osteoclast function and bone mass by RAGE. J Exp Med. 2006 Apr 17;203(4):1067-80

Dattilo BM, Fritz G, Leclerc E, Kooi CW, Heizmann CW, Chazin WJ. The extracellular region of the receptor for advanced glycation end products is composed of two independent structural units. Biochemistry. 2007 Jun 12;46(23):6957-70

Donato R. RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins. Curr Mol Med. 2007 Dec;7(8):711-24

Leclerc E, Fritz G, Weibel M, Heizmann CW, Galichet A. S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains. J Biol Chem. 2007 Oct 26;282(43):31317-31

Logsdon CD, Fuentes MK, Huang EH, Arumugam T. RAGE and RAGE ligands in cancer. Curr Mol Med. 2007 Dec;7(8):777-89

Ostendorp T, Leclerc E, Galichet A, Koch M, Demling N, Weigle B, Heizmann CW, Kroneck PM, Fritz G. Structural and functional insights into RAGE activation by multimeric S100B. EMBO J. 2007 Aug 22;26(16):3868-78

Xie J, Burz DS, He W, Bronstein IB, Lednev I, Shekhtman A. Hexameric calgranulin C (S100A12) binds to the receptor for advanced glycated end products (RAGE) using symmetric hydrophobic target-binding patches. J Biol Chem. 2007 Feb 9;282(6):4218-31

Gebhardt C, Riehl A, Durchdewald M, Németh J, Fürstenberger G, Müller-Decker K, Enk A, Arnold B, Bierhaus A, Nawroth PP, Hess J, Angel P. RAGE signaling sustains inflammation and promotes tumor development. J Exp Med. 2008 Feb 18;205(2):275-85

Hudson BI, Carter AM, Harja E, Kalea AZ, Arriero M, Yang H, Grant PJ, Schmidt AM. Identification, classification, and expression of RAGE gene splice variants. FASEB J. 2008 May;22(5):1572-80

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This article should be referenced as such:

Srikrishna G, Hudson B. AGER (advanced glycosylation end product-specific receptor). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):239-243.

Gene Section Review




ANG (angiogenin, ribonuclease, RNase A family, 5) Shouji Shimoyama

Gastrointestinal Unit, Settlement Clinic, 4-20-7, Towa, Adachi-ku, Tokyo, 120-0003, Japan (SS)


Online updated version : http://AtlasGeneticsOncology.org/Genes/ANGID635ch14q11.html DOI: 10.4267/2042/44976


Identity Other names: ALS9; HEL168; MGC22466;

MGC71966; RNASE4; RNASE5

HGNC (Hugo): ANG

Location: 14q11.2

DNA/RNA

Starts at 2152336 and ends at 2162345 in NCBI reference sequence NT_026437.12. Gene map locus is available at NCBI Nucleotide. Total length of ANG DNA is 10010 nucleotides. The coding region starts at 2162723 and ends at 2162166 including stop codon TAA.

Protein

Description

The amino acid sequence is available at NCBI protein

locus AAA51678. It consists of a signal peptide from

amino acid 1 to 24 and a mature peptide from amino

acid 25 to 147.

ANG is a basic, single chain potent blood-vessel

inducing protein with a molecular weight of 14 kDa

which was originally discovered in conditioned media

of a human colon carcinoma cell line HT-29. ANG

belongs to the RNAse superfamily, being

35% identical and 68% homologous to the pancreatic

RNAse A sequence. The overall crystal structure of

ANG shows a similarity to, but the biological actions of

ANG differ distinctly from those of RNAse A. ANG

possesses two distinct regions: a ribonucleolytic and a

noncatalytic site, both being critical for angiogenic

activity. Besides the ribonucleolytic activity, ANG

differs from RNAse A in noncatalytic activities such as

interactions with endothelial and smooth muscle cells

and subsequent cellular responses in the events of

neovascularization, including basement membrane

degradation, signal transduction, and nuclear

translocation.

Expression

ANG mRNA is expressed in a wide spectrum of cells

including neoplastic cells as well as normal epithelial

cells, fibroblasts, peripheral blood cells, and vascular

endothelial cells.

Localisation

Strikingly, ANG localizes freely in the circulation, and

is translocated into the nucleus. Nuclear translocation

of ANG triggers subsequent cell proliferation.

However, the precise mechanisms for why serum ANG

is inactive and continuous angiogenesis does not take

place remain unknown.

Function

I. Ribonuclease activity

The catalytic activity of ANG is several orders of

magnitude weaker than that of RNAse A, this being

partly due to the partial occupation of the pyrimidine-

binding pocket of RNAse A by glutamine-117 residue

so that the substrate binding is compromised. Key

amino acids for the ribo-

http://www.ncbi.nlm.nih.gov/nuccore/224514933?report=graph&log$=seqview

http://www.ncbi.nlm.nih.gov/nuccore/224514933?report=graph&log$=seqview

http://www.ncbi.nlm.nih.gov/protein/178250

http://www.ncbi.nlm.nih.gov/protein/178250

ANG (angiogenin, ribonuclease, RNase A family, 5) Shimoyama S


The secondary structure elements of human ANG are depicted by purple boxes. The numbers on the upper and lower sides of each element indicate respectively the beginning and end amino acid residue positions (Acharya et al., 1994). The whole amino acid sequences are shown below and the signal peptide sequences (1-24) are enclosed by box. The H elements form helix structure (Acharya et al., 1994). Key amino acids for the ribonucleolytic activity of ANG (His13, Lys40, and His114) are indicated by plus (+) signs, and residues necessary for angiogenesis (60-68 and 109) are underlined.

nucleolytic activity of ANG are His13, Lys40, or

His114 of ANG, a catalytic triad, but mutations of

these amino acids also reduce ANG induced

angiogenesis, suggesting that the ribonucleolytic

activity of ANG, although weak, is necessary for the

angiogenic activity of ANG. Furthermore, several

arginines are essential for ribonucleolytic and

angiogenic activities.

II. Angiogenic activity

In addition to the catalytic activity, cell binding sites

which encompass residues 60-68 of the surface loop as

well as asparagine-109 are necessary for angiogenesis.

The variants undergoing alterations of these residues

lack any angiogenic activity while the enzymatic

activity remains intact. Inversely, replacing the surface

loop in RNAse A (residues 59-73) with the

corresponding region of ANG (residue 57-70) bestows

a neovascularization activity to the RNAse A.

1) Basement membrane degradation

Amino acid residues from Lys60 to Asn68 of the ANG

constitute a cell surface receptor binding site.

Accordingly, a 42 kDa endothelial cell surface protein

was identified as an ANG binding protein, which was

later found to be a smooth muscle type alpha-actin. The

ANG-actin complex dissociates from the cell surface

and activates a tissue type plasminogen activator, thus

accelerating degradation of the basement membrane

and extracellular matrix that allows endothelial cells to

penetrate or migrate through the extracellular matrix

more easily, an initial step of neovascularization.

Furthermore, fibulin-1, an important molecule for

stabilization of the blood vessel wall, binds to ANG,

suggesting that the ANG-fibulin-1 complex modulates

new blood vessel formation and stabilization.

2) Signal transduction

Besides the 42 kDa ANG receptor, a 170 kDa molecule

later found on the endothelial surface is responsible for

signal transduction, an important process leading to cell

proliferation. ANG activates several secondary

message cascades such as extracellular signal related

kinase 1/2 (ERK1 and ERK2), protein kinase B/Akt,

and stress-associated protein kinase/c-Jun N-terminal

kinase (SAPK/JNK).

3) Nuclear translocation

The nuclear mechanisms underlying the function of

ANG remain elusive. Internalization could involve cell

surface ANG binding to proteins as well as to other

molecules such as proteoglycans, followed by

endocytosis. In this event, ANG interacts directly with

intracellular protein alpha-actinin-2 followed by

translocation into the nucleus through the nuclear pore

in a passive manner. After nuclear retention, ANG

binds to carrier proteins through a sequence 29-

IMRRRGL-35 (nuclear localization signal) of ANG

and to the ANG-binding element of ribosomal DNA

(CTCT repeats) and subsequently, stimulates ribosomal

RNA transcription. Nuclear translocation is essential

for cell proliferation since it is considered a third

messenger and promotes gene activation and

transcription events, and inhibition of the nuclear

translocation of angiogenin abolishes ANG-induced

angiogenesis. Interestingly, the expression of cell

surface receptors responsible for internalization as well

as for the nuclear translocation of ANG also depends

on the cell density.



III. Roles of ANG in physiological angiogenesis

The above biological events, which are distinct from

those of RNAse A, are regulated tightly by the cell

density-dependent expression of ANG receptors. The

discovery of the uniquely regulated expression of ANG

receptors provides us with the following conceivable

mechanisms for ANG related angiogenesis. In the

region where neovascularization is required, ANG

binds to the endothelial surface 42 kDa receptor, and

the ANG-42 kDa receptor complex dissociates from the

cell surface and stimulates proteolytic activity, thus

facilitating the penetration of endothelial cells through

the extracellular matrix. After the leading cells migrate

away, the endothelial cell density in the vicinity of

migrating cells might be sparse, and such cell sparsity

triggers the endothelial proliferation machinery that

includes signal transduction, ANG internalization, and

nuclear translocation. A 170 kDa receptor is one of the

receptors responsible for this orchestrated process.

Once the microenvironment is filled up with the

sufficient amount of endothelial cells and the vascular

network is established, such cell proliferating events

diminish. Therefore, the above cell density dependent

biological events are intelligent mechanisms where the

proliferation machinery and subsequent angiogenic

switch are on when neovascularization is needed while

they are off to prevent unwanted angiogenesis.

Homology

Of the 123 amino acids of human ANG, 43 (35%) and

25 are respectively identical to human pancreatic

RNAse or to other RNAse, and 16 are conservative

replacements, constituting an overall homology of

68%.

Implicated in

Various cancers

Note

There is growing evidence that increased ANG

expression in tissue and/or in sera is correlated with

tumor aggressiveness. These facts are explained at least

in part by the hypothesis that ANG in malignancy plays

roles in the proliferation and migration of malignant

cells, mimicking endothelial cell behavior during

physiological angiogenesis. As described earlier, ANG

could activate proteolytic activity, so that ANG-

expressing malignant cells are allowed to invade

through the extracellular matrix and enter into the

bloodstream. In addition, the continuous translocation

of ANG to the nucleus of HeLa cells in a cell density-

independent manner suggests that cancer cells are also

targets for ANG, and that ANG per se is a contributing

factor for sustained cell growth and the constant supply

of ribosomes, a characteristic of malignant cells.

Several ANG antagonists have been introduced and

some have proved to be effective inhibitors for the

establishment or metastasis of human tumors in

athymic mice. These compounds include a monoclonal

antibody, antisense oligonucleotides complementary to

the AUG translational start site region of ANG,

translocation blocker, enzymatic inhibitor targeting

ANG enzymatic active site, ANG binding polypeptide

complementary to the receptor binding site of ANG,

and internalization pathway blocker.

Female breast cancer

Disease

Female breast cancer is globally the most common

cancer with an annual incidence of 1,15 million

worldwide. It is also the leading cause of death, with a

mortality rate of 133 per million. Breast cancer

incidence rates have increased in most geographic

regions.

Prognosis

The ANG level in sera and the roles of ANG in breast

cancer patients seem to be conflicting. Some studies

found significantly increased serum ANG in breast

cancer patients in comparison with normal controls

while other studies failed to find such a difference. The

serum ANG level is significantly decreased after breast

cancer resection, suggesting that the source of ANG is

at least in part the breast cancer cells. However, there is

conflicting evidence concerning the role of ANG. The

correlation between ANG expression in tissue or in sera

and patient survival was inverse, neutral, or even

positive. The absence of any increase in serum ANG

levels in early stage breast cancer patients suggests that

ANG may have clinical implications when breast

cancer progresses to the advanced stage.

Pancreas cancer

Disease

The pancreas is composed of exocrine (acinar glands

and pancreas duct) and endocrine (islets of Langerhans)

components. Both can give rise to malignant

neoplasms, but adenocarcinoma arising from the

pancreatic ducts is representative of all pancreatic

cancers.

Prognosis

Pancreatic cancer is one of the most aggressive diseases

with most cancers already in later stages at

presentation, the 5-year survival rate being around 5%

both in USA and in Europe. Investigations concerning

ANG expression in pancreatic cancer are scarce. ANG

in sera is elevated in pancreatic cancer patients as

compared with healthy volunteers, and increased ANG

mRNA in tissue or increased ANG in sera has been

correlated with cancer aggressiveness. In addition, the

involvement of ANG in the cancer microenvironment

has been suggested by findings of the ANG expression

in chronic pancreatitis adjacent to pancreatic cancer but

not in pure chronic pancreatitis. Cancer derived

fibroblasts also express ANG.



Gastric cancer

Disease

Gastric cancer, the third most common cancer and the

second leading cause of cancer death among men,

arises in an estimated one million new cases in both

sexes worldwide. Gastric cancer is anatomically

classified as noncardia and cardia cancers, and the

former incidence has declined while the latter incidence

has increased. Helicobacter pylori infection is one of

the risk factors for noncardia cancer. Adenocarcinomas

account for a large majority of gastric cancer histologic

diagnoses.

Prognosis

The 5-year survival rate of gastric cancer in Japan is

double that of the United States and Europe. The better

treatment outcomes are ascribed partly to the social

screening program that attempts to capitalize on the

benefit of early detection, and partly to systematic

lymph node dissection. ANG in sera is increased in

gastric cancer patients and is decreased by resection,

suggesting gastric cancer to be a source of ANG.

Increased mRNA expression in gastric cancer tissues or

increased serum ANG levels is correlated with cancer

progression, proliferation ability, and poor patient

prognosis.

Colorectal cancer

Disease

Colon cancer is the fourth most commonly diagnosed

cancer and fourth most frequent cause of cancer death

among men. Colon cancer incidence rates have

increased in most parts of the world. Typically, there is

a pathologic evolution from benign adenomas to cancer

(adenoma-carcinoma sequence), so that colorectal

cancer screening aims to detect lesions at the adenoma

stage and interrupt the adenoma carcinoma sequence,

ultimately reducing colorectal cancer incidence and

mortality.

Prognosis

Colorectal cancer exhibits increased serum ANG

concentration, and the degree of elevation is correlated

with cancer progression. ANG message expression in

colorectal cancer tissue has also been correlated with

poor patient survival.

Cytogenetics

Overexpression of the 14q11.1-14q11.2 product was

observed in a colon adenocarcinoma cell line.

Lung cancer

Disease

Lung cancer is the most frequently diagnosed cancer

among men. The mortality rate is the highest among

men and the second highest among women worldwide.

Cigarette smoking is the most important risk factor for

lung cancer. The main histologic types of lung cancer

are adenocarcinoma, squamous cell carcinoma, large

cell carcinoma, and small cell carcinoma. The stage of

the disease is a strong predictor of survival, suggesting

that early detection is needed for improvement in

treatment outcomes.

Prognosis

Immunoreactivity in lung cancer tissue is correlated

with tumor size and positive nodal involvement.

Recently, the detection of ANG in exhaled breath

condensate has been achieved, and breath based ANG

may help in the early detection of lung cancer.

Cytogenetics

DNA damage in 14q11.2 was found in asbestos-

exposed lung cancer patients.

Liver cancer

Disease

The global incidence and mortality rate of liver cancer

is ranked among top 5 (in men) and top 10 (in women)

cancer types. The hepatitis B virus and hepatitis C virus

are the most important risk factors for liver cancer.

Histologic classification separates hepatocellular

carcinoma (HCC) (liver cell origin) from

cholangiocarcinoma, which arises from intrahepatic

bile ducts. HCC is the most common histology of liver

cancer which is characterized by vigorous

neovascularization.

Prognosis

The serum ANG is increased in hypervascular

hepatocellular carcinoma. The serum ANG level

decreases after therapy but again increases at

recurrence, suggesting the usefulness of serum ANG

measurement for monitoring the disease and prediction

of patient survival. However, other investigators found

a neutral correlation between serum ANG and survival.

The ANG immunoreactivity is correlated with poorer

histological differentiation.

Cytogenetics

14q11.2 is found to be highly amplified in

hepatoblastoma.

Prostate cancer

Disease

Prostate cancer is the second most frequently diagnosed

cancer among men. Prognosis is excellent for early

stage disease while it is poor for those diagnosed with

advanced cases, pointing to the benefit of earlier

diagnosis. Measurement of prostate specific antigen

helps to detect biologically indolent prostate cancer.

Prognosis

The immunoreactivity of ANG is more evident

according to prostate epithelial cells evolution from a

benign to an invasive phenotype. In vitro analyses

using prostate cancer cell line have elucidated that

ANG is one of the elements responsible for

tumorigenicity and tumor growth. Furthermore, serum

ANG is more increased in hormone-refractory patients

than in healthy controls.



Leukemia

Disease

Leukemias are malignancies that affect blood-forming

stem cells in the bone marrow. Leukemia, a

heterogeneous group of malignancies, is classified into

several subtypes according to the major cell type such

as acute lymphoblastic leukemia (ALL), chronic

lymphoblastic leukemia (CLL), acute myeloid

leukemia (AML), chronic myeloid leukemia (CML),

etc. Acute types refer to cancers arising in immature

stem cells while chronic types refer to cancers arising

in mature stem cells.

Prognosis

The ANG level in sera was increased in AML and

CML, although other investigators failed to find such

correlation. In sharp contrast to the clinical significance

of serum ANG in other solid tumors, elevated serum

ANG in AML and CML is correlated with better

patient survival.

Cytogenetics

14q11.2 is one of the risk foci for ALL.

Hodgkin and non-hodgkin lymphomas

Disease

Lymphomas, malignancies of the lymphoid cells, are

divided on the basis of their pathologic features into

Hodgkin lymphoma (HL) and non-Hodgkin lymphoma

(NHL). HL almost always develops in a lymph node or

other lymphoid structure and spread to nearby nodes.

HL is characterized by the presence of Hodgkin Reed

Sternberg cells. It is one of the most common cancers

diagnosed in younger persons. The proportions of

patients being diagnosed below 50 years old accounts

for 60%. NHL occurs in more elderly patients in the

context of HIV-related immunosuppression. NHL with

HIV shows extensively poorer survivals than those

without HIV.

Prognosis

In sharp contrast to the other solid malignancies, serum

ANG concentrations in patients with HL or NHL are

less than or the same as those in healthy controls.

Increased serum ANG renders no or an inverse impact

on survival in NHL patients.

Cytogenetics

One subtype of NHL experiences multiple

translocations at 14q11.2.

Kidney and bladder cancer

Disease

Kidney and bladder cancers are placed among the top

ten cancer types in both sexes. Cancer of the urinary

bladder most commonly originates in the urothelium,

the epithelium that lines the bladder. Bladder cancer

incidence is significantly higher in males than in

females. There are three major histologic types of

bladder cancer: transitional cell carcinoma, squamous

cell carcinoma, and adenocarcinoma, the former being

overwhelmingly the most common. The majority of

cancers of the kidney are renal cell carcinomas, which

arise from renal tubules. On the other hand, cancer of

the renal pelvis designated as transitional cell

carcinoma comprises the minority.

Prognosis

The serum level of ANG is increased in renal cell

carcinoma and bladder cancer; however, the increase in

serum ANG level does not correlate with patient

survival for renal cell carcinoma. On the other hand,

increased serum ANG or ANG message in urothelial

cancer correlates with poor patient survival or cancer

progression. Recently, ANG in the urine has been

found to be increased in bladder cancer patients.

Melanoma

Disease

Melanoma is placed as the leading cause of skin cancer

death and its incidence has dramatically increased over

the last ten years. It is characterized as having a

notorious resistance to currently available therapies so

that early detection and intervention is needed.

Prognosis

Survival clearly worsens with increasing tumor

thickness. Thin lesions exhibit excellent survival

outcomes while the MST of patients with advance

cases is around 8 months. The ANG in sera is increased

in melanoma patients, and increased serum ANG

correlates with malignancy potential, accordingly,

ANG contributes directly to A375 melanoma cell

proliferation. However, other investigators failed to

find such a correlation.

Gynecological cancers

Disease

Cancers of the uterus and ovary are respectively the

second and the sixth most frequently diagnosed cancer

among women. Cancer of the uterus is further

classified as cancer of the cervix and corpus, and

cancer of the cervix uteri shows the highest incidence

among the three gynecological malignancies. Cancer of

the cervix uteri can be attributed to persistent infection

with carcinogenic genotypes of human papilloma virus.

The three most common histological types of cancer of

the cervix uteri are squamous, adenosquamous, and

adenocarcinoma. Adenocarcinoma is the most common

histology of cancer of the corpus uteri and ovary.

Women with ovarian cancer have poorer survival rates

than those with other gynecological cancers.

Prognosis

Serum ANG is significantly increased in ovarian cancer

patients, while other studies failed to find such a

difference. Increased serum ANG concentration is

correlated with cancer progression in ovary and cervix

uteri.

Cytogenetics

Gains on 14q11.2 are associated with chemoresistant

ovarian cancer.



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Leland PA, Staniszewski KE, Park C, Kelemen BR, Raines RT. The ribonucleolytic activity of angiogenin. Biochemistry. 2002 Jan 29;41(4):1343-50

Ni X, Ma Y, Cheng H, Jiang M, Guo L, Ji C, Gu S, Cao Y, Xie Y, Mao Y. Molecular cloning and characterization of a novel human Rab ( Rab2B) gene. J Hum Genet. 2002;47(10):548-51

Olson KA, Byers HR, Key ME, Fett JW. Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody. Int J Cancer. 2002 Apr 20;98(6):923-9

Xu ZP, Tsuji T, Riordan JF, Hu GF. The nuclear function of angiogenin in endothelial cells is related to rRNA production. Biochem Biophys Res Commun. 2002 Jun 7;294(2):287-92

Chao Y, Li CP, Chau GY, Chen CP, King KL, Lui WY, Yen SH, Chang FY, Chan WK, Lee SD. Prognostic significance of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin in patients with resectable hepatocellular carcinoma after surgery. Ann Surg Oncol. 2003 May;10(4):355-62

Hisai H, Kato J, Kobune M, Murakami T, Miyanishi K, et al. Increased expression of angiogenin in hepatocellular carcinoma in correlation with tumor vascularity. Clin Cancer Res. 2003 Oct 15;9(13):4852-9

Majumder PK, Yeh JJ, George DJ, Febbo PG, Kum J, et al. Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: the MPAKT model. Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7841-6

Shimoyama S, Kaminishi M. Angiogenin in sera as an independent prognostic factor in gastric cancer. J Cancer Res Clin Oncol. 2003 Apr;129(4):239-44

Xu ZP, Tsuji T, Riordan JF, Hu GF. Identification and characterization of an angiogenin-binding DNA sequence that stimulates luciferase reporter gene expression. Biochemistry. 2003 Jan 14;42(1):121-8

Giles FJ, Vose JM, Do KA, Johnson MM, Manshouri T, et al. Clinical relevance of circulating angiogenic factors in patients with non-Hodgkin's lymphoma or Hodgkin's lymphoma. Leuk Res. 2004 Jun;28(6):595-604

Molica S, Vitelli G, Levato D, Giannarelli D, Vacca A, Cuneo A, Ribatti D, Digiesi G. Serum angiogenin is not elevated in patients with early B-cell chronic lymphocytic leukemia but is prognostic factor for disease progression. Eur J Haematol. 2004 Jul;73(1):36-42



Musolino C, Alonci A, Bellomo G, Loteta B, Quartarone E, Gangemi D, Massara E, Calabrò L. Levels of soluble angiogenin in chronic myeloid malignancies: clinical implications. Eur J Haematol. 2004 Jun;72(6):416-9

Cho S, Beintema JJ, Zhang J. The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories. Genomics. 2005 Feb;85(2):208-20

Hirukawa S, Olson KA, Tsuji T, Hu GF. Neamine inhibits xenografic human tumor growth and angiogenesis in athymic mice. Clin Cancer Res. 2005 Dec 15;11(24 Pt 1):8745-52

Hu H, Gao X, Sun Y, Zhou J, Yang M, Xu Z. Alpha-actinin-2, a cytoskeletal protein, binds to angiogenin. Biochem Biophys Res Commun. 2005 Apr 8;329(2):661-7

Katona TM, Neubauer BL, Iversen PW, Zhang S, Baldridge LA, Cheng L. Elevated expression of angiogenin in prostate cancer and its precursors. Clin Cancer Res. 2005 Dec 1;11(23):8358-63

Tsuji T, Sun Y, Kishimoto K, Olson KA, Liu S, Hirukawa S, Hu GF. Angiogenin is translocated to the nucleus of HeLa cells and is involved in ribosomal RNA transcription and cell proliferation. Cancer Res. 2005 Feb 15;65(4):1352-60

Zhao H, Grossman HB, Delclos GL, Hwang LY, et al. Increased plasma levels of angiogenin and the risk of bladder carcinoma: from initiation ot recurrence. Cancer. 2005 Jul 1;104(1):30-5

Chen Y, Zhang S, Chen YP, Lin JY. Increased expression of angiogenin in gastric carcinoma in correlation with tumor angiogenesis and proliferation. World J Gastroenterol. 2006 Aug 28;12(32):5135-9

Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol. 2006 May 10;24(14):2137-50

Nymark P, Wikman H, Ruosaari S, Hollmén J, et al. Identification of specific gene copy number changes in asbestos-related lung cancer. Cancer Res. 2006 Jun 1;66(11):5737-43

Song J, Wang J, Yang J, Jiang C, Shen W, Wang L. Influence of angiogenin on the growth of A375 human melanoma cells and the expression of basic fibroblast growth factor. Melanoma Res. 2006 Apr;16(2):119-26

Tas F, Duranyildiz D, Oguz H, Camlica H, Yasasever V, Topuz E. Circulating serum levels of angiogenic factors and vascular endothelial growth factor receptors 1 and 2 in melanoma patients. Melanoma Res. 2006 Oct;16(5):405-11

Yoshioka N, Wang L, Kishimoto K, Tsuji T, Hu GF. A therapeutic target for prostate cancer based on angiogenin-stimulated angiogenesis and cancer cell proliferation. Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14519-24

Kawada M, Inoue H, Arakawa M, Takamoto K, Masuda T, Ikeda D. Highly tumorigenic human androgen receptor-positive prostate cancer cells overexpress angiogenin. Cancer Sci. 2007 Mar;98(3):350-6

Kim HM, Kang DK, Kim HY, Kang SS, Chang SI. Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells. Biochem Biophys Res Commun. 2007 Jan 12;352(2):509-13

Kim SW, Kim JW, Kim YT, Kim JH, Kim S, Yoon BS, Nam EJ, Kim HY. Analysis of chromosomal changes in serous ovarian carcinoma using high-resolution array comparative genomic hybridization: Potential predictive markers of chemoresistant disease. Genes Chromosomes Cancer. 2007 Jan;46(1):1-9

Lones MA, Heerema NA, Le Beau MM, Sposto R, et al. Chromosome abnormalities in advanced stage lymphoblastic lymphoma of children and adolescents: a report from CCG-E08. Cancer Genet Cytogenet. 2007 Jan 1;172(1):1-11

Vihinen P, Kallioinen M, Vuoristo MS, Ivaska J, Syrjänen KJ, Hahka-Kemppinen M, Kellokumpu-Lehtinen PL, Pyrhönen SO. Serum angiogenin levels predict treatment response in patients with stage IV melanoma. Clin Exp Metastasis. 2007;24(7):567-74

Passam FH, Sfiridaki A, Pappa C, Kyriakou D, Petreli E, et al. Angiogenesis-related growth factors and cytokines in the serum of patients with B non-Hodgkin lymphoma; relation to clinical features and response to treatment. Int J Lab Hematol. 2008 Feb;30(1):17-25

Suzuki M, Kato M, Yuyan C, Takita J, Sanada M, et al. Whole-genome profiling of chromosomal aberrations in hepatoblastoma using high-density single-nucleotide polymorphism genotyping microarrays. Cancer Sci. 2008 Mar;99(3):564-70

Zhang H, Gao X, Weng C, Xu Z. Interaction between angiogenin and fibulin 1: evidence and implication. Acta Biochim Biophys Sin (Shanghai). 2008 May;40(5):375-80

Chan HP, Lewis C, Thomas PS. Exhaled breath analysis: novel approach for early detection of lung cancer. Lung Cancer. 2009 Feb;63(2):164-8

Duranyildiz D, Camlica H, Soydinc HO, Derin D, Yasasever V. Serum levels of angiogenic factors in early breast cancer remain close to normal. Breast. 2009 Feb;18(1):26-9

Eissa S, Swellam M, Labib RA, El-Zayat T, El Ahmady O. A panel of angiogenic factors for early bladder cancer detection: enzyme immunoassay and Western blot. J Urol. 2009 Mar;181(3):1353-60

Goon PK, Lip GY, Stonelake PS, Blann AD. Circulating endothelial cells and circulating progenitor cells in breast cancer: relationship to endothelial damage/dysfunction/apoptosis, clinicopathologic factors, and the Nottingham Prognostic Index. Neoplasia. 2009 Aug;11(8):771-9

Ibaragi S, Yoshioka N, Li S, Hu MG, Hirukawa S, Sadow PM, Hu GF. Neamine inhibits prostate cancer growth by suppressing angiogenin-mediated rRNA transcription. Clin Cancer Res. 2009 Mar 15;15(6):1981-8

Jang SH, Song HD, Kang DK, Chang SI, Kim MK, Cho KH, Scheraga HA, Shin HC. Role of the surface loop on the structure and biological activity of angiogenin. BMB Rep. 2009 Dec 31;42(12):829-33

Papaemmanuil E, Hosking FJ, Vijayakrishnan J, et al. Loci on 7p12.2, 10q21.2 and 14q11.2 are associated with risk of childhood acute lymphoblastic leukemia. Nat Genet. 2009 Sep;41(9):1006-10

Yuan Y, Wang F, Liu XH, Gong DJ, Cheng HZ, Huang SD. Angiogenin is involved in lung adenocarcinoma cell proliferation and angiogenesis. Lung Cancer. 2009 Oct;66(1):28-36

Gessner C, Rechner B, Hammerschmidt S, Kuhn H, et al. Angiogenic markers in breath condensate identify non-small cell lung cancer. Lung Cancer. 2010 May;68(2):177-84


Shimoyama S. ANG (angiogenin, ribonuclease, RNase A family, 5). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):244-251.

Gene Section Mini Review




ATF5 (activating transcription factor 5) Arthur KK Ching, Nathalie Wong

Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales

Hospital, Shatin, NT, Hong Kong (AKKC, NW)


Online updated version : http://AtlasGeneticsOncology.org/Genes/ATF5ID50361ch19q13.html DOI: 10.4267/2042/44977


Identity

Other names: ATFX; FLJ34666; HMFN0395

HGNC (Hugo): ATF5

Location: 19q13.33

Local order: Refer to mapping diagram.

DNA/RNA

Description

The ATF5 gene spans a total genomic size of 5219

bases and is composed of four exons.

Transcription

The human ATF5 transcript is 2268 bp in size

(NM_012068.4) and contains 4 exons. Exon 1 and 2

are non-coding exons and the size of open reading

frame is 849 bp.

Protein

Description

ATF5 consists of 282 amino acid with MW of 30.69

kDa (NCBI reference sequence NP_036200.2).

Expression

Northern blot analysis revealed ubiquitous expression

of ATF5, with highest levels in liver, lung, adipose

tissue, heart, and skeletal muscle.

Localisation

Nucleus and cytoplasm.

Mapping diagram. Base on Human Mar. 2006 (NCBI36/hg18) Assembly.

DNA structure diagram. Relative size of the 4 exons of ATF5. Exon 1 and 2 are untranslated exons (NCBI reference sequence NM_012068.4). Blue area is non-coding region and pink is coding region.

ATF5 (activating transcription factor 5) Ching AKK, Wong N


Protein structure diagram.

Function

ATF5 is a member of basic-region leucine zipper

(bZIP) proteins family which binds the cAMP response

element (CRE) consensus sequence:

5'GTGACGT(C/A)(G/A). This sequence is present in

many viral and cellular promoters. ATF within or

between subgroups can form homo- or hetero-dimer

through the bZIP domain and the dimer can then bind

to the DNA through the basic-motif and function as a

transcription factor. Recently, another novel ATF5

consensus DNA binding sequence (CYTCTYCCTTW)

was found in C6 glioma and MCF7 using a cyclic

amplification and selection of targets (CASTing)

approach (Li et al., 2009).

ATF5 is linked to many cellular function including cell

cycle progression, metabolite homeostasis (Al Sarraj et

al., 2005; Watatani et al., 2007), cellular differentiation

and apoptosis. It involves in the proliferation and

differentiation of neural cells (Angelastro et al., 2003;

Angelastro et al., 2005; Mason et al., 2005) and has

been shown to take part in the skeletal development of

mouse limb (Shinomura et al., 2006; Satake at al.,

2009). Data from various groups also suggested that

ATF5 can function as anti-apoptotic factor (Devireddy

et al., 2001; Persengiev et al., 2002; Nishioka et al.,

2009).

Coimmunoprecipitation and GST pull-down analyses

confirmed the association of the C-terminal bZIP motif

of ATF5 with the PRL-1 PTPase domain and adjacent

residues of PTP4A1 in vitro. SDS-PAGE analysis

showed that PRL-1 dephosphorylates ATF5 in vitro

(Peters et al., 2001).

ATF5 has been shown to interact with various proteins

including Cyclin D3 (Liu et al., 2004), GABAB

receptors (White et al., 2000), HTLV-1 viral protein

Tax (Forgacs et al., 2005), E2 ubiguitin-conjugating

enzyme Cdc34, PRL-1 and DISC1 (Morris et al., 2003;

Fujii et al., 2007; Tomppo et al., 2009). It is a target of

Cdc34-dependent ubiquitin-mediated proteolysis (Pati

et al., 1999). Study indicates that during stress

condition, eIF2 is phosphorylated and subsequently

direct ATF5 translation in cell (Watatani et al., 2008;

Zhou et al., 2008). Recent study of the promoter of

ATF5 also suggested that its transcription is regulated

by EBF1 (Wei et al., 2010).

Homology

ATF5 gene is highly conserved in mammals. Protein

identity percentage of human ATF5 compared with

chimpanzee, cow, mouse, and rat is 98.9, 88.0, 87.5

and 88.9 respectively.

Mutations

Germinal

Unknown.

Somatic

Exon2 Leu141Phe, Exon2 Val257Met and Exon2

Arg275Trp.

Implicated in

Various cancers

Note

ATF5 is showed to be overexpressed in various cancers

by TMA (tissue microarray) that includes breast cancer,

glioblastomas, adenocarcinomas, transitional cell

carcinomas, squamous cell carcinomas and metastatic

carcinomas of various origin (Monaco et al., 2007).

However, in hepatocellular carcinoma, ATF5

expression is down-regulated, suggesting that role of

ATF5 in tumor is highly depending on the tumor type.

Glioma

Note

ATF5 has been shown to be highly expressed in

perinecrotic palisades, the most aggressive forms of

malignant gliomas. In a study of 28 tumors without

perinecrotic palisades, the level of ATF5 expression

together with 4 other genes, negatively correlated with

time of patient survival. Interference of ATF5

expression in glioma cell lines causes apoptosis but not

in cultured astrocytes. These findings suggested that

ATF5 plays a role in maintaining cell survival in

glioma.

Cytogenetics

Unknown.

Hybrid/Mutated gene

Unknown.

ATF5 (activating transcription factor 5) Ching AKK, Wong N


Abnormal protein

Unknown.

Hepatocellular carcinoma

Note

A study has showed that ATF5 is down-regulated in 60

out of 77 cases in HCC, as in contrast to adult normal

liver where expression of ATF5 is particularly high.

Gene expression profiling was also done by ectopic re-

expression of ATF5 suggesting cell cycle, actin

skeleton regulation, MAPK signaling and focal

adhesion are the pathways modulated by ATF5. These

findings suggested that ATF5 down regulation may

contribute to the development of HCC. The inactivation

mechanisms of ATF5 involve epigenetic silencing and

chromosome copy number loss.

References Pati D, Meistrich ML, Plon SE. Human Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of cyclic AMP-induced transcription for proteolysis. Mol Cell Biol. 1999 Jul;19(7):5001-13

White JH, McIllhinney RA, Wise A, Ciruela F, Chan WY, Emson PC, Billinton A, Marshall FH. The GABAB receptor interacts directly with the related transcription factors CREB2 and ATFx. Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13967-72

Devireddy LR, Teodoro JG, Richard FA, Green MR. Induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by IL-3 deprivation. Science. 2001 Aug 3;293(5531):829-34

Peters CS, Liang X, Li S, Kannan S, Peng Y, Taub R, Diamond RH. ATF-7, a novel bZIP protein, interacts with the PRL-1 protein-tyrosine phosphatase. J Biol Chem. 2001 Apr 27;276(17):13718-26

Persengiev SP, Devireddy LR, Green MR. Inhibition of apoptosis by ATFx: a novel role for a member of the ATF/CREB family of mammalian bZIP transcription factors. Genes Dev. 2002 Jul 15;16(14):1806-14

Angelastro JM, Ignatova TN, Kukekov VG, Steindler DA, Stengren GB, Mendelsohn C, Greene LA. Regulated expression of ATF5 is required for the progression of neural progenitor cells to neurons. J Neurosci. 2003 Jun 1;23(11):4590-600

Morris JA, Kandpal G, Ma L, Austin CP. DISC1 (Disrupted-In-Schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation. Hum Mol Genet. 2003 Jul 1;12(13):1591-608

Liu W, Sun M, Jiang J, Shen X, Sun Q, Liu W, Shen H, Gu J. Cyclin D3 interacts with human activating transcription factor 5 and potentiates its transcription activity. Biochem Biophys Res Commun. 2004 Sep 3;321(4):954-60

Al Sarraj J, Vinson C, Thiel G. Regulation of asparagine synthetase gene transcription by the basic region leucine zipper transcription factors ATF5 and CHOP. Biol Chem. 2005 Sep;386(9):873-9

Angelastro JM, Mason JL, Ignatova TN, Kukekov VG, et al. Downregulation of activating transcription factor 5 is required for differentiation of neural progenitor cells into astrocytes. J Neurosci. 2005 Apr 13;25(15):3889-99

Dong S, Nutt CL, Betensky RA, Stemmer-Rachamimov AO, Denko NC, Ligon KL, Rowitch DH, Louis DN. Histology-based expression profiling yields novel prognostic markers in human glioblastoma. J Neuropathol Exp Neurol. 2005 Nov;64(11):948-55

Forgacs E, Gupta SK, Kerry JA, Semmes OJ. The bZIP transcription factor ATFx binds human T-cell leukemia virus type 1 (HTLV-1) Tax and represses HTLV-1 long terminal repeat-mediated transcription. J Virol. 2005 Jun;79(11):6932-9

Mason JL, Angelastro JM, Ignatova TN, Kukekov VG, Lin G, Greene LA, Goldman JE. ATF5 regulates the proliferation and differentiation of oligodendrocytes. Mol Cell Neurosci. 2005 Jul;29(3):372-80

Angelastro JM, Canoll PD, Kuo J, Weicker M, Costa A, Bruce JN, Greene LA. Selective destruction of glioblastoma cells by interference with the activity or expression of ATF5. Oncogene. 2006 Feb 9;25(6):907-16

Chow LS, Lam CW, Chan SY, Tsao SW, To KF, Tong SF, Hung WK, Dammann R, Huang DP, Lo KW. Identification of RASSF1A modulated genes in nasopharyngeal carcinoma. Oncogene. 2006 Jan 12;25(2):310-6

Monaco SE, Angelastro JM, Szabolcs M, Greene LA. The transcription factor ATF5 is widely expressed in carcinomas, and interference with its function selectively kills neoplastic, but not nontransformed, breast cell lines. Int J Cancer. 2007 May 1;120(9):1883-90

Watatani Y, Kimura N, Shimizu YI, Akiyama I, Tonaki D, et al. Amino acid limitation induces expression of ATF5 mRNA at the post-transcriptional level. Life Sci. 2007 Feb 6;80(9):879-85

Gho JW, Ip WK, Chan KY, Law PT, Lai PB, Wong N. Re-expression of transcription factor ATF5 in hepatocellular carcinoma induces G2-M arrest. Cancer Res. 2008 Aug 15;68(16):6743-51

Watatani Y, Ichikawa K, Nakanishi N, Fujimoto M, et al. Stress-induced translation of ATF5 mRNA is regulated by the 5'-untranslated region. J Biol Chem. 2008 Feb 1;283(5):2543-53

Zhou D, Palam LR, Jiang L, Narasimhan J, Staschke KA, Wek RC. Phosphorylation of eIF2 directs ATF5 translational control in response to diverse stress conditions. J Biol Chem. 2008 Mar 14;283(11):7064-73

Greene LA, Lee HY, Angelastro JM. The transcription factor ATF5: role in neurodevelopment and neural tumors. J Neurochem. 2009 Jan;108(1):11-22

Li G, Li W, Angelastro JM, Greene LA, Liu DX. Identification of a novel DNA binding site and a transcriptional target for activating transcription factor 5 in c6 glioma and mcf-7 breast cancer cells. Mol Cancer Res. 2009 Jun;7(6):933-43

Satake H, Ito K, Takahara M, Furukawa T, Takagi M, Ogino T, Shinomura T. Spatio-temporal expression of activating transcription factor 5 in the skeletal development of mouse limb. Dev Growth Differ. 2009 Sep;51(7):669-76

Tomppo L, Hennah W, Lahermo P, Loukola A, et al. Association between genes of Disrupted in schizophrenia 1 (DISC1) interactors and schizophrenia supports the role of the DISC1 pathway in the etiology of major mental illnesses. Biol Psychiatry. 2009 Jun 15;65(12):1055-62

Wei Y, Ge Y, Zhou F, Chen H, Cui C, Liu D, Yang Z, et al. Identification and characterization of the promoter of human ATF5 gene. J Biochem. 2010 Aug;148(2):171-8


Ching AKK, Wong N. ATF5 (activating transcription factor 5). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):252-254.

Gene Section Review




BRE (brain and reproductive organ-expressed (TNFRSF1A modulator)) Yiu-Loon Chui, Kenneth Ka-Ho Lee, John Yeuk-Hon Chan

Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong (YLC), School of

Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (KKHL), Key Lab of

Regenerative Medicine, Ministry of Education, Jinan University, Guang Zhou, Guang Dong, China (JYHC)


Online updated version : http://AtlasGeneticsOncology.org/Genes/BREID839ch2p23.html DOI: 10.4267/2042/44978


Identity

Other names: BRCC4; BRCC45

HGNC (Hugo): BRE

Location: 2p23.2

Local order: According to GeneLoc and NCBI Map

Viewer, genes flanking BRE are RBKS 2p23.3

(ribokinase) in the minus strand orientation, and

RPL23AP34 2p23.2 (ribosomal protein L23a

pseudogene 34) in the positive strand orientation.

DNA/RNA

Description

The gene spans 448284 bases, telomere to centromere

orientation. The first exon is non-coding. In humans,

six transcript variants are produced by alternative

splicing predominantly at either end of the gene. All

human cells examined co-express all of the splice

variants, but at different ratios to one another. The

major transcript is αa, also known as variant 3 by NCBI

nomenclature. This transcript encodes the ubiquitous

383-amino-acid protein, designated by NCBI as protein

isoform 2 (NP_954661.1) (Ching et al., 2001).

Functions of all minor transcript variants are

undetermined. In mice, alternative splicing occurs only

at the 5' region of the gene. The major transcript is

variant 5 (NCBI nomenclature), which encodes the

ubiquitous 383-amino acid protein that is 99% identical

to human BRE. The minor transcript variants, unlike

the human counterparts, are expressed differentially

among tissues. Their functions are undetermined

(Ching et al., 2003).

Transcription

Exon 1 is non-coding; its flanking sequences are

embedded in a CpG island of 1216 bases long.

Transcription start varies over the region between 35 to

112 bases upstream of the last base of exon 1, with the

most common site at 40 bases upstream. No TATA or

CAAT box is located within 150 bases upstream of any

of the transcription start sites. BRE mRNA is expressed

ubiquitously, and was initially found to be highly

expressed in brain, and reproductive organs; hence the

name "BRE" (Li et al., 1995). Subsequent screens

using human multiple-tissue RNA dot blot and

Northern blot revealed highest transcript expression in

adrenal and heart (Miao et al., 2001).

Pseudogene

No pseudogene found.

BRE (brain and reproductive organ-expressed (TNFRSF1A modulator)) Chui YL, et al.


Generation of the major transcript variant of human BRE. Human BRE gene (not drawn in scale) is consisted of 15 exons, three of which are alternatively spliced. The light green boxes, X - Z, are alternative exons which are not present in the major transcript. The

asterisked ATG is the translation start. This transcript encodes the major 383-amino-acid BRE protein isoform 2, that has been studied.

Protein

Note

BRE is a 383-amino-acid protein of no identifiable

functional domain by sequence homology. No crystal

structure of BRE is available. This protein has no

paralog. The N-terminal region of 333 residues of

human BRE, which is conserved among vertebrate

orthologs, has been classified as a single unique

domain, pfam06113. It has been recently proprosed that

BRE contains 2 ubiquitin E2 variant (UEV) domains

(Wang et al., 2009).

Description

BRE is an evolutionarily highly conserved protein with

no homolog within the same species. The major protein

isoform is 383 amino-acids long. Based on

bioinformatic analysis, BRE was proposed to have two

ubiquitin-binding UEV (Ubiquitin E2 variant) domains.

One was located in the N-terminal region between

residues 30 and 147. The other one, however, could

only be located in the isoform encoded by a rare

transcript variant 1, as the C-terminal one quarter of the

putative domain is encoded by the alternative exon Y

(Wang et al., 2009). Thus, it is not clear whether the

remaining putative UEV domain sequence from

residues 275 to 363 of the major BRE isoform is

functional.

Expression

BRE is ubiquitously expressed. All mammalian cell

lines examined express high levels of BRE. These cell

lines include Jurkat, KRC/Y, HeLa, HepG2, HL60,

MCF7, NIH3T3, NS0, THP-1, and lymphoblastoid

CB14022 cells. Among mouse tissues, the expression

levels of BRE detected by Western blot analysis

showed the following pattern: lungs = spleen = thymus

> adrenal > testis = kidney > brain > heart = liver.

Human hepatocytes express little BRE as detected by

immumnohistochemistry and Western blot analysis

(Chan et al., 2008).

Localisation

BRE is located in cytoplasm and nucleus.

Function

DNA-repair and anti-apoptosis via regulation of

ubiquitination. BRE was shown able to bind K48- and

K63-linked polyubiquitin chains (Wang et al., 2009).

BRE and its mouse ortholog are expressed in cytosolic

and nuclear compartments (Li et al., 2004). In the

nucleus, BRE is part of the BRCA1-A complex

involved in DNA repair and maintaining G2/M arrest in

response to DNA damage. BRCA1-A complex consists

of BRCA1, BARD1, Abraxas/Abra1/CCDC98, RAP80,

BRCC36, BRE, and MERIT40/NBA1 (Dong et al.,

2003; Sobhian et al., 2007; Feng et al., 2009; Shao et

al., 2009; Wang et al., 2009). BRE interacts strongly

with MERIT40 and is responsible for binding the latter

to the complex of Abraxas, RAP80 and BRCC36 (Feng

et al., 2009). BRE may also regulate the K63

deubiquitinase activity of BRCC36 (Sobhian et al.,

2007). In conjunction with BRCC36, BRE was shown

to potentiate the E3 activity of BRCA1-BARD1

complex (Dong et al., 2003). Furthermore, depletion of

BRE by siRNA sensitized cells to death induced by

ionizing irradiation (Dong et al., 2003; Feng et al.,

2009). This protein also forms multiprotein BRISC

(Brcc36 isopeptidase complex) in the cytoplasm.

BRISC, containing at least 3 proteins,

FAM175B/ABRO1, BRCC36 and MERIT40/NBA1, in

addition to BRE, specifically cleaves K63-linked

polyubiquitin chains (Cooper et al., 2009). It is not

known whether such cytosolic complex is responsible

for attenuating apoptotic response emanating from the

activated death receptors, TNF-R1 and Fas. BRE also

binds to the cytoplasmic region of TNF-R1 and Fas, as

well as the death-inducing signaling complex (DISC)



during apoptotic induction (Gu et al., 1998; Li et al.,

2004). The anti-apoptotic role of BRE has been shown

by the increased apoptotic response to TNF-alpha of

HeLa cell line depleted of BRE by siRNA, and the

attenuated response of HeLa and Jurkat to TNF-alpha

and anti-Fas agonist antibody by over-expression of the

protein. As over-expression of BRE also reduced

intrinsic apoptotic response induced by stress-related

and genotoxic stimuli, it has been proposed that the

death receptor-associating BRE inhibits the recruitment

of mitochondrial apoptotic machinery, which is

necessary for amplifying the death-receptor-initiated

apoptosis of CD95 type II cell types, which include

HeLa, Jurkat, and hepatocytes (Scaffidi et al., 1998;

Engels et al., 2000). Ectopic expression of BRE in

mouse Lewis lung carcinoma cells was shown to

promote tumor growth in footpad injection model, but

have no effect on cell proliferation in culture condition

(Chan et al., 2005). Over-expression of BRE was found

in 74% of 123 samples of human hepatocellular

carcinoma, and the protein expression level correlated

with poor prognosis. Immortalized human cell lines

also uniformly express high levels of BRE regardless of

the tissue origin of these cell lines. Transgenic

expression of BRE in mouse liver attenuated acute

fulminant hepatitis induced by anti-Fas antibody, and

promoted diethylnitrosamine-induced, but not

spontaneous, liver tumors (Chan et al., 2008; Chui et

al., 2010). Thus, it is likely that BRE over-expression

enhances tumor survival through its anti-apoptotic

activity, rather than initiates tumor formation.

Homology

No homologous protein of BRE found within the same

species.

Mutations Note

According to HapMap genotyped SNP data, there is no

SNP polymorphism in any of the coding exons of BRE.

Germinal

According to the current HapMap_rel27 for all the 4

populations (CEU, CHB, JPT and YRI), the number of

nucleotide positions in BRE gene with HapMap

genotyped SNP is 453. Given the size of BRE gene of

448284 bases long, the number of bases with SNP fits

well to the average genome-wide figure of one SNP per

1000 bases (Dutt and Beroukhim, 2007). It is, however,

noteworthy that no SNP has been found in any of the

coding exons. All of the SNPs, except one located in

the 5' UTR, are present in the introns. Two

recombination hotspots are located in the introns, one

of which is from position 28271535 to 28276573,

located between coding exons 7 and 8.

The other one is from position 28338948 to 28341210,

located between coding exons 10 and 11. Copy number

polymorphisms (CNP) involving a large contiguous

region of 163295 bases encompassing the first 3 coding

exons and the upstream sequence of the neighbouring

ribokinase gene and smaller downstream regions have

also been identified (see diagram above) (The

International HapMap Consortium, 2003).

Somatic

One R9L mutation was identified in a lung carcinoma

cell line, NIH-H2126, and a synonymous mutation

S182S in a clear cell renal cell carcinoma sample,

PD2198a.

Implicated in

Hepatocellular carcinoma (HCC)

Note

Immunohistochemical analysis, supplemented by

immunoblotting, has revealed overexpression of BRE

in the tumoral regions of 72% of the 123 human HCC

samples examined. Non-tumoral liver regions, cirrhotic

or otherwise, expressed little BRE (Chan et al., 2008).

Prognosis

The over-expression levels of BRE correlated with

poor differentiation of HCC cells and therefore poor

prognosis.

Cytogenetics

Not determined.

Hybrid/Mutated gene

Not determined.

Abnormal protein

No fusion protein reported.

Oncogenesis

The transgenic mouse model with liver-specific over-

expression of human BRE showed no enhanced

spontaneous tumor development, indicating that BRE

over-expression alone is not tumorigenic. These mice,

however, showed significant attenuation of liver

apoptosis induced by injection anti-Fas agonist

antibody. These findings indicate that the over-

expression of BRE in HCC is related to the anti-

apoptotic activity of the protein which promotes growth

of the carcinoma (Chan et al., 2008).

Copy number polymorphism (CNP) of BRE gene. Regions with CNP are shown in colored boxes. Blue and red indicate copy loss and gain, respectively. Green indicates loss and gain at different segments of the contiguous region. The largest CNP region on the far left



spans the first non-coding and the next 3 coding exons (exons 1, 2, 3 and 4) and extends further upstream into the neighboring ribokinase gene. The copy gain variant at the far right spans the alternative exon Z. Data obtained from HapMap.

Recent work on inducing liver carcinoma to the above

transgenic mice by neonatal injection of

diethylnitrosamine (DEN) confirmed that BRE over-

expression in the liver could only promote growth of

the already initiated tumor, rather than on initiating

tumor formation. Interestingly, the DEN-induced liver

tumors of the non-transgenic controls also showed up-

regulation of endogenous BRE, suggesting that the

BRE is important in liver carcinogenesis through its

anti-apoptotic activity (Chui et al., 2010).

References Li L, Yoo H, Becker FF, Ali-Osman F, Chan JY. Identification of a brain- and reproductive-organs-specific gene responsive to DNA damage and retinoic acid. Biochem Biophys Res Commun. 1995 Jan 17;206(2):764-74

Gu C, Castellino A, Chan JY, Chao MV. BRE: a modulator of TNF-alpha action. FASEB J. 1998 Sep;12(12):1101-8

Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998 Mar 16;17(6):1675-87

Engels IH, Stepczynska A, Stroh C, Lauber K, Berg C, Schwenzer R, Wajant H, Jänicke RU, Porter AG, Belka C, Gregor M, Schulze-Osthoff K, Wesselborg S. Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis. Oncogene. 2000 Sep 21;19(40):4563-73

Ching AK, Li PS, Li Q, Chan BC, Chan JY, Lim PL, Pang JC, Chui YL. Expression of human BRE in multiple isoforms. Biochem Biophys Res Commun. 2001 Nov 2;288(3):535-45

Miao J, Panesar NS, Chan KT, Lai FM, Xia N, Wang Y, Johnson PJ, Chan JY. Differential expression of a stress-modulating gene, BRE, in the adrenal gland, in adrenal neoplasia, and in abnormal adrenal tissues. J Histochem Cytochem. 2001 Apr;49(4):491-500

. The International HapMap Project. Nature. 2003 Dec 18;426(6968):789-96

Ching AK, Li Q, Lim PL, Chan JY, Chui YL. Expression of a conserved mouse stress-modulating gene, Bre: comparison with the human ortholog. DNA Cell Biol. 2003 Aug;22(8):497-504

Dong Y, Hakimi MA, Chen X, Kumaraswamy E, Cooch NS, Godwin AK, Shiekhattar R. Regulation of BRCC, a holoenzyme complex containing BRCA1 and BRCA2, by a signalosome-like

subunit and its role in DNA repair. Mol Cell. 2003 Nov;12(5):1087-99

Li Q, Ching AK, Chan BC, Chow SK, Lim PL, Ho TC, Ip WK, Wong CK, Lam CW, Lee KK, Chan JY, Chui YL. A death receptor-associated anti-apoptotic protein, BRE, inhibits mitochondrial apoptotic pathway. J Biol Chem. 2004 Dec 10;279(50):52106-16

Chan BC, Li Q, Chow SK, Ching AK, Liew CT, Lim PL, Lee KK, Chan JY, Chui YL. BRE enhances in vivo growth of tumor cells. Biochem Biophys Res Commun. 2005 Jan 14;326(2):268-73

Dutt A, Beroukhim R. Single nucleotide polymorphism array analysis of cancer. Curr Opin Oncol. 2007 Jan;19(1):43-9

Sobhian B, Shao G, Lilli DR, Culhane AC, Moreau LA, Xia B, Livingston DM, Greenberg RA. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science. 2007 May 25;316(5828):1198-202

Chan BC, Ching AK, To KF, Leung JC, Chen S, Li Q, Lai PB, Tang NL, Shaw PC, Chan JY, James AE, Lai KN, Lim PL, Lee KK, Chui YL. BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma. Oncogene. 2008 Feb 21;27(9):1208-17

Cooper EM, Cutcliffe C, Kristiansen TZ, Pandey A, Pickart CM, Cohen RE. K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1. EMBO J. 2009 Mar 18;28(6):621-31

Feng L, Huang J, Chen J. MERIT40 facilitates BRCA1 localization and DNA damage repair. Genes Dev. 2009 Mar 15;23(6):719-28

Shao G, Patterson-Fortin J, Messick TE, Feng D, Shanbhag N, Wang Y, Greenberg RA. MERIT40 controls BRCA1-Rap80 complex integrity and recruitment to DNA double-strand breaks. Genes Dev. 2009 Mar 15;23(6):740-54

Wang B, Hurov K, Hofmann K, Elledge SJ. NBA1, a new player in the Brca1 A complex, is required for DNA damage resistance and checkpoint control. Genes Dev. 2009 Mar 15;23(6):729-39

Chui YL, Ching AK, Chen S, Yip FP, Rowlands DK, James AE, Lee KK, Chan JY. BRE over-expression promotes growth of hepatocellular carcinoma. Biochem Biophys Res Commun. 2010 Jan 15;391(3):1522-5


Chui YL, Lee KKH, Chan JYH. BRE (brain and reproductive organ-expressed (TNFRSF1A modulator)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):255-258.





DDX1 (DEAD (Asp-Glu-Ala-Asp) box poly-peptide 1) Takahiko Hara, Kiyoko Tanaka

Stem cell project group, The Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa,

Setagaya-ku, Tokyo 156-8506, Japan (TH, KT)


Online updated version : http://AtlasGeneticsOncology.org/Genes/DDX1ID40283ch2p24.html DOI: 10.4267/2042/44979


Identity

Other names: DBP-RB; UKVH5d

HGNC (Hugo): DDX1

Location: 2p24.3

DNA/RNA

Genomic organization of human DDX1 gene. Boxes and connecting lines indicate exons (omitted in the middle part) and introns, respectively. The ATG transcription initiation site is located in exon 2.

Description

DDX1 gene is located in an approximately 40 kb

chromosomal DNA region of 2p24.3 containing at least

27 exons. There are alternatively used exons for exon

2, 16, 19, 21, 23 and 27.

Transcription

Size of the major mRNA is 2.7 kb.

Protein

Description

DDX1 protein is a putative RNA helicase containing

the characteristic Asp-Glu-Ala-Asp (DEAD) conserved

sequence motif (Linder et al., 1989). It is composed of

740 amino acid residues (82432 Da). Proteins of this

family (more than 30 from bacteria to humans) have

been described to be implicated in a number of cellular

processes involving alteration of RNA secondary

structure such as translational initiation, nuclear and

mitochondrial RNA splicing, and

ribosome/spliceosome assembly (Rocak and Linder,

2004).

Diagram of conserved motifs among DEAD box RNA helicase family proteins.

Expression

DDX1 mRNA is widely expressed in many tissues, but

its expression level is highest in testis (Tanaka et al.,

2009). DDX1 level tends to be higher in tumor-derived

cells than in normal tissues.

Localisation

DDX1 protein is localized both in the cytoplasm and

nucleus of DDX1 gene-amplified neuroblastoma and

retinoblastoma cell lines, but mainly located in the

nucleus of normal fibroblasts (Godbout et al., 1998).

Function

DDX1 is believed to regulate translational initiation,

nuclear hnRNA splicing, ribosome/spliceosome

assembly, and mRNA synthesis as a putative ATP-

dependent RNA helicase. DDX1 is associated with a

pre-mRNA 3'-end cleavage protein CstF-64 (Bleoo et

al., 2001) and heterogeneous nuclear ribonucleoprotein

K (hnRNP K) (Chen et al., 2002). hnRNP-K is

involved in cell migration and cytoplasmic

accumulation of hnRNP-K is crucial for metastasis

(Inoue et al., 2007). It was reported that DDX1

interacts with nuclear diffusion inhibitory signal (NIS)

DDX1 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 1) Hara T, Tanaka K


motif of HIV-1 Rev protein in human astrocytes.

Accumulation of DDX1 in astrocytes changed sub-

cellular distribution of Rev from nuclear to cytoplasmic

(Fang et al., 2005). More recently, it was reported that

DDX1 is recruited to the sites of DNA double strand

breaks in cells exposed to ionizing radiation and

removes single stranded RNAs to facilitate the repair

reaction of transcriptionally active regions of the

genome (Li et al., 2008).

In retinoblastoma and neuroblastoma cell lines, co-

amplification of DDX1 and the proto-oncogene MYCN

has been demonstrated (Godbout and Squire, 1993;

Godbout et al., 1998). Similar gene amplification of the

genomic region containing DDX1 and MYCN was

observed in alveolar rhabdomyosarcoma samples (Barr

et al., 2009) and Wilms tumor-derived cells (Noguera

et al., 2010). Elevated expression of DDX1 mRNA was

reported to be a prognostic marker for early recurrence

in primary breast cancer (Germain et al., 2010).

Recently, it was demonstrated that DDX1 is essential

for the solid tumor formation of a human testicular

tumor cell line NEC8 in nude mice (Tanaka et al.,

2009). In this case, DDX1 directly bound to the -348

and -329 promoter region of the cyclin-D2 gene and

enhanced its transcription. Furthermore, siRNA-

mediated knockdown of DDX1 resulted in coordinated

down-regulation of stem cell-associated genes located

in chromosomal region 12p13. Therefore, DDX1 may

function as an essential transcriptional activator for the

tumorigenic capacity of testicular germ line tumor-

derived cells. In agreement, DDX1 promotes the

proliferation of JC virus via the transcriptional

activation of its viral promoter (Sunden et al., 2007). It

was also reported that DDX1 acts as a co-activator to

enhance NF-kappaB-mediated transcription (Ishaq et

al., 2009).

Implicated in

Retinoblastoma and neuroblastoma

Note

Coamplication of DDX1 and MYCN genes frequently

occurs in both retinoblastoma and neuroblastoma cell

lines. This is because DDX1 gene is located to

chromosome 2p24, 400-kb telomeric to MYCN gene.

This type of gene amplification has also been reported

in alveolar rhabdomyosarcoma samples and Wilms

tumor-derived cells. However, prognostic significance

of the DDX1 gene amplification for the clinical

outcome is not clear.

Testicular tumors

Note

In testicular tumors including seminoma and

nonseminoma, significantly higher levels of DDX1

mRNA are expressed. In a nonseminoma-derived cell

line NEC8, siRNA-mediated knockdown of DDX1

abrogated their anchorage-independent growth in a

semisolid medium and in vivo tumor formation in nude

mice.

Breast cancer

Note

Expression of DDX1 mRNA and cytoplasmic DDX1

levels are significantly elevated in relapsed breast

cancer samples. Thus, DDX1 can be a prognostic

biomarker for early recurrence in primary breast

cancer.

References Linder P, Lasko PF, Ashburner M, Leroy P, Nielsen PJ, Nishi K, Schnier J, Slonimski PP. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121-2

Godbout R, Squire J. Amplification of a DEAD box protein gene in retinoblastoma cell lines. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7578-82

Godbout R, Packer M, Bie W. Overexpression of a DEAD box protein (DDX1) in neuroblastoma and retinoblastoma cell lines. J Biol Chem. 1998 Aug 14;273(33):21161-8

Bléoo S, Sun X, Hendzel MJ, Rowe JM, Packer M, Godbout R. Association of human DEAD box protein DDX1 with a cleavage stimulation factor involved in 3'-end processing of pre-MRNA. Mol Biol Cell. 2001 Oct;12(10):3046-59

Chen HC, Lin WC, Tsay YG, Lee SC, Chang CJ. An RNA helicase, DDX1, interacting with poly(A) RNA and heterogeneous nuclear ribonucleoprotein K. J Biol Chem. 2002 Oct 25;277(43):40403-9

Rocak S, Linder P. DEAD-box proteins: the driving forces behind RNA metabolism. Nat Rev Mol Cell Biol. 2004 Mar;5(3):232-41

Fang J, Acheampong E, Dave R, Wang F, Mukhtar M, Pomerantz RJ. The RNA helicase DDX1 is involved in restricted HIV-1 Rev function in human astrocytes. Virology. 2005 Jun 5;336(2):299-307

Inoue A, Sawata SY, Taira K, Wadhwa R. Loss-of-function screening by randomized intracellular antibodies: identification of hnRNP-K as a potential target for metastasis. Proc Natl Acad Sci U S A. 2007 May 22;104(21):8983-8

Sunden Y, Semba S, Suzuki T, Okada Y, Orba Y, Nagashima K, Umemura T, Sawa H. DDX1 promotes proliferation of the JC virus through transactivation of its promoter. Microbiol Immunol. 2007;51(3):339-47

Li L, Monckton EA, Godbout R. A role for DEAD box 1 at DNA double-strand breaks. Mol Cell Biol. 2008 Oct;28(20):6413-25

Barr FG, Duan F, Smith LM, Gustafson D, Pitts M, Hammond S, Gastier-Foster JM. Genomic and clinical analyses of 2p24 and 12q13-q14 amplification in alveolar

rhabdomyosarcoma: a report from the Children's Oncology Group. Genes Chromosomes Cancer. 2009 Aug;48(8):661-72

Ishaq M, Ma L, Wu X, Mu Y, Pan J, Hu J, Hu T, Fu Q, Guo D. The DEAD-box RNA helicase DDX1 interacts with RelA and enhances nuclear factor kappaB-mediated transcription. J Cell Biochem. 2009 Feb 1;106(2):296-305

Tanaka K, Okamoto S, Ishikawa Y, Tamura H, Hara T. DDX1 is required for testicular tumorigenesis, partially through the transcriptional activation of 12p stem cell genes. Oncogene. 2009 May 28;28(21):2142-51

DDX1 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 1) Hara T, Tanaka K


Germain DR, Graham K, Glubrecht DD, Hugh JC, Mackey JR, Godbout R. DEAD box 1: a novel and independent prognostic marker for early recurrence in breast cancer. Breast Cancer Res Treat. 2010 May 25;

Noguera R, Villamón E, Berbegall A, Machado I, Giner F, Tadeo I, Navarro S, Llombart-Bosch A. Gain of MYCN region in a Wilms tumor-derived xenotransplanted cell line. Diagn Mol Pathol. 2010 Mar;19(1):33-9


Hara T, Tanaka K. DDX1 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):259-261.

Gene Section Review




DIO2 (deiodinase, iodothyronine, type II) Ana Luiza Maia, Simone Magagnin Wajner, Leonardo B Leiria

Thyroid Section, Endocrine Division, Hospital de Clinicas de Porto Alegre (HCPA), Universidade Federal

do Rio Grande do Sul, Porto Alegre, RS, Brazil (ALM, SMW, LBL)


Online updated version : http://AtlasGeneticsOncology.org/Genes/DIO2ID44390ch14q31.html DOI: 10.4267/2042/44980


Identity

Other names: 5DII; D2; SelY; TXDI2

HGNC (Hugo): DIO2

Location: 14q31.1

DNA/RNA

Description

The Dio2 gene is composed of 3 exons comprising

14656 bp of the genomic DNA.

Transcription

The length of transcribed mRNA is about 6,8 kb and

generates three variants of mRNA. Transcript variant 1

represents the longest transcript and encodes isoform a.

Transcript variant 2 differs in the 5'UTR when

compared to variant 1. Both variants 1 and 2 encode

isoform a. Transcript variant 3 includes an alternate in-

frame exon in the coding region, compared to variant 1.

Variant 3 encodes isoform b, which is longer than

isoform a.

Pseudogene

No pseudogene have been described.

Protein

Description

The protein encoded by this gene belongs to the

iodothyronine deiodinase family. This enzyme activates

thyroid hormone by converting the prohormone

thyroxine (T4) by outer ring deiodination to bioactive

3,3',5-triiodothyronine (T3). It is highly expressed in

the thyroid, and may contribute significantly to the

relative increase in thyroidal T3 production in patients

with Graves' disease and thyroid adenomas. This

protein contains selenocysteine (Sec) residues encoded

by the UGA codon, which often signals the end of

process of translation. The 3'UTR of Sec-containing

genes have a common stem-loop structure, the sec

insertion sequence (SECIS), which is necessary for the

recognition of UGA as a Sec codon rather than a stop

signal. Alternative splicing results in multiple transcript

variants encoding different isoforms. Ubiquitination

can also regulate proteins by transiently inactivating

enzymatic function through conformational change in a

dimeric enzyme, which can be reversed upon

deubiquitination (post-translational).

Organization of the Dio2 gene: Yellow bars represent the coding region (exon) and red bars, the untranslated region.

DIO2 (deiodinase, iodothyronine, type II) Maia AL, et al.


Schematic representation of D2 peptide structure (not on scale). Isoform a (273 aa) and Isoform b (309 aa). In deep green transmembran domain (position 10-34). In yellow active site (position 133). In deep blue alternative sequence isoform b (position 74).

Expression

Ohba et al. (2001) identified 2 alternatively spliced

DIO2 transcripts that include intronic sequences

between the 2 invariant DIO2 exons. These splice

variants showed tissue-specific expression in brain,

thyroid, liver, thymus, anterior pituitary gland and

brown adipose tissue. In mesothelioma cell lysates,

Curcio et al. (2001) determined that endogenous DIO2

gene had an apparent molecular mass of 31 kD. In

normal tissues, D2 activity/mRNA ratio is variable, but

the enzyme is expressed in rodents in the developing

and adult testis, heart, muscle, thyroid, BAT, brain,

pituitary, thymus, skin, spinal cord, placenta, liver and

pancreas. In humans D2 is expressed in brain, BAT,

heart, thyroid, muscle, placenta, skin and vascular

smooth muscle cells.

Localisation

Immuno location of the protein in cells showed D2 as

an endoplasmic reticulum resident protein.

Function

Type 2 deiodinase converts intracellular pro-hormone-

3,3',5,5'-tetraiodothyronine (T4) into the active thyroid

hormone 3,3',5-triiodothyronine (T3) thereby

regulating intracellular levels of active T3 in target

tissues.

Thermogenesis

The expression of D2 is increased in response to cold

stimulation in brown adipocytes isolated from mice.

Dio2 activation in the brown adipose tissue (BAT) of

human newborns and rodents is known to play a role in

adaptive energy expenditure during cold exposure.

Development

D2 activity is present in human placenta through all

pregnancy, and is highly expressed during the first

trimester. The level of activity is low in the non-

pregnant uterus, but in pregnancy the level rises

progressively to a maximum at gestation day 17 when

it is increased threefold.

Homology

Several homologues of Dio2 have been identified in

Pan troglodytes and Macaca mulatta (100%). The

chicken and mouse have similar domain structures with

human Dio2 (97%). Human Dio2 homology with D3 is

expressed in Sus scrofia, Equus caballus, Cricetus

cricetus, Oryctolagus cuniculus, Pituophis deppei (92%

similarity) and limited domains with human D3 and D1.

Mutations

Note

No germinal or somatic mutations has been described.

However, the polymorphism Thr92Ala in Dio2 gene is

associated with increased risk of mental retardation,

insulin resistance in type 2 diabetic patients, reduced

glucose availability in obese women, symptomatic

osteoarthritis, Graves' disease and arterial hypertension.

Implicated in

Various cancers

Note

Although not completely understood, Dio2 gene

expression and activity is altered in some tumors. It is

under-expressed in papillary thyroid carcinomas (PTC).

In follicular tumors, D2 activity is similar or elevated

when compared to non tumoral tissues, and augmented

in follicular adenomas. D2 is also highly expressed in

medullar thyroid carcinoma. A higher expression of the

Dio2 gene was also described in gliosarcoma,

oligoastrocytoma, glioblastoma, oligodendroglioma

and pituitary tumors. In contrast, meningioma does not

express D2 activity. These differences might be related

to the embrionary tumor origin. Mesothelioma

expresses higher activity of D2, whereas osteosarcoma

has diminished D2 activity.

Insulin resistance

Note

Dio2 polymorphism Thr92Ala interacts with a

polymorphism in PPAR gamma 2 gene and is

associated with insulin resistance in diabetic patients.

This Dio2 polymorphism is associated with a ~20%

lower rate of glucose disposal in obese women than in

non-obese women. Although the association between

those two genes occurs in patients with insulin

resistance, these results are contradictory in non

diabetic population.



Hypothyroidism

Note

Disruption in mouse Dio2 gene is associated with

alterations in T4/T3 balance with elevated TSH levels,

which demonstrates that the Dio2 gene is of critical

importance in the feedback regulation of TSH

secretion.

Graves' disease

Note

It is suggested that the Thr92Ala variant of the Dio2

gene is associated or might be in linkage disequilibrium

with a functional DIO2 polymorphism which involves

the development of Graves' disease in a Russian

population.

Mental retardation

Note

A case control study in Chinese patients demonstrated

that two allelic intronic SNPs (rs225010 (T/C) and

rs225012 (A/G)) in the DIO2 gene could affect the

amount of T3 available and in an iodine-deficient

environment and partially determine on augmented risk

of mental retardation. They found a positive association

with mental retardation and the two intronic Dio2

polymorphisms but not with Dio2 Thr92Ala alone and

concluded that the genetic variation in Dio2 determine

the risk of development of mental retardation that could

be due to alterations in the local amount of T3 available

in the brain.

Bone metabolism

Note

Dio2 is expressed in human and mouse osteoblast cells.

In patients with differentiated thyroid carcinoma, the

Dio2 Thr92Ala polymorphism is associated with a

decreased femoral neck bone mineral density and

higher bone turnover independent of serum thyroid

hormone levels.

Cardiomyopathy and arterial hypertension

Note

Dio2 gene expression is also markedly up-regulated in

hearts of mice that develops hypothyroidism or

eccentric hypertrophy after myocardial infarction. The

Dio2 polymorphism Thr92Ala is also associated with

increased risk for the development of hypertension.

References Campos-Barros A, Hoell T, Musa A, Sampaolo S, Stoltenburg G, Pinna G, Eravci M, Meinhold H, Baumgartner A. Phenolic and tyrosyl ring iodothyronine deiodination and thyroid hormone concentrations in the human central nervous system. J Clin Endocrinol Metab. 1996 Jun;81(6):2179-85

Croteau W, Davey JC, Galton VA, St Germain DL. Cloning of the mammalian type II iodothyronine deiodinase. A selenoprotein differentially expressed and regulated in human and rat brain and other tissues. J Clin Invest. 1996 Jul 15;98(2):405-17

Salvatore D, Tu H, Harney JW, Larsen PR. Type 2 iodothyronine deiodinase is highly expressed in human thyroid. J Clin Invest. 1996 Aug 15;98(4):962-8

Buettner C, Harney JW, Larsen PR. The 3'-untranslated region of human type 2 iodothyronine deiodinase mRNA contains a functional selenocysteine insertion sequence element. J Biol Chem. 1998 Dec 11;273(50):33374-8

Celi FS, Canettieri G, Yarnall DP, Burns DK, Andreoli M, Shuldiner AR, Centanni M. Genomic characterization of the coding region of the human type II 5'-deiodinase gene. Mol Cell Endocrinol. 1998 Jun 25;141(1-2):49-52

Araki O, Murakami M, Morimura T, Kamiya Y, Hosoi Y, Kato Y, Mori M. Assignment of type II iodothyronine deiodinase gene (DIO2) to human chromosome band 14q24.2-->q24.3 by in situ hybridization. Cytogenet Cell Genet. 1999;84(1-2):73-4

Bartha T, Kim SW, Salvatore D, Gereben B, Tu HM, Harney JW, Rudas P, Larsen PR. Characterization of the 5'-flanking and 5'-untranslated regions of the cyclic adenosine 3',5'-monophosphate-responsive human type 2 iodothyronine deiodinase gene. Endocrinology. 2000 Jan;141(1):229-37

Campos-Barros A, Amma LL, Faris JS, Shailam R, Kelley MW, Forrest D. Type 2 iodothyronine deiodinase expression in the cochlea before the onset of hearing. Proc Natl Acad Sci U S A. 2000 Feb 1;97(3):1287-92

Murakami M, Araki O, Morimura T, Hosoi Y, Mizuma H, Yamada M, Kurihara H, Ishiuchi S, Tamura M, Sasaki T, Mori M. Expression of type II iodothyronine deiodinase in brain tumors. J Clin Endocrinol Metab. 2000 Nov;85(11):4403-6

Curcio C, Baqui MM, Salvatore D, Rihn BH, Mohr S, Harney JW, Larsen PR, Bianco AC. The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line. J Biol Chem. 2001 Aug 10;276(32):30183-7

Mizuma H, Murakami M, Mori M. Thyroid hormone activation in human vascular smooth muscle cells: expression of type II iodothyronine deiodinase. Circ Res. 2001 Feb 16;88(3):313-8

Murakami M, Araki O, Hosoi Y, Kamiya Y, Morimura T, Ogiwara T, Mizuma H, Mori M. Expression and regulation of type II iodothyronine deiodinase in human thyroid gland. Endocrinology. 2001 Jul;142(7):2961-7

Murakami M, Kamiya Y, Morimura T, Araki O, Imamura M, Ogiwara T, Mizuma H, Mori M. Thyrotropin receptors in brown adipose tissue: thyrotropin stimulates type II iodothyronine deiodinase and uncoupling protein-1 in brown adipocytes. Endocrinology. 2001 Mar;142(3):1195-201

Ohba K, Yoshioka T, Muraki T. Identification of two novel splicing variants of human type II iodothyronine deiodinase mRNA. Mol Cell Endocrinol. 2001 Feb 14;172(1-2):169-75

Schneider MJ, Fiering SN, Pallud SE, Parlow AF, St Germain DL, Galton VA. Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol. 2001 Dec;15(12):2137-48

Zaninovich AA. [Thyroid hormones, obesity and brown adipose tissue thermogenesis]. Medicina (B Aires). 2001;61(5 Pt 1):597-602

Mentuccia D, Proietti-Pannunzi L, Tanner K, Bacci V, Pollin TI, Poehlman ET, Shuldiner AR, Celi FS. Association between a novel variant of the human type 2 deiodinase gene Thr92Ala and insulin resistance: evidence of interaction with the Trp64Arg variant of the beta-3-adrenergic receptor. Diabetes. 2002 Mar;51(3):880-3

Curcio-Morelli C, Zavacki AM, Christofollete M, Gereben B, de Freitas BC, Harney JW, Li Z, Wu G, Bianco AC.



Deubiquitination of type 2 iodothyronine deiodinase by von Hippel-Lindau protein-interacting deubiquitinating enzymes regulates thyroid hormone activation. J Clin Invest. 2003 Jul;112(2):189-96

Wagner MS, Morimoto R, Dora JM, Benneman A, Pavan R, Maia AL. Hypothyroidism induces type 2 iodothyronine deiodinase expression in mouse heart and testis. J Mol Endocrinol. 2003 Dec;31(3):541-50

Chistiakov DA, Savost'anov KV, Turakulov RI. Screening of SNPs at 18 positional candidate genes, located within the GD-1 locus on chromosome 14q23-q32, for susceptibility to Graves' disease: a TDT study. Mol Genet Metab. 2004 Nov;83(3):264-70

Guo TW, Zhang FC, Yang MS, Gao XC, Bian L, Duan SW, Zheng ZJ, Gao JJ, Wang H, Li RL, Feng GY, St Clair D, He L. Positive association of the DIO2 (deiodinase type 2) gene with mental retardation in the iodine-deficient areas of China. J Med Genet. 2004 Aug;41(8):585-90

Arnaldi LA, Borra RC, Maciel RM, Cerutti JM. Gene expression profiles reveal that DCN, DIO1, and DIO2 are underexpressed in benign and malignant thyroid tumors. Thyroid. 2005 Mar;15(3):210-21

Bianco AC, Maia AL, da Silva WS, Christoffolete MA. Adaptive activation of thyroid hormone and energy expenditure. Biosci Rep. 2005 Jun-Aug;25(3-4):191-208

Canani LH, Capp C, Dora JM, Meyer EL, Wagner MS, Harney JW, Larsen PR, Gross JL, Bianco AC, Maia AL. The type 2 deiodinase A/G (Thr92Ala) polymorphism is associated with decreased enzyme velocity and increased insulin resistance in patients with type 2 diabetes mellitus. J Clin Endocrinol Metab. 2005 Jun;90(6):3472-8

Galton VA. The roles of the iodothyronine deiodinases in mammalian development. Thyroid. 2005 Aug;15(8):823-34

Gouveia CH, Christoffolete MA, Zaitune CR, Dora JM, Harney JW, Maia AL, Bianco AC. Type 2 iodothyronine selenodeiodinase is expressed throughout the mouse skeleton and in the MC3T3-E1 mouse osteoblastic cell line during differentiation. Endocrinology. 2005 Jan;146(1):195-200

Maia AL, Kim BW, Huang SA, Harney JW, Larsen PR. Type 2 iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans. J Clin Invest. 2005 Sep;115(9):2524-33

Morimura T, Tsunekawa K, Kasahara T, Seki K, Ogiwara T, Mori M, Murakami M. Expression of type 2 iodothyronine deiodinase in human osteoblast is stimulated by thyrotropin. Endocrinology. 2005 Apr;146(4):2077-84

Canani LH, Leie MA, Machado WE, Capp C, Maia AL. Type 2 deiodinase Thr92Ala polymorphism is not associated with arterial hypertension in type 2 diabetes mellitus patients. Hypertension. 2007 Jun;49(6):e47; author reply e48

Fiorito M, Torrente I, De Cosmo S, Guida V, Colosimo A, Prudente S, Flex E, Menghini R, Miccoli R, Penno G, Pellegrini F, Tassi V, Federici M, Trischitta V, Dallapiccola B. Interaction of DIO2 T92A and PPARgamma2 P12A polymorphisms in the modulation of metabolic syndrome. Obesity (Silver Spring). 2007 Dec;15(12):2889-95

Gumieniak O, Perlstein TS, Williams JS, Hopkins PN, Brown NJ, Raby BA, Williams GH. Ala92 type 2 deiodinase allele

increases risk for the development of hypertension. Hypertension. 2007 Mar;49(3):461-6

Maia AL, Dupuis J, Manning A, Liu C, Meigs JB, Cupples LA, Larsen PR, Fox CS. The type 2 deiodinase (DIO2) A/G polymorphism is not associated with glycemic traits: the Framingham Heart Study. Thyroid. 2007 Mar;17(3):199-202

Sagar GD, Gereben B, Callebaut I, Mornon JP, Zeöld A, da Silva WS, Luongo C, Dentice M, Tente SM, Freitas BC, Harney JW, Zavacki AM, Bianco AC. Ubiquitination-induced conformational change within the deiodinase dimer is a switch regulating enzyme activity. Mol Cell Biol. 2007 Jul;27(13):4774-83

Wajner SM, dos Santos Wagner M, Melo RC, Parreira GG, Chiarini-Garcia H, Bianco AC, Fekete C, Sanchez E, Lechan RM, Maia AL. Type 2 iodothyronine deiodinase is highly expressed in germ cells of adult rat testis. J Endocrinol. 2007 Jul;194(1):47-54

Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Simonides WS, Zeöld A, Bianco AC. Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling. Endocr Rev. 2008 Dec;29(7):898-938

Maia AL, Hwang SJ, Levy D, Larson MG, Larsen PR, Fox CS. Lack of association between the type 2 deiodinase A/G polymorphism and hypertensive traits: the Framingham Heart Study. Hypertension. 2008 Apr;51(4):e22-3

Meyer EL, Goemann IM, Dora JM, Wagner MS, Maia AL. Type 2 iodothyronine deiodinase is highly expressed in medullary thyroid carcinoma. Mol Cell Endocrinol. 2008 Jul 16;289(1-2):16-22

Watanabe M, Yamamoto T, Mori C, Okada N, Yamazaki N, Kajimoto K, Kataoka M, Shinohara Y. Cold-induced changes in gene expression in brown adipose tissue: implications for the activation of thermogenesis. Biol Pharm Bull. 2008 May;31(5):775-84

Williams AJ, Robson H, Kester MH, van Leeuwen JP, Shalet SM, Visser TJ, Williams GR. Iodothyronine deiodinase enzyme activities in bone. Bone. 2008 Jul;43(1):126-34

Panicker V, Saravanan P, Vaidya B, Evans J, Hattersley AT, Frayling TM, Dayan CM. Common variation in the DIO2 gene predicts baseline psychological well-being and response to combination thyroxine plus triiodothyronine therapy in hypothyroid patients. J Clin Endocrinol Metab. 2009 May;94(5):1623-9

Heemstra KA, Hoftijzer H, van der Deure WM, Peeters RP, Hamdy NA, Pereira A, Corssmit EP, Romijn JA, Visser TJ, Smit JW. The type 2 deiodinase Thr92Ala polymorphism is associated with increased bone turnover and decreased femoral neck bone mineral density. J Bone Miner Res. 2010 Jun;25(6):1385-91

Wang YY, Morimoto S, Du CK, Lu QW, Zhan DY, Tsutsumi T, Ide T, Miwa Y, Takahashi-Yanaga F, Sasaguri T. Up-regulation of type 2 iodothyronine deiodinase in dilated cardiomyopathy. Cardiovasc Res. 2010 Sep 1;87(4):636-46


Maia AL, Wajner SM, Leiria LB. DIO2 (deiodinase, iodothyronine, type II). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):262-265.

Gene Section Review




GFI1B (growth factor independent 1B transcription repressor) Lothar Vassen, Tarik Möröy

Research Unit Hematopoiesis and Cancer, Institut de recherches cliniques de Montreal, 110 avenue des Pins

Ouest, Montreal (Quebec) H2W 1R7, Canada (TM, LV)


Online updated version : http://AtlasGeneticsOncology.org/Genes/GFI1BID40707ch9q34.html DOI: 10.4267/2042/44981


Identity

HGNC (Hugo): GFI1B

Location: 9q34.13

Local order: The human GFI1B gene is telomeric to

TSC1 (tuberous sclerosis 1 protein) and centromeric to

GTF3C5 (general transcription factor 3C polypeptide

5).

DNA/RNA

Description

The GFI1B gene structure is composed of at least 7

exons (ranging from 120 to 824 bp), six of which are

coding. Spliced ESTs and human mRNAs may define

up to eight additional 5'exons which possibly point to

an alternative promoter close to the promoter of TSC1.

Transcription

Two human mRNA transcripts arise from alternative

splicing. GFI1B mRNA variant one encodes the more

frequent full length GFI1B (330 aa), while variant two

lacks the in frame exon 5 (according to the NCBI

RefSeq for hGFI1B) leading to a shorter isoform (284

aa) lacking zinc-finger two and parts of zinc-finger one

and three. The residual parts of Znf one and three are

joined to form a new zinc-finger (see mRNA and

protein below).

Pseudogene

Unknown.

Protein

Note

The longer GFI1B variant 1 is the protein refered to in

most cases. GFI1B variant 2 shows only a restricted

expression in normal cells and could be preferentially

associated with leukemic diseases. Functional

differences between both proteins are not described yet.

Description

GFI1B (isoform 1) is a protein of 330 aa residues and

has a predicted molecular mass of 37492.38 Da.

Isoelectric point: 9.3076, charge: 25.0, average residue

weight: 113613.

GFI1B is composed of a 20-amino-acid N-terminal

SNAG (SNAIL-GFI) transcription repressor domain,

and intermediary domain of largely unknown function

and six c-terminal C2H2 zinc-finger domains

encompassing residues 163-327. Zinc-fingers 3-5 are

involved in sequence specific DNA binding and

recognize a taAATCaca/tgca/t core motif. The bases

flanking the AATC core motif seem to be poorly

conserved. Predictions of true GFI1B binding sites in

the genome based only on this sequence have to be

validated independently.

GFI1B (growth factor independent 1B transcription repressor) Möröy T, Vassen L


Model for the generation of the two major GFI1B isoforms. GFI1B-V2 is translated from a shorter mRNA splice variant where exon 9 is skipped (aa 171-216 missing). There are many potential 5' transcriptional start sites, but the major start site seems to be in exon 5, corresponding to exon 1 (122 bp) in the RefSeq database. SD: SNAG domain necessary for repression of transcription by GFI1B. ID: Intermediary domain with very low homology to the GFI1B homolog GFI1 and unknown function, which is presumably important for specific protein-protein interactions unique to GFI1B. Znf 1-6: C2H2 zinc-finger domains which are highly conserved between all members of the GFI1 protein family. Znf 3-5 bind to the major groove of target DNA. Znf 4-5 of GFI1B (almost identical to GFI1) recognize a AATC DNA core sequence in the GFI1/GFI1B predicted binding site.

Expression

GFI1B is mainly expressed in the fetal and adult

hematopoietic system, where it is detected in

hematopoietic stem cells, megakaryocyte/erythroid

precursors (MEP), common myeloid precursors (CMP),

erythroblasts and early erythrocytes, megakaryocytes

and megakaryocyte precursors, B-cell precursors and a

small subset of T-cell precursors (Vassen et al., 2007).

GFI1B is lso detected in fetal thymus and testes. GFI1B

expression varies throughout the maturation of these

cells, with the highest expression levels in MEPs,

megakaryocytes and erythrocytes and seems to be

tightly regulated. The shorter isoform 2 of GFI1B is

lowly expressed in normal cells, but upregulated in

several types of leukemia (e.g. chronic myelogenous

leukemia, acute myeloid leukemia, erythroleukemia,

megakaryocytic leukemia). The expression of GFI1B is

described to be positively regulated by GATA-1, NF-

Y, E2-alpha/TCF3, and HMGB2 and to be repressed by

Oct1, GFI1B and GFI1. GFI1B expression is down-

regulated by erythropoietin (EPO) in a signal-

transducer-and-activator-of-transcription-5 (STAT5)

dependent manner.

Localisation

Almost exclusively nuclear, frequently accumulating in

foci of pericentric heterochromatin.

Function

Negative regulation of transcription. GFI1B is an

essential factor in erythroid and megakaryocytic

development and differentiation, very likely with proto-

oncogenic potential. GFI1B deficiency leads to

embryonic lethality in mice due to failure to produce

functional erythrocytes and megakaryocytes and

increases the apoptosis rate in leukemic cell lines. The

GFI1B gene locus can be autoregulated by

autorepression of its own promoter in hematopoietic

cells (Vassen et al., 2005; Anguita et al., 2010), most

likely by interaction with GATA1 (GATA binding

protein 1) (Huang et al., 2005), an activator of GFI1B

transcription that is also essential for erythroid and

megakaryocytic development. GFI1B and its homolog

GFI1 show cross-repression, resulting in an enhanced

expression of the respective counterpart, when one of

these genes is deleted. The repressory activity of

GFI1B is achieved by recruiting histone deacetylases

(HDAC1 and HDAC2), lysine specific demethylase 1

(LSD1 or KDM1) and the REST corepressor

(CoREST) to target DNA sequences (Saleque et al.,

2007). GFI1B alters histone methylation at target gene

promoters and is associated with sites of gamma-

satellite containing heterochromatin (Vassen et al.,

2006). GFI1B interacts also with the histone

methyltransferases G9a and SUV39H1 and a role in



heterochromatin formation is hypothesized (Vassen et

al., 2006). GFI1B target genes (e.g. BCL2L1, SOCS1,

SOCS3, CDKN1A, GATA3) are frequently also

GATA1 target genes (e.g. GFI1B, GATA2, Myb, Myc)

and GFI1B is overrepresented at sites where GATA1

binds to repress its target genes (Yu et al., 2009).

GATA2 needs to be repressed by GATA1 in

developing erythroid cells pointing to an involvement

of a GATA1-GFI1B repressory complex in this

process. GATA1 and GFI1B have been found in a

complex with SUZ12, a member of the polycomb

repressory complex 2 (PRC2, SUZ12 and Eed) on

repressed genes in MEL (erythroleukemia) cells (Yu et

al., 2009). Since GFI1B is also expressed in

hematopoietic stem cells (HSC), the existence of such a

repressory complex might point to a role of GFI1B in

maintaining HSC self renewal, where PCR complexes

play a major role. Consistent with this hypothesis,

GFI1B can functionally replace GFI1 during

hematopoiesis but not in the development of inner ear

hair cells, where GFI1 exerts a critical survival function

(Fiolka et al., 2006; Wallis et al., 2003). An implication

of GFI1B in sensory epithelial cells similar to GFI1

remains to be elucidated. In megakaryocytes GFI1B is

found in a complex with GATA1 and ETO2, another

corepressor protein that is also implicated in human

leukemogenesis (Hamlett et al., 2008), but how GFI1B

regulates megakaryocytic development is not clear yet.

GFI1B regulates TGF-beta signaling in bipotent

erythroid-megakaryocytic progenitors (Randrianarison-

Huetz, 2010), which is involved in the control of their

differentiation. Finally, GFI1B regulates the expression

of GATA3 in T-cell lymphomas, a critical factor for

survival of lymphomas and T-cell progenitors (Wei and

Kee, 2007).

Positive regulation of transcription. GFI1B can

activate transcription from a promoter containing four

GFI1 consensus-sites in the erythroid cell line K562

(Osawa et al., 2002).

Homology

GFI1B is highly homologous to its closest relative

GFI1. Highly conserved GFI1(B) proteins have been

detected in many species from C. elegans to drosophila

and human.

Mutations

Germinal

A single base mutation in the GFI1B promoter (T-C)

was detected affecting a potential Oct-1 binding site in

an acute lymphoblastic leukemia patient. The mutation

was shown to affect the promoter activity, leading to an

increased expression of GFI1B. (Hernández et al.,

2010).

Somatic

A natural variant (p.R231H) was detected in a

colorectal cancer sample. A GFI1B promoter mutation

was detected in an acute myeloid leukemia M5a

patient, affecting a GATA1 binding site which was

previously shown to be involved in the regulation of

GFI1B expression. (Hernández et al., 2010).

Implicated in

Leukemia

Note

GFI1B was shown to be highly overexpressed in

various leukemias (Elmaagacli et al., 2007; Vassen et

al., 2009). Knock down of GFI1B in leukemia cell lines

markedly increased the apoptosis rate of these cells

(Elmaagacli et al., 2007), pointing to a role of GFI1B in

protection against apoptosis. Overexpression of GFI1B

in human CD34+ hematopoietic progenitors induced an

expansion of erythroblasts, independent from

erythropoietin, pointing to a role of GFI1B in

regulation of proliferation (Osawa et al., 2002). Since

reduced apoptosis and enhanced proliferation both are

involved in leukemogenesis, GFI1B may play an

important role in these diseases. Additionally, the

GFI1B locus was found in a retroviral insertion

mutagenesis screen for factors, cooperating with EGR1

haploinsufficiency to induce myeloid leukemias in the

mouse (Quian et al., 2010).

Disease

Chronic myeloid leukemia (CML), essential

thrombocythemia (ET), myelodysplastic syndrome

(MDS), myeloproliferative syndrome (MPS), B-cell

acute lymphocytic leukemia (B-ALL), acute myeloid

leukemia (AML), erythroleukemia (EL),

megakaryocytic/megakaryoblastic leukemias.

Breakpoints

Note

Translocated along with ABL1 in chronic myeloid

leukemia with translocation t(9:22).

References Rödel B, Wagner T, Zörnig M, Niessing J, Möröy T. The human homologue (GFI1B) of the chicken GFI gene maps to chromosome 9q34.13-A locus frequently altered in hematopoietic diseases. Genomics. 1998 Dec 15;54(3):580-2

Tong B, Grimes HL, Yang TY, Bear SE, Qin Z, Du K, El-Deiry WS, Tsichlis PN. The Gfi-1B proto-oncoprotein represses p21WAF1 and inhibits myeloid cell differentiation. Mol Cell Biol. 1998 May;18(5):2462-73

Jegalian AG, Wu H. Regulation of Socs gene expression by the proto-oncoprotein GFI-1B: two routes for STAT5 target gene induction by erythropoietin. J Biol Chem. 2002 Jan 18;277(3):2345-52

Osawa M, Yamaguchi T, Nakamura Y, Kaneko S, Onodera M, Sawada K, Jegalian A, Wu H, Nakauchi H, Iwama A. Erythroid expansion mediated by the Gfi-1B zinc finger protein: role in normal hematopoiesis. Blood. 2002 Oct 15;100(8):2769-77

Wallis D, Hamblen M, Zhou Y, Venken KJ, Schumacher A, Grimes HL, Zoghbi HY, Orkin SH, Bellen HJ. The zinc finger



transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival. Development. 2003 Jan;130(1):221-32

Huang DY, Kuo YY, Lai JS, Suzuki Y, Sugano S, Chang ZF. GATA-1 and NF-Y cooperate to mediate erythroid-specific transcription of Gfi-1B gene. Nucleic Acids Res. 2004;32(13):3935-46

Garçon L, Lacout C, Svinartchouk F, Le Couédic JP, Villeval JL, Vainchenker W, Duménil D. Gfi-1B plays a critical role in terminal differentiation of normal and transformed erythroid progenitor cells. Blood. 2005 Feb 15;105(4):1448-55

Huang DY, Kuo YY, Chang ZF. GATA-1 mediates auto-regulation of Gfi-1B transcription in K562 cells. Nucleic Acids Res. 2005;33(16):5331-42

Rodriguez P, Bonte E, Krijgsveld J, Kolodziej KE, Guyot B, Heck AJ, Vyas P, de Boer E, Grosveld F, Strouboulis J. GATA-1 forms distinct activating and repressive complexes in erythroid cells. EMBO J. 2005 Jul 6;24(13):2354-66

Vassen L, Fiolka K, Mahlmann S, Möröy T. Direct transcriptional repression of the genes encoding the zinc-finger proteins Gfi1b and Gfi1 by Gfi1b. Nucleic Acids Res. 2005;33(3):987-98

Fiolka K, Hertzano R, Vassen L, Zeng H, Hermesh O, Avraham KB, Dührsen U, Möröy T. Gfi1 and Gfi1b act equivalently in haematopoiesis, but have distinct, non-overlapping functions in inner ear development. EMBO Rep. 2006 Mar;7(3):326-33

Vassen L, Fiolka K, Möröy T. Gfi1b alters histone methylation at target gene promoters and sites of gamma-satellite containing heterochromatin. EMBO J. 2006 Jun 7;25(11):2409-19

Elmaagacli AH, Koldehoff M, Zakrzewski JL, Steckel NK, Ottinger H, Beelen DW. Growth factor-independent 1B gene (GFI1B) is overexpressed in erythropoietic and megakaryocytic malignancies and increases their proliferation rate. Br J Haematol. 2007 Jan;136(2):212-9

Kuo YY, Chang ZF. GATA-1 and Gfi-1B interplay to regulate Bcl-xL transcription. Mol Cell Biol. 2007 Jun;27(12):4261-72

Saleque S, Kim J, Rooke HM, Orkin SH. Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1. Mol Cell. 2007 Aug 17;27(4):562-72

Vassen L, Okayama T, Möröy T. Gfi1b:green fluorescent protein knock-in mice reveal a dynamic expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1. Blood. 2007 Mar 15;109(6):2356-64

Wickrema A, Crispino JD. Erythroid and megakaryocytic transformation. Oncogene. 2007 Oct 15;26(47):6803-15

Xu W, Kee BL. Growth factor independent 1B (Gfi1b) is an E2A target gene that modulates Gata3 in T-cell lymphomas. Blood. 2007 May 15;109(10):4406-14

Hamlett I, Draper J, Strouboulis J, Iborra F, Porcher C, Vyas P. Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation. Blood. 2008 Oct 1;112(7):2738-49

Koldehoff M, Zakrzewski JL, Klein-Hitpass L, Beelen DW, Elmaagacli AH. Gene profiling of growth factor independence 1B gene (Gfi-1B) in leukemic cells. Int J Hematol. 2008 Jan;87(1):39-47

Tsiftsoglou AS, Vizirianakis IS, Strouboulis J. Erythropoiesis: model systems, molecular regulators, and developmental programs. IUBMB Life. 2009 Aug;61(8):800-30

Vassen L, Khandanpour C, Ebeling P, van der Reijden BA, Jansen JH, Mahlmann S, Dührsen U, Möröy T. Growth factor independent 1b (Gfi1b) and a new splice variant of Gfi1b are highly expressed in patients with acute and chronic leukemia. Int J Hematol. 2009 May;89(4):422-30

Yu M, Riva L, Xie H, Schindler Y, Moran TB, Cheng Y, Yu D, Hardison R, Weiss MJ, Orkin SH, Bernstein BE, Fraenkel E, Cantor AB. Insights into GATA-1-mediated gene activation versus repression via genome-wide chromatin occupancy analysis. Mol Cell. 2009 Nov 25;36(4):682-95

Anguita E, Villegas A, Iborra F, Hernández A. GFI1B controls its own expression binding to multiple sites. Haematologica. 2010 Jan;95(1):36-46

Hernández A, Villegas A, Anguita E. Human promoter mutations unveil Oct-1 and GATA-1 opposite action on Gfi1b regulation. Ann Hematol. 2010 Aug;89(8):759-65

Laurent B, Randrianarison-Huetz V, Maréchal V, Mayeux P, Dusanter-Fourt I, Duménil D. High-mobility group protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription. Blood. 2010 Jan 21;115(3):687-95

Qian Z, Joslin JM, Tennant TR, Reshmi SC, Young DJ, Stoddart A, Larson RA, Le Beau MM. Cytogenetic and genetic pathways in therapy-related acute myeloid leukemia. Chem Biol Interact. 2010 Mar 19;184(1-2):50-7

Randrianarison-Huetz V, Laurent B, Bardet V, Blobe GC, Huetz F, Duménil D. Gfi-1B controls human erythroid and megakaryocytic differentiation by regulating TGF-beta signaling at the bipotent erythro-megakaryocytic progenitor stage. Blood. 2010 Apr 8;115(14):2784-95

Yue P, Forrest WF, Kaminker JS, Lohr S, Zhang Z, Cavet G. Inferring the functional effects of mutation through clusters of mutations in homologous proteins. Hum Mutat. 2010 Mar;31(3):264-71


Möröy T, Vassen L. GFI1B (growth factor independent 1B transcription repressor). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):266-269.

Gene Section Review




LRP5 (low density lipoprotein receptor-related protein 5) Zhendong Alex Zhong, Bart O Williams

Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, Michigan

49503-2518, USA (ZAZ, BOW)


Online updated version : http://AtlasGeneticsOncology.org/Genes/LRP5ID44282ch11q13.html DOI: 10.4267/2042/44982


Identity Other names: BMND1, EVR1, EVR4, HBM, LR3,

LRP7, OPPG, OPS, OPTA1, VBCH2

HGNC (Hugo): LRP5

Location: 11q13.2

DNA/RNA

Description

Genomic size: 136636; genomic sequence: (chr11: 67

836 684-67 973 319).

Transcription

5161 bp mRNA; (NM_002335, 05-oct-2009).

Pseudogene

Homo sapiens low density lipoprotein receptor-related

protein 5-like (LRP5L), transcript variant 1,

Aliases: DKFZp434O0213,

NCBI Reference Sequence: NM_182492.2,

Location: 22q11.23,

HGNC ID: HGNC:25323.

Protein

Description

LRP5 contains a large extracellular domain (ECD)

making up over 85% of the approximately 1600-amino-

acid protein. At the amino terminus of the ECD, four

beta-propeller motifs and four epidermal growth factor

(EGF)-like repeats create the binding sites for

extracellular ligands. These domains are followed by

three LDLR type A (LA) domains. The intracellular

domain of LRP5 contains 5 PPPSP motifs, to which

Axin preferentially binds after phosphorylation of the

PPPSP motif induced by Wnt ligands. Tamai et al.

showed that Wnt activates LRP5's homologue, LRP6,

by inducing LRP6 phosphorylation at the PPP(S/T)P

motifs, which serve as inducible docking sites for Axin,

thereby recruiting Axin to the plasma membrane.

Expression

Widely expressed, with the highest level of expression

in the liver.

Exon count: 23; coding exon count: 23.

LRP5 (low density lipoprotein receptor-related protein 5) Zhong ZA, Williams BO


Schematic diagram of human LRP5, 1615 aa. (from He et al., Development. 2004 Apr;131(8):1663-77).

Post-translational modification: Phosphorylation of the

PPPSP motif creates an inducible docking site for

Axin. Palmitoylation is required for LRP6 to exit the

endoplasmic reticulum (ER).

Localisation

Membrane; single-pass type I membrane protein.

Function

Involved in the Wnt/beta catenin signaling pathway,

acting as a co-receptor together with Frizzled for Wnt

ligands.

Mutations

Germinal

The heterozygous LRP5V171 mutation cosegregated

with high bone density. This gain-of-function mutation

in LRP5 causes an autosomal dominant disorder

characterized by high bone density, torus palatinus, and

a wide, deep mandible.

In 2001, Gong et al. reported that they identified a total

of six different homozygous frame-shift or nonsense

mutations in affected offspring from consanguineous

families affected by osteoporosis pseudoglioma

syndrome. They also found homozygous missense

mutations in affected patients from two other

consanguineous families and heterozygous nonsense,

frame-shift, and missense mutations in affected patients

from four nonconsanguineous families. Many patients

with this syndrome are also born with severe disruption

of the ocular structure, phthisis bulbi.

Jiao et al. reported that homozygous mutations R570Q,

R752G, and E1367K in LRP5 cosegregated with

familial exudative vitreoretinopathy (FEVR).

There are many other papers reporting LRP5 gene

mutations and SNP polymorphisms that are associated

with bone density variation, familial exudative

vitreoretinopathy, obesity, etc.

Somatic

Westin's group reported that the tumor-associated

shorter transcript of LRP5 containing an in-frame

deletion of 142 amino acids (D666-809) was strongly

implicated in deregulated activation of the Wnt/beta-

catenin signaling pathway in hyperparathyroid tumors

and mammary gland tumorigenesis.

Schematic representation of LRP5 mutations; those associated with osteoporosis pseudoglioma (OPPG) syndrome, autosomal-dominant familial exudative vitreoretinopathy (FEVR), and various high-bone-density diseases are shown in red, purple, and green, respectively. Arrows indicate mutation locations: *, nonsense mutation; fs, frame-shift mutation. (from He et al., Development. 2004 Apr;131(8):1663-77).



Implicated in

Hyperparathyroid tumors, breast cancer

Note

According to Bjorklund's reports, the internally

truncated human LRP5 receptor is strongly implicated

in deregulated activation of the Wnt/beta-catenin

signaling pathway in hyperparathyroid tumors and

mammary gland tumorigenesis, and thus presents a

potential target for therapeutic intervention.

The truncation version of LRP5 (LRP5Δ666-809) missed the

last 93 bp of exon 9, all 227 bp of exon 10, and the first 106 bp of exon 11.

Oncogenesis

Reverse transcription PCR and Western blot analysis

showed expression of truncated LRP5 in 32 out of 37

primary hyperparathyroidism (pHPT) tumors (86%)

and 20 out of 20 secondary hyperparathyroidism

(SHPT) tumors (100%).

Truncated LRP5 frequently expressed in breast tumors

of different cancer stages (58-100%), including

carcinoma in situ and metastatic carcinoma. Truncated

LRP5 was required in MCF7 breast cancer cells for the

nonphosphorylated active beta-catenin level,

transcription activity of beta-catenin, cell growth in

vitro, and breast tumor growth in a xenograft SCID

mouse model.

Other cancers

Note

LRP5 is required for maintaining the basal lineage of

mouse mammary tissue (Badders et al., 2009) and for

mammary ductal stem cell activity and Wnt1-induced

tumorigenesis (Lindvall et al., 2006).

LRP5 is a novel marker for disease progression in high-

grade osteosarcoma (Hoang et al., 2004). Dominant

negative LRP5 showed inhibition of osteosarcoma

tumorigenicity and metastasis in mouse model (Guo et

al., 2008).

Osteoporosis-pseudoglioma syndrome (OPPG)

Note

Children with the autosomal recessive disorder

osteoporosis pseudoglioma syndrome (OPPG) (Gong et

al., 1996) have very low bone mass and are prone to

developing fractures and deformation. In addition to the

skeletal phenotype, many individuals with OPPG have

eye involvement in the form of severe disruption of the

ocular structure, called phthisis bulbi.

Cytogenetics

Gong et al. found that OPPG carriers have reduced

bone mass when compared with age- and gender-

matched controls. They demonstrated LRP5 expression

by osteoblasts in situ and showed that LRP5 can

transduce Wnt signaling in vitro via the canonical

pathway. They also showed that a mutant secreted form

of LRP5 can reduce bone thickness in mouse calvarial

explant cultures. These data indicate that Wnt-mediated

signaling via LRP5 affects bone accrual during growth

and is important for the establishment of peak bone

mass.

Ai et al. sequenced the coding exons of LRP5 in 37

probands suspected of having OPPG on the basis of the

co-occurrence of severe congenital or childhood-onset

visual impairment and bone fragility or osteoporosis

recognized by young adulthood. They measured the

ability of wild-type and mutant LRP5 to transduce Wnt

and Norrin signals ex vivo. Each of the seven OPPG

mutations tested had reduced signal transduction

relative to wild-type controls. These results indicate

that early bilateral vitreoretinal eye pathology coupled

with skeletal fragility is a strong predictor of LRP5

mutation and that mutations in LRP5 cause OPPG by

impairing Wnt and Norrin signal transduction.

In 2008, Yadav et al. identified Tph1, which encodes

the rate-limiting enzyme in serotonin synthesis, as the

most highly overexpressed gene in LRP5-/-

mice. Tph1

expression was also elevated in LRP5-/-

duodenal cells.

Decreasing serotonin blood levels normalized bone

formation and bone mass in LRP5-/-

mice, and gut-

specific LRP5 inactivation decreased bone formation in

a beta-catenin-independent manner. They concluded

that LRP5 inhibits bone formation by inhibiting

serotonin production in the gut.

Cheung et al. identified a family with osteoporosis

pseudoglioma syndrome due to compound

heterozygosity of two novel mutations in the LRP5

gene (W478R and W504C).

In 2007, Drenser et al. found familial exudative

vitreoretinopathy and osteoporosis pseudoglioma

syndrome caused by a mutation in the LRP5 gene.

Xiong et al. found that LRP5 gene polymorphisms are

associated with bone mass density in both Chinese and

whites. The Chinese sample consisted of 733 unrelated

subjects and the white sample was made up of 1873

subjects from 405 nuclear families.

The most frequently studied polymorphisms in LRP5

are two amino acid substitutions, Val667Met and

Ala1330Val. A common variant of LRP6, Ile1062Val,

contributes to fracture risk in elderly men, and is linked

to coronary heart disease and low BMD. In 2008, Joyce

et al. confirmed that the two common LRP5 variants

are consistently associated with BMD and fracture risk

across different white populations, but the LRP6

variant is not.



High bone mass (HBM)

Note

Bone mass density (BMD) and fracture rates vary

among women of differing ethnicities. Most reports had

suggested that BMD is highest in African Americans,

lowest in Asians, and intermediate in Caucasians, yet

Asians have lower fracture rates than Caucasians.

Finkelstein et al. (2002) assessed lumbar spine and

femoral neck BMD by dual-energy x-ray

absorptiometry in 2277 (lumbar) and 2330 (femoral)

premenopausal or early perimenopausal women (mean

age, 46.2 yr) participating in the Study of Women's

Health Across the Nation. When BMD was assessed in

a subset of women weighing less than 70 kg and then

adjusted for covariates, lumbar spine BMD was similar

in African American, Chinese, and Japanese women

and was lowest in Caucasian women. Femoral neck

BMD was highest in African Americans and similar in

Chinese, Japanese, and Caucasians. They also

suggested that these findings may explain why

Caucasian women have higher fracture rates than

African Americans and Asians.

Cytogenetics

Little et al. also identified the same Gly171Val

mutation in the LRP5 gene (G171V; 603506.0013) that

results in an autosomal dominant high bone mass trait.

Van Wesenbeeck et al. performed mutation analysis of

the LRP5 gene in 10 families or isolated patients with

various conditions of an increased bone density,

including endosteal hyperostosis. Direct sequencing of

the LRP5 gene revealed 19 sequence variants. Six

novel missense mutations (D111Y, G171R, A214T,

A214V, A242T, and T253I) are located in the amino-

terminal part of the gene, before the first epidermal

growth factor-like domain, which is the same as for the

G171V mutation that causes the high-bone-mass

phenotype and most likely is disease-causing.

Boyden et al. found that the expression of LRP5V171 did

not activate signaling in the absence of Wnt-1. The

activation of the signaling pathway in response to Wnt-

1 was the same with normal and mutant LRP5.They

also tested the action of the endogenous antagonist of

Wnt signaling, Dkk-1. Although Dkk-1 inhibited Wnt

signaling in conjunction with wild-type LRP5, Dkk-1

inhibition of Wnt signaling was virtually abolished in

cells expressing LRP5V171. These findings indicated

that the mutation G171V, located in the first YWTD

repeat of LRP5, results in increased Wnt signaling

because of loss of Dkk antagonism to LRP5.

However, Zhang et al. found that the third YWTD

repeat (but not the first repeat domain) was required for

DKK1-mediated antagonism. They found that the

G171V mutation disrupted the interaction of LRP5

with Mesd, a chaperone protein for LRP5/6 that is

required for transport of the co-receptors to cell

surfaces, resulting in fewer LRP5 molecules on the cell

surface. So they think that the G171V mutation may

cause an increase in Wnt activity in osteoblasts by

reducing the number of targets for paracrine DKK1 to

antagonize without affecting the activity of autocrine

Wnt.

Ai et al. expressed seven different HBM-LRP5

missense mutations, including G171V, to delineate the

mechanism by which they alter Wnt signaling. Each

mutant receptor was able to reach the cell surface,

albeit in differing amounts, and transduce exogenously

supplied Wnt1 and Wnt3a signals. The affinities

between the mutant forms of LRP5 and Mesd did not

correlate with their abilities to reach the cell surface.

All HBM mutant proteins had reduced physical

interaction with and reduced inhibition by DKK1.

These data suggest that HBM mutant proteins can

transit to the cell surface in sufficient quantity to

transduce Wnt signal and that the likely mechanism for

the HBM mutations' physiologic effects is via reduced

affinity to and inhibition by DKK1.

Semenov further showed that LRP5 HBM mutant

proteins exhibit reduced binding to a secreted bone-

specific LRP5 antagonist, SOST, and consequently are

more refractory to inhibition by SOST. Further, Bhat

used structure-based mutation analysis to show the

importance of LRP5 beta-propeller 1 in modulating

Dkk1-mediated inhibition of Wnt signaling.

Familial exudative vitreoretinopathy (FEVR)

Note

Familial exudative vitreoretinopathy (FEVR) is a well-

defined inherited disorder of retinal vessel development

(Benson, 1995). It is reported to have a penetrance of

100%, but clinical features can be highly variable even

within the same family. Severely affected patients may

be legally blind during the first decade of life, whereas

mildly affected individuals may not even be aware of

symptoms and may receive a diagnosis only by use of

fluorescein angiography.

Cytogenetics

As reported by Toomes et al., mutations in LRP5

within the EVR1 locus can cause FEVR, accounting for

15% of the patients and indicating that other

unidentified FEVR genes may be a more significant

cause of the disease than previously thought.

Jiao et al. studied three consanguineous families of

European descent in which autosomal recessive FEVR

was diagnosed in multiple individuals. Sequencing of

LRP5 showed, in all three families, homozygosity for

mutation in LRP5: R570Q, R752G, and E1367K. Thus,

mutations in the LRP5 gene can cause autosomal

recessive as well as autosomal dominant FEVR.

Qin et al. screened 56 unrelated patients with FEVR

(31 familial and 25 simplex cases) for possible

mutations in LRP5 and Frizzled 4 (FZD4). Six novel

mutations in either LRP5 or FZD4 were identified in

six familial cases. Four novel mutations in LRP5 and

one known mutation in FZD4 were detected in three

simplex cases, and two of these patients carried



compound heterozygous mutations in LRP5. They also

demonstrated that reduced bone density is a common

feature in patients with FEVR who harbor LRP5

mutations.

Obesity

Note

Obesity is a growing health care problem and a risk

factor for common diseases such as diabetes, heart

disease, and hypertension.

LRP5 is highly expressed in many tissues, including

hepatocytes and pancreatic beta cells. Some evidence

has shown that LRP5 can bind apolipoprotein E (apoE),

which raises the possibility that LRP5 plays a role in

the hepatic clearance of apoE-containing chylomicron

remnants, a major plasma lipoprotein carrying diet-

derived cholesterol.

Using LRP5 knock-out mice model, Fujino et al.

showed that LRP5-deficient islets had a marked

reduction in the levels of intracellular ATP and Ca2+

in

response to glucose, and thereby glucose-induced

insulin secretion was decreased. The intracellular

inositol 1,4,5-trisphosphate (IP3) production in

response to glucose was also reduced in LRP5-/-

islets.

The authors suggested that Wnt/LRP5 signaling

contributes to the glucose-induced insulin secretion in

the islets.

Cytogenetics

Guo et al. performed genotyping of 27 single

nucleotide polymorphisms (SNPs), spaced 5 kb apart

on average and covering the full transcript length of the

LRP5 gene, using samples of 1873 Caucasian people

from 405 nuclear families. They found that SNP4

(rs4988300) and SNP6 (rs634008), located in block 2

(intron 1), showed significant associations with obesity

and BMI after Bonferroni correction (SNP4: p < 0.001

and p = 0.001, respectively; SNP6: p = 0.002 and

0.003, respectively). The common allele A for SNP4

and minor allele G for SNP6 were associated with an

increased risk of obesity. Significant associations were

also observed between the common haplotype A-G-G-

G of block 2 and obesity, BMI, fat mass, and PFM,

with global empirical values of p < 0.001, p < 0.001, p

= 0.003 and p = 0.074, respectively. They concluded

that intronic variants of the LRP5 gene are markedly

associated with obesity, possibly due to the role of

LRP5 in the Wnt signaling pathway or lipid

metabolism.

References Benson WE. Familial exudative vitreoretinopathy. Trans Am Ophthalmol Soc. 1995;93:473-521

Gong Y, Vikkula M, Boon L, Liu J, Beighton P, Ramesar R, Peltonen L, Somer H, Hirose T, Dallapiccola B, De Paepe A, Swoboda W, Zabel B, Superti-Furga A, Steinmann B, Brunner HG, Jans A, Boles RG, Adkins W, van den Boogaard MJ, Olsen BR, Warman ML. Osteoporosis-pseudoglioma

syndrome, a disorder affecting skeletal strength and vision, is assigned to chromosome region 11q12-13. Am J Hum Genet. 1996 Jul;59(1):146-51

Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med. 2002 May 16;346(20):1513-21

Hsieh JC, Lee L, Zhang L, Wefer S, Brown K, DeRossi C, Wines ME, Rosenquist T, Holdener BC. Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity. Cell. 2003 Feb 7;112(3):355-67

Van Wesenbeeck L, Cleiren E, Gram J, Beals RK, Bénichou O, Scopelliti D, Key L, Renton T, Bartels C, Gong Y, Warman ML, De Vernejoul MC, Bollerslev J, Van Hul W. Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet. 2003 Mar;72(3):763-71

Ferrari SL, Deutsch S, Choudhury U, Chevalley T, Bonjour JP, Dermitzakis ET, Rizzoli R, Antonarakis SE. Polymorphisms in the low-density lipoprotein receptor-related protein 5 (LRP5) gene are associated with variation in vertebral bone mass, vertebral bone size, and stature in whites. Am J Hum Genet. 2004 May;74(5):866-75

He X, Semenov M, Tamai K, Zeng X. LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development. 2004 Apr;131(8):1663-77

Hoang BH, Kubo T, Healey JH, Sowers R, Mazza B, Yang R, Huvos AG, Meyers PA, Gorlick R. Expression of LDL receptor-related protein 5 (LRP5) as a novel marker for disease progression in high-grade osteosarcoma. Int J Cancer. 2004 Mar;109(1):106-11

Jiao X, Ventruto V, Trese MT, Shastry BS, Hejtmancik JF. Autosomal recessive familial exudative vitreoretinopathy is associated with mutations in LRP5. Am J Hum Genet. 2004 Nov;75(5):878-84

Tamai K, Zeng X, Liu C, Zhang X, Harada Y, Chang Z, He X. A mechanism for Wnt coreceptor activation. Mol Cell. 2004 Jan 16;13(1):149-56

Toomes C, Bottomley HM, Jackson RM, Towns KV, Scott S, Mackey DA, Craig JE, Jiang L, Yang Z, Trembath R, Woodruff G, Gregory-Evans CY, Gregory-Evans K, Parker MJ, Black GC, Downey LM, Zhang K, Inglehearn CF. Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q. Am J Hum Genet. 2004 Apr;74(4):721-30

Zhang Y, Wang Y, Li X, Zhang J, Mao J, Li Z, Zheng J, Li L, Harris S, Wu D. The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd. Mol Cell Biol. 2004 Jun;24(11):4677-84

Ai M, Holmen SL, Van Hul W, Williams BO, Warman ML. Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass-associated missense mutations in LRP5 affect canonical Wnt signaling. Mol Cell Biol. 2005 Jun;25(12):4946-55

Cheung WM, Jin LY, Smith DK, Cheung PT, Kwan EY, Low L, Kung AW. A family with osteoporosis pseudoglioma syndrome due to compound heterozygosity of two novel mutations in the LRP5 gene. Bone. 2006 Sep;39(3):470-6

Guo YF, Xiong DH, Shen H, Zhao LJ, Xiao P, Guo Y, Wang W, Yang TL, Recker RR, Deng HW. Polymorphisms of the low-density lipoprotein receptor-related protein 5 (LRP5) gene are associated with obesity phenotypes in a large family-based association study. J Med Genet. 2006 Oct;43(10):798-803



Lindvall C, Evans NC, Zylstra CR, Li Y, Alexander CM, Williams BO. The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis. J Biol Chem. 2006 Nov 17;281(46):35081-7

Semenov MV, He X. LRP5 mutations linked to high bone mass diseases cause reduced LRP5 binding and inhibition by SOST. J Biol Chem. 2006 Dec 15;281(50):38276-84

Bhat BM, Allen KM, Liu W, Graham J, Morales A, Anisowicz A, Lam HS, McCauley C, Coleburn V, Cain M, Fortier E, Bhat RA, Bex FJ, Yaworsky PJ. Structure-based mutation analysis shows the importance of LRP5 beta-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling. Gene. 2007 Apr 15;391(1-2):103-12

Björklund P, Akerström G, Westin G. An LRP5 receptor with internal deletion in hyperparathyroid tumors with implications for deregulated WNT/beta-catenin signaling. PLoS Med. 2007 Nov 27;4(11):e328

Drenser KA, Trese MT. Familial exudative vitreoretinopathy and osteoporosis-pseudoglioma syndrome caused by a mutation in the LRP5 gene. Arch Ophthalmol. 2007 Mar;125(3):431-2

Mani A, Radhakrishnan J, Wang H, Mani A, Mani MA, Nelson-Williams C, Carew KS, Mane S, Najmabadi H, Wu D, Lifton RP. LRP6 mutation in a family with early coronary disease and metabolic risk factors. Science. 2007 Mar 2;315(5816):1278-82

Xiong DH, Lei SF, Yang F, Wang L, Peng YM, Wang W, Recker RR, Deng HW. Low-density lipoprotein receptor-related protein 5 (LRP5) gene polymorphisms are associated with

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Guo Y, Rubin EM, Xie J, Zi X, Hoang BH. Dominant negative LRP5 decreases tumorigenicity and metastasis of osteosarcoma in an animal model. Clin Orthop Relat Res. 2008 Sep;466(9):2039-45

Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, Schütz G, Glorieux FH, Chiang CY, Zajac JD, Insogna KL, Mann JJ, Hen R, Ducy P, Karsenty G. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell. 2008 Nov 28;135(5):825-37

Badders NM, Goel S, Clark RJ, Klos KS, Kim S, Bafico A, Lindvall C, Williams BO, Alexander CM. The Wnt receptor, Lrp5, is expressed by mouse mammary stem cells and is required to maintain the basal lineage. PLoS One. 2009 Aug 12;4(8):e6594

Björklund P, Svedlund J, Olsson AK, Akerström G, Westin G. The internally truncated LRP5 receptor presents a therapeutic target in breast cancer. PLoS One. 2009;4(1):e4243

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Zhong ZA, Williams BO. LRP5 (low density lipoprotein receptor-related protein 5). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):270-275.

Gene Section Review




MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)) Jan-Philipp Bach, Michael Bacher, Richard Dodel

Department of Neurology, Philipps-University Marburg, Rudolf-Bultmann-Strasse 8, 35039 Marburg,

Germany (JPB, MB, RD)


Online updated version : http://AtlasGeneticsOncology.org/Genes/MIFID41365ch22q11.html DOI: 10.4267/2042/44983


Identity

Other names: GIF, GLIF, MMIF

HGNC (Hugo): MIF

Location: 22q11.23

DNA/RNA

Description

0,84 kb; mRNA: 561 bp; 3 Exons.

Transcription

The promoter region contains no TATA box.

Pseudogene

There are no MIF pseudogenes in the human genome.

In contrast 5 pseudogenes have been described in the

murine genome.

Protein

Description

MIF is comprised of 115 amino acids with a molecular

weight of 12,5 kDa (Weiser et al., 1989). In addition,

research on the secondary structure revealed the

existence of two antiparallel alpha-helices and six beta-

pleated sheets with a high degree of similarity to MHC

molecules (Suzuki et al., 1996). MIF acts as a pro-

inflammatory protein, exists as a homo-trimer and

displays enzymatic action (Rosengren et al., 1996).

Expression

Widely.

Localisation

Intracellular, cytoplasm, cytosolic, near the plasma

membrane, perinuclear.

Function

MIF monomers are able to align in order to form a

homotrimeric molecule that is homologous to the

enzyme D-Dopachrome-tautomerase (Sun et al., 1996).

From this structural analysis, some researches

suggested that MIF may also display enzymatic activity

(Rosengren et al., 1996). To date, the physiological

importance of this enzymatic activity has not yet been

revealed. Interestingly, using ISO-1, a known inhibitor

of the enzyme D-Dopachrome-tautomerase led to

reduced activity of MIF (Lubetsky et al., 2002).

Therefore, it was hypothesized that this enzymatic

activity may be related to its proper functioning. The

protein MIF is involved in inducing angiogenesis,

promoting cell cycle progression, inhibiting apoptosis

and inhibiting lysing of tumor cells by NK cells

(Takahashi et al., 1998; Shimizu et al., 1999; Mitchell

and Bucala, 2000; Morrison et al., 2001; Fingerle-

Rowson et al., 2003). Recently, the CD74 molecule has

been suggested to act as a potential receptor for MIF

(Leng et al., 2003).

Homology

MIF shows homology to the enzyme D-Dopachrome

tautomerase.

Mutations

Note

Not known.

MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)) Bach JP, et al.


Implicated in

Colon cancer

Disease

MIF is upregulated in tumors as well as precancerous

lesions (Wilson et al., 2005). They examined patients

suffering from adenomas. In addition, an animal model

of adenomatous polyposis coli was used. In both

settings, the authors describe an increase in MIF

mRNA levels in the diseases group when this was

compared to healthy controls. Interestingly, in a mouse

model of intestinal tumorigenesis, MIF deletion leads

to a significant decrease in tumor size. In addition,

reduced angiogenesis was taking place. The

involvement of MIF in angiogenesis was also shown by

Ogawa et al., using a mouse model of colon cancer

(Ogawa et al., 2000). The application of MIF

antibodies leads to suppression of angiogenesis in this

disease model. Further work by Sun et al. demonstrated

an involvement of MIF in tumor cell migration (Sun et

al., 2005). They used siRNA technique and were able

to show inhibition of cell migration after addition of

siRNA directed against MIF. In an animal model, they

injected colon cancer cells into mice portal vein after

pretreatment with siRNA. The number of liver

metastases was significantly reduced in the pretreated

model. In summary, there is sufficient evidence to

assume a role of MIF in both angiogenesis and tumor

cell migration in colon cancer (Bach et al., 2009).

Prognosis

MIF expression is correlated with outcome (Legendre

et al., 2003). Legendre et al. (2003) examined MIF

distribution in 99 specimens of colorectal cancer.

Primarily, they applied immunohistochemistry. They

describe that the expression of MIF (and also galectin-

3) were increased in tumor tissue compared to normal

tissue. For MIF, they could provide evidence that in

Dukes C or D tumors with high concentration of MIF,

this was associated with significantly better prognosis

than in tumors with low MIF concentrations. They

suggest that MIF could be used to identify patients at

risk needing more aggressive treatment strategies.

Melanoma

Disease

MIF inhibits lysis of melanoma cells by NK cells

(Repp et al., 2000). This was shown by Apte et al., who

demonstrated that NK cells are prevented from cell

lysis of melanoma cells (Apte et al., 1998). This

underlines the influence of MIF on the immune system.

In addition, it enhances tumor cell proliferation.

Prognosis

To date, there is no clear association between MIF

concentration and prognosis in melanoma cells.

Prostate cancer

Disease

MIF was shown to be abundant in prostate cancer

(Meyer-Siegler et al., 1998). In addition, MIF was

shown to influence cell viability and invasiveness.

Specifically in prostate cancer, androgen independent

cancer cells relied on MIF activated pathways in order

to grow and for their invasiveness. Androgen

dependent tumor cells did not require these signal

transduction pathways. Meyer-Siegler et al. were able

to show that CD74, a potential MIF surface receptor,

was abundant in androgen-independent tumor cells

(Meyer-Siegler et al., 2006). Receptor blockage as well

as strategies to reduce MIF resulted in decreased cell

proliferation, MIF secretion and invasion of tumor

cells.

Prognosis

MIF expression clearly correlates with disease

progression (Meyer-Siegler et al., 2002). In androgen

independent prostate cancer, cells require MIF

activated signal transduction pathways for invasion and

growth, in contrast to androgen dependent tumor cells.

In a study by Meyer-Siegler, serum MIF concentration

was measured in dependence of Gleason score in

patients with prostate cancer (Meyer-Siegler et al.,

2002). They were able to show that increased MIF

concentration is positively associated with a Gleason

Score greater than 5. In addition, even in patients with

normal prostate-specific antigen, MIF concentration

was increased. From their data, they conclude that MIF

may be a suitable biomarker for prostate cancer.

Lung adenocarcinoma

Note

Treatment with siRNA leads to a significant reduction

in cell invasiveness and cell migration (Rendon et al.,

2007), paralled by a reduction of a Rho GTPase. MIF

overexpression led to adverse effects.

Prognosis

Kamimura and colleagues analysed the role of MIF

with respect to prognosis in lung cancer (Kamimura et

al., 2000). They used immunofluorescence staining in

primary lung tissue that had been obtained surgically.

They were able to demonstrate a diffuse staining

pattern within the cytoplasm. Furthermore, sometimes,

a nuclear staining was also observed. Interestingly, the

authors were able to demonstrate a correlation between

a lack of nuclear staining and a poorer prognosis.

From this observation, they concluded that MIF might

play different roles with respect to subcellular

localization. It needs to be mentioned however, that

only thirty-eight cases were reviewed.

Glioblastoma

Note

MIF is involved in angiogenesis and cell cycle

regulation. It was demonstrated that MIF expression is

induced following hypoxia and hypoglycemia (Bacher



et al., 2003), which are both considered activators of

angiogenesis. Using immunofluorescence techniques,

MIF could be demonstrated around necrotic tissue and

in blood vessels surrounding tumor cells. Therefore it

was concluded that neovascularization is enhanced by

MIF. In line with this observation is the work published

by Munaut et al. (2002). They demonstrated that there

is a correlation between MIF expression and the

expression of vascular endothelial growth factor

(VEGF). They analysed primary glioblastoma tissue

using RT-PCR and immmunohistochemistry and they

were able to show a strong correlation between MIF

expression and VEGF mRNA concentration. >From

these data, it was suggested that there may be a

common triggering factor. Inactivation of p53 may be

of central importance, as it is a common event in

glioblastoma progression (Bach et al., 2009). In

addition, p53 inactivation is also involved in increased

VEGF expression (Kieser et al., 1994). Since the

angiogenic potential of a tumor is vital for formation of

metastases, it may be a potential target for antitumor

therapy.

Hepatocellular carcinoma

Note

MIF expression and increased angiogenesis were also

demonstrated in hepatocellular carcinoma following

hypoxia (Hira et al., 2005).

Prognosis

MIF overexpression correlates with negative prognosis

(Hira et al., 2005). 56 samples of hepatocellular

carcinoma were analysed using Western blot and

correlated these values with clinical parameters. In

addition, they used immunohistochemistry. They

showed that MIF expression was correlated with high

alpha fetoprotein level and the recurrence of hepatic

tumor. In addition, tumor free survival was reduced

when MIF expression was increased. In addition,

increased mRNA concentrations can be seen (Ren et

al., 2003).

References Weiser WY, Temple PA, Witek-Giannotti JS, Remold HG, Clark SC, David JR. Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7522-6

Kieser A, Weich HA, Brandner G, Marmé D, Kolch W. Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene. 1994 Mar;9(3):963-9

Rosengren E, Bucala R, Aman P, Jacobsson L, Odh G, Metz CN, Rorsman H. The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction. Mol Med. 1996 Jan;2(1):143-9

Sun HW, Swope M, Cinquina C, Bedarkar S, Bernhagen J, Bucala R, Lolis E. The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer. Protein Eng. 1996 Aug;9(8):631-5

Suzuki M, Sugimoto H, Nakagawa A, Tanaka I, Nishihira J, Sakai M. Crystal structure of the macrophage migration inhibitory factor from rat liver. Nat Struct Biol. 1996 Mar;3(3):259-66

Apte RS, Sinha D, Mayhew E, Wistow GJ, Niederkorn JY. Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege. J Immunol. 1998 Jun 15;160(12):5693-6

Meyer-Siegler K, Fattor RA, Hudson PB. Expression of macrophage migration inhibitory factor in the human prostate. Diagn Mol Pathol. 1998 Feb;7(1):44-50

Takahashi N, Nishihira J, Sato Y, Kondo M, Ogawa H, Ohshima T, Une Y, Todo S. Involvement of macrophage migration inhibitory factor (MIF) in the mechanism of tumor cell growth. Mol Med. 1998 Nov;4(11):707-14

Shimizu T, Abe R, Nakamura H, Ohkawara A, Suzuki M, Nishihira J. High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis. Biochem Biophys Res Commun. 1999 Nov 2;264(3):751-8

Kamimura A, Kamachi M, Nishihira J, Ogura S, Isobe H, Dosaka-Akita H, Ogata A, Shindoh M, Ohbuchi T, Kawakami Y. Intracellular distribution of macrophage migration inhibitory factor predicts the prognosis of patients with adenocarcinoma of the lung. Cancer. 2000 Jul 15;89(2):334-41

Mitchell RA, Bucala R. Tumor growth-promoting properties of macrophage migration inhibitory factor (MIF). Semin Cancer Biol. 2000 Oct;10(5):359-66

Ogawa H, Nishihira J, Sato Y, Kondo M, Takahashi N, Oshima T, Todo S. An antibody for macrophage migration inhibitory factor suppresses tumour growth and inhibits tumour-associated angiogenesis. Cytokine. 2000 Apr;12(4):309-14

Repp AC, Mayhew ES, Apte S, Niederkorn JY. Human uveal melanoma cells produce macrophage migration-inhibitory factor to prevent lysis by NK cells. J Immunol. 2000 Jul 15;165(2):710-5

Morrison H, Sherman LS, Legg J, Banine F, Isacke C, Haipek CA, Gutmann DH, Ponta H, Herrlich P. The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44. Genes Dev. 2001 Apr 15;15(8):968-80

Lubetsky JB, Dios A, Han J, Aljabari B, Ruzsicska B, Mitchell R, Lolis E, Al-Abed Y. The tautomerase active site of macrophage migration inhibitory factor is a potential target for discovery of novel anti-inflammatory agents. J Biol Chem. 2002 Jul 12;277(28):24976-82

Meyer-Siegler KL, Bellino MA, Tannenbaum M. Macrophage migration inhibitory factor evaluation compared with prostate specific antigen as a biomarker in patients with prostate carcinoma. Cancer. 2002 Mar 1;94(5):1449-56

Munaut C, Boniver J, Foidart JM, Deprez M. Macrophage migration inhibitory factor (MIF) expression in human glioblastomas correlates with vascular endothelial growth factor (VEGF) expression. Neuropathol Appl Neurobiol. 2002 Dec;28(6):452-60

Bacher M, Schrader J, Thompson N, Kuschela K, Gemsa D, Waeber G, Schlegel J. Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme. Am J Pathol. 2003 Jan;162(1):11-7

Fingerle-Rowson G, Petrenko O, Metz CN, Forsthuber TG, Mitchell R, Huss R, Moll U, Müller W, Bucala R. The p53-



dependent effects of macrophage migration inhibitory factor revealed by gene targeting. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9354-9

Legendre H, Decaestecker C, Nagy N, Hendlisz A, Schüring MP, Salmon I, Gabius HJ, Pector JC, Kiss R. Prognostic values of galectin-3 and the macrophage migration inhibitory factor (MIF) in human colorectal cancers. Mod Pathol. 2003 May;16(5):491-504

Leng L, Metz CN, Fang Y, Xu J, Donnelly S, Baugh J, Delohery T, Chen Y, Mitchell RA, Bucala R. MIF signal transduction initiated by binding to CD74. J Exp Med. 2003 Jun 2;197(11):1467-76

Ren Y, Tsui HT, Poon RT, Ng IO, Li Z, Chen Y, Jiang G, Lau C, Yu WC, Bacher M, Fan ST. Macrophage migration inhibitory factor: roles in regulating tumor cell migration and expression of angiogenic factors in hepatocellular carcinoma. Int J Cancer. 2003 Oct 20;107(1):22-9

Hira E, Ono T, Dhar DK, El-Assal ON, Hishikawa Y, Yamanoi A, Nagasue N. Overexpression of macrophage migration inhibitory factor induces angiogenesis and deteriorates prognosis after radical resection for hepatocellular carcinoma. Cancer. 2005 Feb 1;103(3):588-98

Sun B, Nishihira J, Yoshiki T, Kondo M, Sato Y, Sasaki F, Todo S. Macrophage migration inhibitory factor promotes

tumor invasion and metastasis via the Rho-dependent pathway. Clin Cancer Res. 2005 Feb 1;11(3):1050-8

Wilson JM, Coletta PL, Cuthbert RJ, Scott N, MacLennan K, Hawcroft G, Leng L, Lubetsky JB, Jin KK, Lolis E, Medina F, Brieva JA, Poulsom R, Markham AF, Bucala R, Hull MA. Macrophage migration inhibitory factor promotes intestinal tumorigenesis. Gastroenterology. 2005 Nov;129(5):1485-503

Meyer-Siegler KL, Iczkowski KA, Leng L, Bucala R, Vera PL. Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells. J Immunol. 2006 Dec 15;177(12):8730-9

Rendon BE, Roger T, Teneng I, Zhao M, Al-Abed Y, Calandra T, Mitchell RA. Regulation of human lung adenocarcinoma cell migration and invasion by macrophage migration inhibitory factor. J Biol Chem. 2007 Oct 12;282(41):29910-8

Bach JP, Deuster O, Balzer-Geldsetzer M, Meyer B, Dodel R, Bacher M. The role of macrophage inhibitory factor in tumorigenesis and central nervous system tumors. Cancer. 2009 May 15;115(10):2031-40


Bach JP, Bacher M, Dodel R. MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):276-279.





NEU3 (sialidase 3 (membrane sialidase)) Kazunori Yamaguchi, Taeko Miyagi

Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan (KY), Cancer

Glycosylation Research, Tohoku Pharmaceutical University, Sendai 981-8558, Japan (TM)


Online updated version : http://AtlasGeneticsOncology.org/Genes/NEU3ID44505ch11q13.html DOI: 10.4267/2042/44984


Identity

Other names: FLJ12388, SIAL3

HGNC (Hugo): NEU3

Location: 11q13.4

DNA/RNA

Description

The NEU3 gene spans 22 kb, consists of 4 exons and 3

introns. It is a member of sialidase family consisting of

NEU1, NEU2, NEU3, and NEU4.

Transcription

Northern blot analysis reveals 2.5 kb and 7 kb

transcripts of NEU3 gene, possessing a common open

reading frame of 1284 bp. NEU3 gene expression is

diversely regulated by Sp1/Sp3 transcription factors

which are recently considered to play critical roles in

regulating the transcription of genes involved in cell

growth and tumorigenesis.

Protein

Description

Deduced amino acid sequence of human NEU3

comprises of 428 amino acids and contains RIP box at

N-terminal region and three Asp boxes in the center of

the amino acid sequence. These motifs are commonly

found in sialidases of microorganisms and vertebrates.

The RIP motif is a part of active site and mutation of

this motif led to decreased enzymatic activity. The Asp

box is thought to participate in proper 3D structure

formation of NEU3.

Expression

The gene is ubiquitously expressed with relatively

higher levels in skeletal muscle, heart, and testis. The

expression is upregulated during tumorigenesis,

neuronal differentiation, T cell activation, and

monocyte differentiation. Abnormal upregulation of

NEU3 is observed in various

NEU3 (sialidase 3 (membrane sialidase)) Yamaguchi K, Miyagi T


human neoplasms including colon, renal, ovarian and

prostate cancers, except for the down-regulation in

acute lymphoblastic leukemia.

It is interesting to note that the transgenic mice

ectopically expressing human NEU3 develop impaired

insulin signaling and insulin-resistant diabetes mellitus

by 18-22 weeks.

Localisation

Biochemical fractionation shows NEU3 to be localized

in membrane fractions, especially in raft or caveolae

membrane microdomains. In immunofluorescence

studies, the bovine and mouse Neu3 sialidases are

mostly detected on the cell surface, but the human

NEU3 may exist also in other cellular membrane

components and can mobilize to membrane ruffles

together with Rac-1 in response to growth stimuli such

as EGF, enhancing cell movement.

Function

NEU3 sialidase removes sialic acid moiety of

gangliosides. It hardly acts on glycoproteins,

oligosaccharides, and an artificial substrate 4MU-

NeuAc. NEU3 alters gangliosides composition of

tissues and cells, and leads to modulation of signal

transduction such as EGFR (epidermal growth factor

receptor) and IR (insulin receptor) signaling. Besides,

NEU3 has been shown to associate with signaling

molecules including EGFR, Grb2, caveolin-1, and Rac.

In addition to the catalytic reaction as a sialidase, the

interaction with the protein molecules may also give an

influence on the NEU3 function. Disturbance of

signaling by abnormal upregulation of NEU3 is likely a

possible cause of tumorigenesis or diabetes mellitus.

Homology

The NEU3 amino acid sequence shows 28% identity to

NEU1, 42% to NEU2, and 45% to NEU4. NEU3

orthologs are identified in bovine, mouse, and rat.

Implicated in

Colon cancer

Note

NEU3 shows higher enzymatic activity and mRNA

level in colon tumors as compared to adjacent normal

mucosa. In tumor cells, lactosylceramide, one of the

products of NEU3 enzymatic reaction, accumulates in

tumor cells. Over expression of NEU3 or exogenous

addition of lactosylceramide to the cell culture confer

resistance to sodium butyrate-induced apoptosis. On

the other hand, silencing of NEU3 by siRNA causes

induction of apoptosis in cancer cells accompanied

with suppression of EGFR signaling, suggesting that

survival of tumor cells is addictive to NEU3

expression. Interestingly, NEU3-knock down in normal

cells including primary culture of keratinocytes or

fibroblasts does not cause growth arrest nor apoptosis.

Transgenic mice ectopically expressing human NEU3

shows upregulation of EGFR signaling, lower

induction of apoptosis, and high incidence of ACF

(aberrant crypt foci) formation in colon mucosa cells

upon administration of azoxymethane (AOM), a

carcinogen for colonic tumorigenesis.

Renal cell carcinoma

Note

Renal cell carcinoma shows upregulation of NEU3

along with high expression of IL6. IL6 appears to

increase NEU3 gene expression in ACHN cells, and the

increase brings about enhanced activation of PI3K and

Akt upon IL6 administration, resulting in suppression

of apoptosis.

Type II diabetes mellitus

Note

NEU3 transgenic mice develop impaired insulin

signaling and insulin-resistant diabetes mellitus by 18-

22 weeks, associated with hyper-insulinemia, islet

hyperplasia and increase in the beta-cell mass. As

compared to the wild type, insulin-stimulated

phosphorylation of the insulin receptor and insulin

receptor substrate I is significantly reduced, and

activities of phosphatidylinositol 3-kinase and glycogen

synthase are also decreased. In muscle extracts,

association of tyrosine-phosphorylated NEU3 with

Grb2 occurs in response to insulin, together with

accumulation of ganglioside GM1 and GM2.

Involvement of NEU3 in cancer progression and

development of diabetes suggests that these diseases

might be closely related to each other in pathogenesis,

given the recent epidemiological reports of higher

cancer risk in diabetic patients than in controls.

References Wada T, Yoshikawa Y, Tokuyama S, Kuwabara M, Akita H, Miyagi T. Cloning, expression, and chromosomal mapping of a human ganglioside sialidase. Biochem Biophys Res Commun. 1999 Jul 22;261(1):21-7

Monti E, Bassi MT, Papini N, Riboni M, Manzoni M, Venerando B, Croci G, Preti A, Ballabio A, Tettamanti G, Borsani G. Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane. Biochem J. 2000 Jul 1;349(Pt 1):343-51

Kakugawa Y, Wada T, Yamaguchi K, Yamanami H, Ouchi K, Sato I, Miyagi T. Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10718-23

Monti E, Preti A, Venerando B, Borsani G. Recent development in mammalian sialidase molecular biology. Neurochem Res. 2002 Aug;27(7-8):649-63

Wang Y, Yamaguchi K, Wada T, Hata K, Zhao X, Fujimoto T, Miyagi T. A close association of the ganglioside-specific sialidase Neu3 with caveolin in membrane microdomains. J Biol Chem. 2002 Jul 19;277(29):26252-9

Ueno S, Saito S, Wada T, Yamaguchi K, Satoh M, Arai Y, Miyagi T. Plasma membrane-associated sialidase is up-regulated in renal cell carcinoma and promotes interleukin-6-

NEU3 (sialidase 3 (membrane sialidase)) Yamaguchi K, Miyagi T


induced apoptosis suppression and cell motility. J Biol Chem. 2006 Mar 24;281(12):7756-64

Wada T, Hata K, Yamaguchi K, Shiozaki K, Koseki K, Moriya S, Miyagi T. A crucial role of plasma membrane-associated sialidase in the survival of human cancer cells. Oncogene. 2007 Apr 12;26(17):2483-90

Miyagi T. Aberrant expression of sialidase and cancer progression. Proc Jpn Acad Ser B Phys Biol Sci. 2008;84(10):407-18

Miyagi T, Wada T, Yamaguchi K. Roles of plasma membrane-associated sialidase NEU3 in human cancers. Biochim Biophys Acta. 2008 Mar;1780(3):532-7

Miyagi T, Wada T, Yamaguchi K, Hata K, Shiozaki K. Plasma membrane-associated sialidase as a crucial regulator of transmembrane signalling. J Biochem. 2008 Sep;144(3):279-85

Miyagi T, Wada T, Yamaguchi K, Shiozaki K, Sato I, Kakugawa Y, Yamanami H, Fujiya T. Human sialidase as a cancer marker. Proteomics. 2008 Aug;8(16):3303-11

Shiozaki K, Yamaguchi K, Sato I, Miyagi T. Plasma membrane-associated sialidase (NEU3) promotes formation of

colonic aberrant crypt foci in azoxymethane-treated transgenic mice. Cancer Sci. 2009 Apr;100(4):588-94

Tringali C, Lupo B, Cirillo F, Papini N, Anastasia L, Lamorte G, Colombi P, Bresciani R, Monti E, Tettamanti G, Venerando B. Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3. Cell Death Differ. 2009 Jan;16(1):164-74

Mandal C, Tringali C, Mondal S, Anastasia L, Chandra S, Venerando B, Mandal C. Down regulation of membrane-bound Neu3 constitutes a new potential marker for childhood acute lymphoblastic leukemia and induces apoptosis suppression of neoplastic cells. Int J Cancer. 2010 Jan 15;126(2):337-49

Yamaguchi K, Koseki K, Shiozaki M, Shimada Y, Wada T, Miyagi T. Regulation of plasma-membrane-associated sialidase NEU3 gene by Sp1/Sp3 transcription factors. Biochem J. 2010 Aug 15;430(1):107-17


Yamaguchi K, Miyagi T. NEU3 (sialidase 3 (membrane sialidase)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):280-282.

Gene Section Review




NPY1R (neuropeptide Y receptor Y1) Massimiliano Ruscica, Elena Dozio, Luca Passafaro, Paolo Magni

Dipartimento di Endocrinologia, Fisiopatologia e Biologia Applicata, Universita degli Studi di Milano, Italy

(MR, LP, PM), Dipartimento di Morfologia Umana e Scienze Biomediche "Citta Studi", Universita degli

Studi di Milano, Italy (ED)


Online updated version : http://AtlasGeneticsOncology.org/Genes/NPY1RID44260ch4q32.html DOI: 10.4267/2042/44985


Identity

Other names: NPYR

HGNC (Hugo): NPY1R

Location: 4q32.2

DNA/RNA

Note

History: The human NPY1R cDNA was cloned from a

human brain cDNA library. The NPY1R was the first

to be characterized, when the expression pattern of an

orphan receptor was recognized to overlap with the

distribution of NPY in brain. NPY receptors belong to

the large superfamily of G-protein-coupled receptors.

Many of these receptor genes lack introns, supporting

the proposition that they were created via RNA-

mediated transpositional events. Differently from the

other NPY receptor isoforms NPY1R is the only one

containing a single 97-base pairs (bp) intron in the

coding region following the fifth transmembrane

domain.

Description

A 14-kilobase pair (kb) region of genomic DNA

encoding the human neuropeptide Y Y1-receptor gene

including 3'- and 5'- flanking sequences is localized to

chromosome 4. It encompasses 8632 bp of DNA

(4q32.2) between 164245117 and 164253748 bp. The

overall sequence of the gene consists of approximately

10 kb. The genomic structure presents a 6-kb intron

situated approximately 150 bp upstream of the start

codon within the 5'-untranslated region (5'UTR), as

well as a small intron within the coding region.

The human NPY1R gene is divided into three exons:

exon 1 (115 bp), exon 2 (850 bp), and exon 3 (1749

bp). In particular, the NPY1R gene contains three

alternative exon 1 sequences (80, 110, and 106 bp)

located 6.4, 18.4, and 23.9 kb upstream of exon 2. Exon

1A is located 6.4 kb upstream of exon 2; exon 1B was

found a further 12 kb upstream exon 1A, and exon 1C

another 5.5 kb upstream of exon 1B. These alternative

5' exons allow the regulation of tissue-specific

expression of the receptor. The first 57 nucleotides of

the 5'UTR of the human NPY1R mRNA are separated

by a 6-kb intron from the second exon. The second

intron 97 bp, containing an in-frame stop codon, is

located at nucleotide 908 in the protein coding region

after the fifth transmembrane domain between exon 2

and 3. Moreover, as shown by Nakamura, mouse

NPY1R gene contains an alternate exon 4 located over

15 kb downstream of exon 3.

Transcription

The human cDNA encodes a protein of 384 amino-

acids (aa) in lenght that is preceded by approximately

200 bp of 5'UTR sequence.

Exon/intron structure and splice sites in the 5'UTR of the human NPY1R gene.

NPY1R (neuropeptide Y receptor Y1) Ruscica M, et al.


Figure A. Y1R affinity for various PP-family hormones and their C-terminal sequences. Figure B. Homo sapiens Neuropeptide Y receptor type 1 (384 aa).

Protein

Note

The NPY Y1 receptor subtype (Y1R) was the first to be

cloned in the rat, and subsequently in human and

mouse. This receptor is as conserved as its ligand

(NPY) throughout evolution and mammalian and non-

mammalian species. The complete NPY1-36 molecule is

necessary for NPY to bind to Y1R. Any proteolytic

process leading to alterations in the NH2-terminal

domain essentially abolishes the ability of NPY to bind

to Y1R. Therefore, NH2-terminally truncated NPY

fragments such as NPY2-36, or NPY3-36 have little or no

affinity for the Y1R. Modification of COOH-terminal

residues does not affect agonist binding. Thus, it has

been established that the NH2 terminus is essential for

NPY to activate Y1R. The pharmacological profile of

the Y1R is characterized by high affinity for NPY,

PYY and the corresponding analogs containing Pro34

and low affinity for the N-terminally truncated analogs

and for PP.

Description

Y1R has seven putative transmembrane domains

associated with G-protein (GPCR). In the N-terminal

portion Y1R presents potential sites of glycosylation

and in the second extracellular loop, four extracellular

cysteines in position 33, 113, 198 and 296 which may

form two disulfide bridges (Cys 33 and 296; Cys 133

and 198). Phosphorylation sites are present in the

intracellular domain (cysteine in the C-terminal portion

at position 338). These cysteines may also explain the

capability of palmitate residues to bind to the receptor.

As observed for many GPCR, Y1R is internalized

together with its ligand into endosomes and recycled to

the cell surface within 60 minutes upon agonist

stimulation. Moreover, Y1R is able to form

homodimers.

Expression

The expression of the human NPY Y1 receptor has

been studied extensively by using

immunohistochemical methods, in situ hybridization

experiments and reverse-transcription polymerase chain

reaction (RT-PCR; mRNA detection). The human

NPY1R is expressed in both central nervous system

(i.e., cerebral cortex, thalamus and amigdala) and

periphery (i.e., heart, kidneys, gastrointestinal tract, as

well as blood vessels).

Nervous system

The NPY Y1R is widely distributed in the central

nervous system. A study conducted on four normal

human brains revealed that high levels of Y1R receptor

mRNA were expressed in cortical areas and in the

claustrum, while moderate levels were present in the

nucleus accumbens, caudate nucleus, putamen,

amygdaloid nuclei and arcuate and paraventricular

nuclei of the hypothalamus. Moreover, a study

conducted on prefrontal cortex of subjects affected by

bipolar disorder, major depression, or schizophrenia

revealed a progressive age-related decline in the

expression of Y1R mRNA associated with a lack of

coexpression with NPY neurons. Interestingly, there

was no significant effect of suicide as a cause of death

on Y1R mRNA expression levels. In fact, subjects with

suicide as a cause of death tended to have higher Y1R

mRNA expression levels, but these individuals were

among the youngest ones (45 years old) in the

population studied.

Periphery

Peripherally, Y1Rs are expressed mainly in arteries and

veins, where they are associated with vasoconstriction

and potentiation of other vasoconstrictors of neurogenic

origin. Although limited, there is evidence of

prejunctional Y1R inhibition of neurotransmitter

release. Nonetheless, NPY Y1R is primarily located

postjunctionally on vascular smooth muscle cells.

1) Colon

In vitro receptor autoradiography ([125

I]PYY)

performed on normal human colonic tissue obtained

from nine patients showed that Y1R is distributed only

in vessels. No measurable levels of subtype Y1 was

identified in smooth muscle, mucosa, muscularis

mucosae, as well as in lymphoid follicles, myoenteric

and submucosal plexus.

2) Heart

A study conducted on 20-week old fetal human hearts

showed that Y1R is present on right ventricular

endocardial endothelial cells. In particular, it is highly



expressed at the level of the nucleus specifically at the

perinucleoplasm and nuclear membrane levels, while

lower levels were detected in the cytoplasm and the

plasma membrane.

3) Dental pulp

NPY Y1R proteins were present in solubilized

membrane preparations of both healthy and inflamed

human gingival tissue by Western blotting. Major

immunoreactive bands were detected at approximately

55 kDa due to a glycosylated form of the native

receptor protein. By using the SwissProt glycosylation

prediction packages NetNGlyc and NetOGly, authors

confirmed that the human Y1R has potential N- and O-

glycosylation sites. The expression of Y1R protein in

both healthy and inflamed gingival tissue suggests that

NPY could act via the Y1R to exert its tonic effects.

Moreover, Y1R was expressed in human dental pulp

with evidence of increased expression in carious

compared with noncarious teeth. Y1R were localized to

nerve fibres and inflammatory cells in the dental pulp

of carious teeth.

4) Achilles tendons

Y1R is expressed in the tenocytes in the Achilles

tendon. Specifically, Y1R is present within the smooth

muscle of the blood vessel walls, but not in the

endothelial layer of calcaneal tendons.

5) Skin

In human tissues, RT-PCR and immunocytochemistry

studies suggested that Y1R is the primary receptor in

human cutaneous circulation, supporting the findings

that local non-noradrenergic mechanisms are entirely

Y1R-based. Skin blood flow in humans is controlled

through two branches of the sympathetic nervous

system: a vasoconstrictor system and an active

vasodilator system of uncertain neurotransmitter. In

this context, NPY showed a vasoconstrictor effect in

human subcutaneous arteries that had been dissected

out of the abdominal regions from patients who

underwent nonvascular disease surgeries (e.g., hernia).

NPY decreased cutaneous blood flow via Y1R, with

evidence for the additional involvement of

postjunctional Y2R. This ability of NPY and Y1R to

affect skin vascular conductance varies in accordance

with relative innervations at specific sites.

Localisation

NPY Y1R is a seven transmembrane receptor which

has all the characteristics of the GPCR family including

potential glycosylation sites in the N-terminal portion

and in the second extra-cellular loop.

Function

NPY has been demonstrated to be involved in

mitogenic pathways and stimulate cell proliferation via

the Y1R. The activation of Y1R is generally associated

with reduction of cAMP accumulation, increase of

intracellular free calcium concentration ([Ca2+

]i), and

modulation of the MAPK pathway via several signaling

molecules, including the protein kinase C (PKC).

Y1R has been involved in several NPY-induced

responses, such as activation of neuroendocrine axes,

vasoconstriction, anxiolysis, as well as the stimulation

of food intake. Moreover, Y1R mediates emotional

behavior, stress response, and ethanol consumption.

The prototype of NPY Y1R-mediated responses is

vasoconstriction. Specifically, the physiological role of

the Y1R subtype was demonstrated in mice lacking

Y1R expression, which show no blood pressure

response to NPY, but a normal response to

norepinephrine. Y1R knockout mice have normal blood

pressure, suggesting that the Y1R does not play a

crucial role in maintaining blood pressure homeostasis

in unstimulated conditions. However, Y1R has been

also involved in other NPY-induced responses, such as

stimulation of food intake and activation of

neuroendocrine axes. In particular, Y1R and Y5R, both

expressed in hypothalamic regions involved in the

control of feeding, represent the most likely candidates

for mediating the appetite stimulatory capacity of NPY.

Mice lacking Y1R showed an increased body weight

due to a low-energy expenditure rather than high-

energy intake. In fact, these mice had a decreased

metabolic rate secondary to decreased locomotor

activity and movement associated thermogenesis.

Homology

The human Y1R subtype shares closest aa identity with

the Y4R subtype (42%) and the non-active, human

form of the y6 subtype (51%).

Mutations

Note

In 2004 Ramanathan described a case of autism in

which a 19 megabase on chromosome 4q, spanning

4q32 to 4q34, was detected. Being involved in the

deletion, those genes which are abundantly expressed

in the brain, Y1R and Y5R resulted implicated. In this

context, being the neuroproliferative effect of NPY in

the hippocampus mediated through the neuropeptide Y

Y1R, the authors postulate that the effect of NPY on

learning and memory may be mediated through NPY

neurogenesis.

Okahisa et al. described that genetic variants of

rs7687423 of the NPY1R gene may alter the subjective

effects of methamphetamine and result in susceptibility

to dependence. Because NPY1R mRNA changes were

observed in peripheral tissues and the brain in

schizophrenia patients, these findings may also indicate

that the NPY1R gene is involved in vulnerability to

methamphetamine-induced psychosis because almost

all of the analyzed subjects with methamphetamine

dependence had comorbid methamphetamine

psychosis.



Implicated in

Various tumors

Note

NPY receptors are mainly expressed in specific

endocrine tumors and epithelial malignancies as well as

in embryonal tumors. In endocrine tumors, NPY

receptors are present in steroid hormone producing

tumors, namely adrenal cortical adenomas, carcinomas,

ovarian granulosa cell tumors, Sertoli-Leydig cell

tumors, and in catecholamine producing tumors, i.e.

pheochromocytomas and paragangliomas. Based on

pharmacological displacement experiments, in addition

to tumor cells, intra- and peritumoral blood vessels

express Y1Rs. The Y1R-expressing blood vessels are

mainly small and medium-sized arteries.

Prostate cancer

Note

Prostate cancer represents one of the most common

malignant diseases among men in the Western world. It

is initially androgen dependent and it may later

progress to the androgen-independent stage, which is

associated with a lack of efficacy of the available

hormonal therapy. This tumoral progression appears to

be promoted at least in part by several growth factors

and neurohormones. Within this context, we showed

that Y1R protein is expressed in three human prostate

cancer cell lines (LNCaP -androgen dependent-,

DU145 and PC3 -androgen independent-) and that

NPY treatment reduced the proliferation of LNCaP and

DU145 cells and increased that of PC3 cells.

Interestingly, the Y1R antagonist BIBP3226 abolished

such effects, suggesting a mandatory role of Y1-R in

this process. Moreover, these effects are associated

with a clone-specific pattern of intracellular signaling

activation, including a peculiar time-course of

MAPK/ERK1/ERK2 phosphorylation (long-lasting in

DU145 and transient in PC3 cells).

Breast cancer

Note

Breast cancer accounts for almost 1/3 of all incident

cases of cancer in women. Interestingly, the expression

of NPY-Rs has been found in 85% of primary breast

cancer in a series of 95 cases, and in 100% of lymph

node metastases of receptor-positive primaries, where

Y1R expression predominated and was often present in

high density and great homogeneity. In normal breast

tissue, however, Y1R was only found in a minority of

the cases and concomitantly with Y2R, which seemed

to be predominant in non-neoplastic breast. The

neoplastic condition of breast tissue may thus induce a

switch of expression from Y2R to Y1R.

Moreover, a functional interplay between estrogen and

Y1R has been shown in a human breast cancer cell line

responsive to this steroid, where estrogen was found to

increase Y1R expression, which in turn negatively

regulated estrogen-stimulated cell proliferation.

Pheochromocytoma and paraganglioma

Note

The frequency of NPY receptors (NPYRs) expression

in pheochromocytomas and paragangliomas was found

to be 35% and 61%, respectively. Both Y1R and Y2R

are expressed, with a higher density of Y2R in

paragangliomas than in pheochromocytomas, whereas

the density of Y1R is comparably low in both tumor

categories. NPYRs, mainly Y1R and Y5R, are also

expressed in the Ewing's sarcoma family of tumors,

other related neural crest-derived tumors, where

activation of these receptors has been reported to

regulate cell proliferation, as shown in the SK-N-MC

cell line, an Ewing's sarcoma family of tumors

expressing Y1R, where NPY has been shown to inhibit

cell growth.

References Soares MB, Schon E, Henderson A, Karathanasis SK, Cate R, Zeitlin S, Chirgwin J, Efstratiadis A. RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon. Mol Cell Biol. 1985 Aug;5(8):2090-103

Wahlestedt C, Yanaihara N, Håkanson R. Evidence for different pre-and post-junctional receptors for neuropeptide Y and related peptides. Regul Pept. 1986 Feb;13(3-4):307-18

Eva C, Oberto A, Sprengel R, Genazzani E. The murine NPY-1 receptor gene. Structure and delineation of tissue-specific expression. FEBS Lett. 1992 Dec 21;314(3):285-8

Grundemar L, Jonas SE, Mörner N, Högestätt ED, Wahlestedt C, Håkanson R. Characterization of vascular neuropeptide Y receptors. Br J Pharmacol. 1992 Jan;105(1):45-50

Larhammar D, Blomqvist AG, Yee F, Jazin E, Yoo H, Wahlested C. Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Y1 type. J Biol Chem. 1992 Jun 5;267(16):10935-8

Herzog H, Hort YJ, Shine J, Selbie LA. Molecular cloning, characterization, and localization of the human homolog to the reported bovine NPY Y3 receptor: lack of NPY binding and activation. DNA Cell Biol. 1993 Jul-Aug;12(6):465-71

Ball HJ, Shine J, Herzog H. Multiple promoters regulate tissue-specific expression of the human NPY-Y1 receptor gene. J Biol Chem. 1995 Nov 10;270(45):27272-6

Nakamura M, Sakanaka C, Aoki Y, Ogasawara H, Tsuji T, Kodama H, Matsumoto T, Shimizu T, Noma M. Identification of two isoforms of mouse neuropeptide Y-Y1 receptor generated by alternative splicing. Isolation, genomic structure, and functional expression of the receptors. J Biol Chem. 1995 Dec 15;270(50):30102-10

Ammar DA, Eadie DM, Wong DJ, Ma YY, Kolakowski LF Jr, Yang-Feng TL, Thompson DA. Characterization of the human type 2 neuropeptide Y receptor gene (NPY2R) and localization to the chromosome 4q region containing the type 1 neuropeptide Y receptor gene. Genomics. 1996 Dec 15;38(3):392-8

Jacques D, Tong Y, Dumont Y, Shen SH, Quirion R. Expression of the neuropeptide Y Y1 receptor mRNA in the human brain: an in situ hybridization study. Neuroreport. 1996 Apr 10;7(5):1053-6



Blomqvist AG, Herzog H. Y-receptor subtypes--how many more? Trends Neurosci. 1997 Jul;20(7):294-8

Cabrele C, Beck-Sickinger AG. Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family. J Pept Sci. 2000 Mar;6(3):97-122

Caberlotto L, Hurd YL. Neuropeptide Y Y(1) and Y(2) receptor mRNA expression in the prefrontal cortex of psychiatric subjects. Relationship of Y(2) subtype to suicidal behavior. Neuropsychopharmacology. 2001 Jul;25(1):91-7

Rettenbacher M, Reubi JC. Localization and characterization of neuropeptide receptors in human colon. Naunyn Schmiedebergs Arch Pharmacol. 2001 Oct;364(4):291-304

Reubi JC, Gugger M, Waser B, Schaer JC. Y(1)-mediated effect of neuropeptide Y in cancer: breast carcinomas as targets. Cancer Res. 2001 Jun 1;61(11):4636-41

Balasubramaniam A. Neuropeptide Y (NPY) family of hormones: progress in the development of receptor selective agonists and antagonists. Curr Pharm Des. 2003;9(15):1165-75

Dinger MC, Bader JE, Kobor AD, Kretzschmar AK, Beck-Sickinger AG. Homodimerization of neuropeptide y receptors investigated by fluorescence resonance energy transfer in living cells. J Biol Chem. 2003 Mar 21;278(12):10562-71

Pheng LH, Dumont Y, Fournier A, Chabot JG, Beaudet A, Quirion R. Agonist- and antagonist-induced sequestration/internalization of neuropeptide Y Y1 receptors in HEK293 cells. Br J Pharmacol. 2003 Jun;139(4):695-704

Körner M, Waser B, Reubi JC. High expression of neuropeptide y receptors in tumors of the human adrenal gland and extra-adrenal paraganglia. Clin Cancer Res. 2004 Dec 15;10(24):8426-33

Pedrazzini T. Importance of NPY Y1 receptor-mediated pathways: assessment using NPY Y1 receptor knockouts. Neuropeptides. 2004 Aug;38(4):267-75

Ramanathan S, Woodroffe A, Flodman PL, Mays LZ, Hanouni M, Modahl CB, Steinberg-Epstein R, Bocian ME, Spence MA, Smith M. A case of autism with an interstitial deletion on 4q leading to hemizygosity for genes encoding for glutamine and glycine neurotransmitter receptor sub-units (AMPA 2, GLRA3, GLRB) and neuropeptide receptors NPY1R, NPY5R. BMC Med Genet. 2004 Apr 16;5:10

Jemal A, Ward E, Thun MJ. Contemporary lung cancer trends among U.S. women. Cancer Epidemiol Biomarkers Prev. 2005 Mar;14(3):582-5

Kitlinska J, Abe K, Kuo L, Pons J, Yu M, Li L, Tilan J, Everhart L, Lee EW, Zukowska Z, Toretsky JA. Differential effects of neuropeptide Y on the growth and vascularization of neural crest-derived tumors. Cancer Res. 2005 Mar 1;65(5):1719-28

Amlal H, Faroqui S, Balasubramaniam A, Sheriff S. Estrogen up-regulates neuropeptide Y Y1 receptor expression in a human breast cancer cell line. Cancer Res. 2006 Apr 1;66(7):3706-14

Ruscica M, Dozio E, Boghossian S, Bovo G, Martos Riaño V, Motta M, Magni P. Activation of the Y1 receptor by neuropeptide Y regulates the growth of prostate cancer cells. Endocrinology. 2006 Mar;147(3):1466-73

Ruscica M, Dozio E, Motta M, Magni P. Relevance of the neuropeptide Y system in the biology of cancer progression. Curr Top Med Chem. 2007;7(17):1682-91

El Karim IA, Lamey PJ, Linden GJ, Lundy FT. Neuropeptide Y Y1 receptor in human dental pulp cells of noncarious and carious teeth. Int Endod J. 2008 Oct;41(10):850-5

Bjur D, Alfredson H, Forsgren S. Presence of the neuropeptide Y1 receptor in tenocytes and blood vessel walls in the human Achilles tendon. Br J Sports Med. 2009 Dec;43(14):1136-42

Hodges GJ, Jackson DN, Mattar L, Johnson JM, Shoemaker JK. Neuropeptide Y and neurovascular control in skeletal muscle and skin. Am J Physiol Regul Integr Comp Physiol. 2009 Sep;297(3):R546-55

Lundy FT, El Karim IA, Linden GJ. Neuropeptide Y (NPY) and NPY Y1 receptor in periodontal health and disease. Arch Oral Biol. 2009 Mar;54(3):258-62

Okahisa Y, Ujike H, Kotaka T, Morita Y, Kodama M, Inada T, Yamada M, Iwata N, Iyo M, Sora I, Ozaki N, Kuroda S. Association between neuropeptide Y gene and its receptor Y1 gene and methamphetamine dependence. Psychiatry Clin Neurosci. 2009 Jun;63(3):417-22


Ruscica M, Dozio E, Passafaro L, Magni P. NPY1R (neuropeptide Y receptor Y1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):283-287.

Gene Section Review




REPS2 (RALBP1 associated Eps domain containing 2) Salvatore Corallino, Luisa Castagnoli

Department of Biology, University of Rome Tor Vergata, via ricerca scientifica, 00133 Rome, Italy (SC, LC)


Online updated version : http://AtlasGeneticsOncology.org/Genes/REPS2ID44120chXp22.html DOI: 10.4267/2042/44986


Identity

Other names: POB1

HGNC (Hugo): REPS2

Location: Xp22.2

Local order: Forward strand: before 16804550-

16862642 CXorf15 (ENSG00000086712) and after

17300683-17301216. Known pseudogene RP11-

674N8.1 (ENSG00000214321).

Note: This gene is a member of the human CCDS set:

CCDS14180, CCDS43919.

DNA/RNA

Description

18 exons in REPS2/POB1 gene.

Transcription

The transcript length of REPS2/POB1 is 7945 bp.

REPS2 is not differentially expressed in monozygotic

twins. Northern blot analysis reveal

strong expression in rat cerebrum, cerebellum, lung,

and testis, with weak expression in kidney and no

expression in heart, thymus, liver, spleen, or adrenal

gland. Relatively highly expressed in androgen-

dependent as compared to androgen-independent

prostate cancer cell lines. REPS2/POB1 is down-

regulated during progression of prostate cancer.

Protein

Description

REPS2/POB1, Swiss-Prot Q8NFH8, is expressed as

two isoforms, the short isoform is 521 residues long,

while the 660 residues one differs by having a 139

amino acid extension at the amino-terminus. The most

prominent structural/functional features, which are

common to both isoforms, include an amino-terminal

EH (Eps15 homology) domain, a central region

containing two adjacent proline-rich regions and a

carboxy-terminal portion mediating the binding to

RalBP1.

REPS2/POB1: synopsis of protein structure, interactors, functions.

REPS2 (RALBP1 associated Eps domain containing 2) Corallino S, Castagnoli L


In the figure (not in scale), are described the main

motifs and domains of the long isoform of

REPS2/POB1, the interacting proteins and the cellular

functions, that are described in the text.

Expression

The POB1 mRNA is expressed in cerebrum,

cerebellum, lung, weakly in kidney, and testis (Ikeda et

al., 1998). It is relatively highly expressed in androgen-

dependent as compared to androgen-independent

prostate cancer cell lines and xenografts and it is down-

regulated during progression of prostate cancer

(Oosterhoff et al., 2003).

Localisation

REPS2/POB1 localizes in the cytosol, in different sub

cellular compartments: it colocalizes with clathrin CHC

in coated pits, with CD63 in late endosomes, with

GM130 in golgi and with LAMP2 in lysosomes.

Localization is not EGF dependent and POB1 doesn't

localize with EEA1 in early endosomes (Tomassi et al.,

2008).

Function

REPS2/POB1 is part of a protein complex that

regulates the endocytosis and down regulation of

growth factor receptors. Its expression can negatively

affect growth factor signaling. Multiple transcript

variants encoding different isoforms have been found

for this gene and posttranslational modifications have

been described, such as phosphorylation of Ser493 and

Ser549 (Oppermann et al., 2009). The REPS2/POB1

has two amino-terminal EH domains. The structure of

the second EH domain that extends from 265 to 367

has been solved by NMR and consists of two EF-hand

structures, and the second one binds a calcium ion

(Koshiba et al., 1999). The EH domain binds epsin,

Eps15 and NF-kappaB p65, and it is associated to

endocytosis and apoptosis. The central proline rich

region of POB1/REPS2 binds to 14-3-3, amphiphysin

II and Grb2 and it is associated to receptor

downregulation and signaling. The carbossi-terminal

proline rich binds PAG2 and influences paxillin

localization in focal adhesion. POB1 C-terminus (514-

660) directly interacts with a GTPase activating protein

that functions downstream of the small G protein Ral,

RalBP1. Their interaction induces apoptosis.

REPS2 downregulates receptor signaling and

endocytosis: REPS2/POB1 interacts with Eps15, epsin

EPN1, 14-3-3 isoforms, Grb2, amphiphysin

The presence of EH domains in REPS2/POB1 is

symptomatic of a role of this gene in receptor

endocytosis. In fact, REPS2/POB1 EH domain binds to

Eps15 and to epsin that are both proteins present in

clathrin-coated pits, involved in receptor endocytosis

and receptor down regulation. The EH domain interacts

specifically with the three Asn-Pro-Phe (NPF) motifs in

the C-terminal region of epsin and their binding

regulates receptor-mediated endocytosis (Morinaka et

al., 1999).

Augmented expression of full-length POB1 in A431

cells does not affect either binding or internalization of

EGF, on the contrary, over-expression of either the EH

domain or the C-terminal region of REPS2/POB1

affects the ligand dependent internalization pathway of

EGF and insulin without interfering with the

constitutive transferrin pathway (Nakashima et al.,

1999).

Santonico et al. have demonstrated that the EH domain

of REPS2/POB1 binds Eps15 through an

unconventional recognition specificity, since it binds to

both NPF and DPF (Asp-Prp-Phe) motifs. The region

of Eps15 responsible for the interaction with the EH

domain of REPS2/POB1 maps within a 18 amino acid

peptide (residues 623-640) that includes three DPF

repeats. Accordingly, the authors identify a cluster of

solvent exposed Lys residues, which are only found in

the EH domain of REPS2/POB1, and influence binding

to both NPF and DPF motifs (Santonico et al., 2007).

RalBP1, REPS2/POB1, epsin, and Eps15 form a

complex with alpha-adaptin of AP-2 in Chinese

hamster ovary cells and this complex is reduced in

mitotic phase, when REPS2/POB1 and epsin are found

phosphorylated. They are both phosphoryated by p34

cdc2 kinase, in vitro. POB1 is found phosphorylated in

Ser551 and Ser493, in vivo. Phosphorylation of epsin

in Ser 357 inhibits binding to POB1, causing

disassembly of the complex, thus inhibiting receptor

mediated endocytosis (Kariya et al., 2000). This data

explains the contribution of the EH domain of POB1 to

the formation of a protein complex that favours

receptor internalization and that it is dismantled in

mitosis. It is suggested that EGF stimulation induces

also tyrosine-phosphorylation of POB1 and subsequent

formation of a EGFR-POB1 complex in COS cells

(Ikeda et al., 1998).

REPS2/POB1 shorter isoform2 is downregulated

during human prostate cancer progression from

androgen-dependent to androgen-independent

(Oosterhoff et al., 2003). It was observed that a high

level of REPS2 correlates with reduced EGF-

internalisation and signaling since the induced

expression of REPS2 exerts an inhibiting effect on

several EGF-responsive genes (EGF-receptor, EGR-1,

Fos and Jun) (Oosterhoff et al., 2005). Accordingly,

increased expression of POB1 isoform 2 correlates with

a decrease of EGF-induced phosphorylation of Erk1-

Erk2 and Shc (Tomassi et al., 2008).

From these experiments, it can be concluded that

increased REPS2/POB1 expression negatively affects

EGF receptor internalisation and subsequent signaling.

Therefore, the decreased REPS2 expression observed

during prostate cancer progression, results in enhanced

EGF receptor expression and signaling, which could

add to the androgen-independent state of advanced

prostate cancer.

The central region of REPS2/POB1 plays a regulatory

role in epidermal growth factor receptor endocytosis



and signaling. Overexpression of the central region of

POB1 (447-504), inhibits EGF endocytosis, titrating

essential proteins away, thus depauperating the receptor

down-regulation machinery. In fact, this region of

POB1/REPS2 plays its regulatory role in EGFR

endocytosis by binding: (i) to 14-3-3 proteins in a

phosphorylation dependent way (i.e., phospho-Ser493

of POB1), (ii) to the C-SH3 domain of Grb2 and (iii) to

the SH3 of amphiphysin II. The target of the SH3

domain of amphiphysin and of the carboxy-terminal

SH3 of Grb2 is a short peptide flanking Arg483 in

POB1. These interactions are not EGF dependent and

are probably exclusive, since the binding motifs are

only nine amino acids apart. These findings suggest

that 14-3-3 could work by bridging the EGF receptor

and the scaffold protein POB1/REPS2. The 14-3-3

binding motif HSRASSLD, flanking the Ser493 of

POB1, is conserved in the mouse orthologs and in the

14-3-3 binding motif that flanks the Ser510 of human

REPS1 protein, found phosphorylated in vivo in A431

cells (Stover DR et al. Phosphosite). The POB1 Ser493

is predicted to be phosphorylated by Akt. In agreement,

when cells are treated with PI3K/Akt inhibitor

wortmannin, 14-3-3 binding to REPS2/POB1 is

abolished (Tomassi et al., 2008). The 14-3-3 zeta has

already been reported as associating with the EGFR,

epidermal growth factor receptor, cytoplasmic tail and

co-localizing along the plasma membrane with EGFR

upon EGF stimulation (Jin et al., 2004). Thus a 14-3-3

dimer could bridge REPS2/POB1 to the EGFR upon

EGF induction.

Cell migration and paxillin localization: REPS2/POB1

antagonises PAG2/ASAP1

POB1 forms a complex with PAG2/ASAP1 in intact

cells. PAG2 is a paxillin-associated protein with ADP-

ribosylation factor GTPase-activating protein activity,

also called ASAP1 (ArfGAP with SH3 domain, ankyrin

repeat and PH domain UniProtKB: Q9ULH1). The

SH3 of PAG2 binds the proline motif (562PSKPIR567)

at the carboxyl-terminal region of POB1. This motif is

essential for PAG2-POB1 interaction since substitution

of the two proline residues with alanines in mutant

POB1(PA), impaired its binding to PAG2. POB1 may

therefore form a complex with paxillin through PAG2.

Paxillin is a focal adhesion-associated scaffolding

protein that recruits signaling molecules to the focal

adhesions and forms protein complexes that coordinate

signaling, cell spreading and motility. PAG2

overexpression causes loss of endogenous paxillin

recruitment to focal contacts and also impaires cell

migratory activities. The ability to suppress fibronectin-

dependent migration depends on the ArfGAP domain

of PAG2, but not on the POB1-binding domain, of

PAG2. On the other end, POB1, but not POB1(PA),

can suppress the inhibitory action of PAG2 on paxillin

localization to focal adhesion (Oshiro et al., 2002).

These results suggest that POB1, by binding to PAG2,

suppresses the inhibitory action of PAG2 on the

paxillin recruitment to focal contacts. This suggests that

POB1 may function as a scaffold protein that interacts

with proteins involved in endocytosis and migration,

thus regulating signaling and motility. PAG2/ASAP1

gene was found associated with prostate cancer

metastasis since it is up-regulated in a human

metastatic prostate subline and immunohistochemistry

of xenograft sections show a significantly strong

cytoplasmic ASAP1 protein staining in tumor-

nonmetastatic PCa2 tissue, compared to a non-staining

in benign tissue, and an even stronger staining in PCa1-

metastatic tissue. Moreover, additional ASAP1 gene

copies are detected in 58% of the primary prostate

cancer clinical specimens. A small interfering RNA

reducing ASAP1 protein expression, can suppress in

vitro PC-3 cell migration and matrigel invasion.

Therefore PAG2/ASAP1 represents a therapeutic target

and a biomarker for metastatic disease (Lin et al., 2008)

while REPS2/POB1 can suppress PAG2 oncogenic-

metastatic activity.

Mutations

Somatic

- S324F, cds mutation 971C>T heterozygous in

glioblastoma multiforme (Parsons et al., 2008).

- V67M, cds mutation 199G>A homozygous in

malignant melanoma.

- No high level gene amplification (>7), 1 homozygous

deletion in breast cancer, 559 LOH (Loss of

Heterozygosity).

Implicated in

Non-small cell lung cancer (NSLC) and prostate cancer

- Apoptosis in non-small cell lung cancer (NSLC) and

prostate cancer: REPS2 binds and inhibits RalBP1.

Ikeda et al. (1998) cloned POB1 (partner of Ralbp1) as

the first known binding partner of Ralbp1 by the yeast

two-hybrid method using Ralbp1/RLIP76 as bait and

clearly demonstrated specific binding and complex

formation between Ralbp1 and REPS2/POB1. The

binding to RalBP1 did not affect the GTPase activating

activity of RalBP1. The interaction of POB1 with

RalBP1 induces cell death in human prostate cancer

cell ines LNCaP-FGC and LNCaP-LNO. Oosterhoff et

al. show that REPS2/POB1-induces apoptosis in 45%

of transfected cells, within 48 hours. When prostate

cancer cell lines are transfected with a deletion mutant

of REPS2/POB1, lacking the RalBP1 binding domain,

only 30-40% of the transfected cells became apoptotic

after 72-96 hours (Oosterhoff et al., 2003).

REPS2/POB1 (514-660) binds RalBP1 C-terminal

amino acids, 499-655, while an almost overlapping

region of RalBP1 (440-655) binds the heat shock factor

1 Hsf-1 (Hu and Mivechi, 2003). Shingal et al. show a

ternary complex formation between RalBP1, Hsf-1, and

REPS2/POB1. RalBP1, Hsf1, HSP90 and tubulin make

http://www.phosphosite.org/



a complex in cell (Singhal et al., 2008). Hsf-1 and

REPS2/POB1 induce drug sensitivity and apoptosis by

inhibiting RalBP1.

Binding of REPS2/POB1 to RALBP1 inhibits the

transport activity of the Ral-binding nucleotidase,

which functions as an energy-dependent transporter for

GSH-conjugates as well as unrelated xenobiotics.

RALBP1 (RLIP76) is the major transporter of the

anthracycline antibiotic, doxorubicin, which is one of

the most widely used anticancer drugs (Singhal et al.,

2007). Therefore, REPS2/POB1 is a specific and

saturable inhibitor of the glutathione-electrophile

conjugates and of the doxorubicin transport activity of

RalBP1. Yadav et al. show that REPS2/POB1 can

regulate the transport function of RalBP1/RLIP76 and,

in agreement with previous studies, show that

inhibition of RalBP1 induces apoptosis in cancer cells

through the accumulation of endogenously formed

GSH-conjugates (Yadav et al., 2005). Hence,

REPS2/POB1 over-expression inhibits RalBP1-

mediated transport of glutathione-conjugates thus

promoting apoptosis and can influence drug-efflux

mechanisms that cause resistance to cancer treatment.

Hsf-1 also causes specific and saturable inhibition of

the transport activity of RalBP1. The combined

augmentation of Hsf-1 and REPS2/POB1 causes nearly

complete inhibition of RalBP1 and a dramatic

apoptosis in NSLC (non-small cell lung cancer) cell

line H358 through Ralbp1 binding (Singhal et al.,

2008). The marked apoptotic effect caused by the

increase of Hsf-1 and REPS2/POB1 in lung cancer

cells, suggests a novel targeted therapy in which

liposomally encapsulated Hsf-1 and POB1 could be

used clinically as a therapeutic agent.

- Apoptosis in prostate cancer cells: REPS2/POB1

counteracts the apoptosis inhibitor NF-kappaB p65

The NF-kappaB subunit p65 is identified as a human

REPS2/POB1 protein partner, since the NPF-motif in

p65 acts as binding site for the EH domain in REPS2.

However, in cultured prostate cancer cells, the REPS2-

p65 interaction is triggered upon stimulation with the

phorbol ester, phorbol-12-myristate-13-acetate (PMA).

During prostate cancer progression from androgen-

dependent to androgen-independent growth, the

observed downregulation of REPS2 is accompanied by

upregulation of NF-kappaB activity, that inhibits

apoptosis (Penninkhof et al., 2004). Hence, the authors

suggest that a decreased expression of REPS2 might be

a key factor in causing prostate cancer cells to avoid

apoptosis.

Breast cancer

Note

Doolan et al. (2009) suggest that REPS2 mRNAs may

be useful as favourable prognostic and predictive

markers for breast cancer. Univariate and multivariate

analyses were used to identify associations between

expression of these transcripts and patients'

clinicopathological and survival data.

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Koshiba S, Kigawa T, Iwahara J, Kikuchi A, Yokoyama S. Solution structure of the Eps15 homology domain of a human POB1 (partner of RalBP1). FEBS Lett. 1999 Jan 15;442(2-3):138-42

Morinaka K, Koyama S, Nakashima S, Hinoi T, Okawa K, Iwamatsu A, Kikuchi A. Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis. Oncogene. 1999 Oct 21;18(43):5915-22

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Singhal SS, Yadav S, Drake K, Singhal J, Awasthi S. Hsf-1 and POB1 induce drug sensitivity and apoptosis by inhibiting Ralbp1. J Biol Chem. 2008 Jul 11;283(28):19714-29

Tomassi L, Costantini A, Corallino S, Santonico E, Carducci M, Cesareni G, Castagnoli L. The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth

factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins. BMC Biochem. 2008 Jul 22;9:21

Doolan P, Clynes M, Kennedy S, Mehta JP, Germano S, Ehrhardt C, Crown J, O'Driscoll L. TMEM25, REPS2 and Meis 1: favourable prognostic and predictive biomarkers for breast cancer. Tumour Biol. 2009;30(4):200-9

Oppermann FS, Gnad F, Olsen JV, Hornberger R, Greff Z, Kéri G, Mann M, Daub H. Large-scale proteomics analysis of the human kinome. Mol Cell Proteomics. 2009 Jul;8(7):1751-64


Corallino S, Castagnoli L. REPS2 (RALBP1 associated Eps domain containing 2). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):288-292.





XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) Sabina Pucci, Maria Josè Zonetti

Lab of Molecular Pathology, Dept of Biopathology, University of Rome "Tor Vergata", Via Montpellier,

00133 Rome, Italy (SP, MJZ)


Online updated version : http://AtlasGeneticsOncology.org/Genes/XRCC6ID246ch22q13.html DOI: 10.4267/2042/44987


Identity Other names: CTC75, CTCBF, G22P1, KU70, ML8,

TLAA

HGNC (Hugo): XRCC6

Location: 22q13.2

DNA/RNA

Description

The KU70 gene is composed of 13 exons.

Transcription

2156 bp mRNA.

Protein

Description

The Ku70 protein is 609 amino acid long and its

molecular weight is 69.8 kDa. It is composed of 3

domains: an amino (N) amino-terminal alpha/beta

domain, a central beta-barrel domain and a helical C-

terminal arm (Rivera-Calzada et al., 2007). The C-

terminal region consists of a 5 kDa SAP domain

(Ku70-SAP) which involved in DNA binding during

NHEJ reaction.

Expression

Ku70 is ubiquitously expressed. Changes in Ku70

expression correlated to a pathological state.

Localisation

Ku was originally reported to be a nuclear protein,

consistent with its functions as a subunit of DNA-PK

involved in DNA double strand breaks repair.

However, several studies have revealed the cytoplasmic

or cell surface localization of Ku proteins in various

cell types (Prabhakar et al., 1990). Recently, it has been

demonstrated that the shift from the nucleus to the

cytoplasm of the Ku70/Ku80 proteins in tumor cells

could represents a mechanism to inhibit cell death

through the Ku70-Bax-sCLU interactions, giving rise

to a new chemoresistant clone with a more aggressive

phenotype.

Function

Ku is a heterodimeric protein composed of two

subunits with molecular weight of 70 and 86 kDa. Ku

forms a complex with the DNA-dependent protein

kinase catalytic subunit (DNA-PKcs) to form the full

DNA-dependent protein kinase, DNA-PK, consisting

of 470 kDa and required for the non-homologous end

joining (NHEJ) pathway of DNA repair. The Ku

heterodimer binds the ends of various types of DNA

discontinuity, and is involved in the repair of DNA

breaks caused by an incorrect DNA replication, V(D)J

recombination, physiological oxidations, ionizing

irradiation, and some chemotherapeutic drug effects

(Featherstone and Jackson, 1999).

XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) Pucci S, Zonetti MJ


Ku70/80-CLU-Bax interactions. (A) Bax is localized inactive in the cytoplasm in normal, undamaged cell interacting with the Ku70 protein C-terminus. sCLU stabilizes the Ku70-Bax interaction in the cytoplasm acting as cytoprotectant. (B) After DNA damage inducing DNA double-strand breaks repair (UV treatment, ionizing radiation, etc.) Ku70 allows the translocation of Bax to the mitochondria inducing apoptosis (Mazzarelli et al., 2009).

The principal role of Ku proteins is to take care of the

homeostasis of the genome being involved in telomere

maintenance, specific gene transcription, DNA

replication, cell-cycle regulation and regulation of

apoptosis induction. Ku70 has been shown to bind to

the pro-apoptotic protein BAX in the cytoplasm in

normal, undamaged cell. After DNA damage inducing

DNA double-strand breaks repair (UV treatment,

ionizing radiation, etc.) Ku70 allows the translocation

of Bax to the mitochondria leading to the release of

death-promoting factors, such as cytochrome c, in the

cytoplasmic compartment.

In normal cells, after an irreversible cell damage, nCLU

cooperates with Ku70 to induce apoptotic death,

activating the translocation of Bax to mitochondria

whereas the sCLU protein stabilizes the Ku70-Bax

interaction in the cytoplasm acting as cytoprotectant.

The Ku70-Bax-sCLU interaction in the cytoplasm

seems to play an important role in cell survival

pathways and in cell death escape, that in pathological

condition could lead to the survival of the aberrant cell

clone. Overall, the dynamic interactions among CLU,

Ku70, and Bax seems to have an important role in both

tumor insurgence and its progression (Pucci et al.,

2009a; Pucci et al., 2009b).

Implicated in

Colon cancer

Note

The colon cancer expression and the localization of

Ku70 and Ku80 are related to tumor progression in

colon cancer. DNA repair is inhibited in high

infiltrative colon carcinoma by Ku80 loss and Ku70

cell compartment shift (from the nucleus to the

cytoplasm).

Moreover in colorectal carcinoma was demonstrated a

very important role of Ku70 expression, localization,

and physical interaction with CLU and Bax. In fact the

Ku70-CLU-Bax colocalization in the cytoplasm and an

increase in Ku70-CLU-Bax binding were observed in

highly aggressive human colon cancer (Pucci et al.,

2004; Pucci et al., 2009c), confirming that these

interactions regulate the Bax-dependent cell death.

Breast cancer

Note

Experimental data further reported an inactivation of

Ku DNA-binding activity, essential for genomic

stability in breast and in bladder carcinomas. A

dysfunction of this protective activity let the aberrant

cell clone growing. In highly infiltrative and metastatic

tumors of the breast and bladder, the impaired DNA-

repair activity is due to the loss of Ku86 (Pucci et al.,

2001) and to the Ku70 shifting from the nucleus to the

cytoplasm. The shift from the nucleus to the cytoplasm

of the Ku70/80 proteins in tumor cells could represents

a mechanism to inhibit cell death through the

cooperative interaction with sCLU, giving rise to a new

chemoresistant clone with a more aggressive

phenotype.



Tumor-specific modulation of Ku70/80 in human colon cancer. Ku70 staining was strongly positive in the nuclei of normal mucosa. In node-negative carcinomas (pT3N0) Ku70 expression slightly decreased and it localized mainly in the nucleus. In node-positive carcinomas (pT3N1) Ku70 staining was distributed mainly in cytoplasm. The expression of Ku86 was positive in the nuclei of control tissues (normal mucosa). Nuclear Ku86 expression was strongly decreased in node-negative tumors (pT3N0). No staining for Ku86 was found in the nucleus or in the cytoplasm of node-positive carcinomas (pT3N1).



Ku70-Bax-CLU pathological interaction. Apoptosis escaping. The shift of clusterin forms production, the loss of Ku80, and the cytoplasmic relocalization of Ku70 are related to cell death inhibition and cancer progression.

References Prabhakar BS, Allaway GP, Srinivasappa J, Notkins AL. Cell surface expression of the 70-kD component of Ku, a DNA-binding nuclear autoantigen. J Clin Invest. 1990 Oct;86(4):1301-5

Featherstone C, Jackson SP. Ku, a DNA repair protein with multiple cellular functions? Mutat Res. 1999 May 14;434(1):3-15

Pucci S, Mazzarelli P, Rabitti C, Giai M, Gallucci M, Flammia G, Alcini A, Altomare V, Fazio VM. Tumor specific modulation of KU70/80 DNA binding activity in breast and bladder human tumor biopsies. Oncogene. 2001 Feb 8;20(6):739-47

Pucci S, Bonanno E, Pichiorri F, Angeloni C, Spagnoli LG. Modulation of different clusterin isoforms in human colon tumorigenesis. Oncogene. 2004 Mar 25;23(13):2298-304

Rivera-Calzada A, Spagnolo L, Pearl LH, Llorca O. Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs. EMBO Rep. 2007 Jan;8(1):56-62

Mazzarelli P, Pucci S, Spagnoli LG. CLU and colon cancer. The dual face of CLU: from normal to malignant phenotype. Adv Cancer Res. 2009;105:45-61

Pucci S, Bonanno E, Sesti F, Mazzarelli P, Mauriello A, Ricci F, Zoccai GB, Rulli F, Galatà G, Spagnoli LG. Clusterin in stool: a new biomarker for colon cancer screening? Am J Gastroenterol. 2009 Nov;104(11):2807-15

Pucci S, Mazzarelli P, Nucci C, Ricci F, Spagnoli LG. CLU "in and out": looking for a link. Adv Cancer Res. 2009;105:93-113

Pucci S, Mazzarelli P, Sesti F, Boothman DA, Spagnoli LG. Interleukin-6 affects cell death escaping mechanisms acting on Bax-Ku70-Clusterin interactions in human colon cancer progression. Cell Cycle. 2009 Feb 1;8(3):473-81


Pucci S, Zonetti MJ. XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):293-296.

Leukaemia Section Short Communication




t(6;22)(p21;q11) Jean-Loup Huret

Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France

(JLH)


Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0622p21q11ID1563.html DOI: 10.4267/2042/44988


Identity

t(6;22)(p21;q11) G-banding and FISH - Courtesy Claudia Haferlach.

t(6;22)(p21;q11) Huret JL


Clinics and pathology

Disease

Hematological malignancies.

Note

So far, the t(6;22)(p21;q11) is heterogeneous, and its

significance remains problematic.

Phenotype/cell stem origin

Four cases availble: one case was a myeloid type blast

crisis of chronic myeloid leukemia (CML) in a 36-year-

old male patient with a t(9;22)(q34;q11); another case

was a CML aberrant translocation t(6;22)(p21;q11)

without apparent involvement of chromosome 9 in a

44-year-old male patient; a third case was that of a B-

cell precursor (CD10+) L1- acute lymphoblastic

leukemia in a 2-year-old girl who was still in complete

remission 72 months after diagnosis. A cryptic 5' ETV6

- 3' RUNX1 was found; there were accompanying

anomalies, of which a +10 and a +21; the last case was

a chronic lymphocytic leukemia (CLL) stage C with

also a del(11q), and a del(13q).

Genes involved and proteins

Note

Genes involved, if any, are unknown.

References Gödde-Salz E, Schmitz N, Bruhn HD. Philadelphia chromosome (Ph) positive chronic myelocytic leukemia (CML): frequency of additional findings. Cancer Genet Cytogenet. 1985 Jan 15;14(3-4):313-22

Fears S, Vignon C, Bohlander SK, Smith S, Rowley JD, Nucifora G. Correlation between the ETV6/CBFA2 (TEL/AML1) fusion gene and karyotypic abnormalities in children with B-cell precursor acute lymphoblastic leukemia. Genes Chromosomes Cancer. 1996 Oct;17(2):127-35

Tanaka H, Tanaka K, Oguma N, Ito K, Ito T, Kyo T, Dohy H, Kimura A. Effect of interferon-alpha on chromosome abnormalities in treated chronic myelogenous leukemia patients. Cancer Genet Cytogenet. 2004 Sep;153(2):133-43

Mayr C, Speicher MR, Kofler DM, Buhmann R, Strehl J, Busch R, Hallek M, Wendtner CM. Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia. Blood. 2006 Jan 15;107(2):742-51


Huret JL. t(6;22)(p21;q11). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):297-298.

Solid Tumour Section Short Communication




t(11;22)(q24;q12) in giant cell tumour of bone Jean-Loup Huret


(JLH)


Online updated version : http://AtlasGeneticsOncology.org/Tumors/t1122q24q12GiantCellID6283.html DOI: 10.4267/2042/44989



Disease

Giant cell tumour of bone is a locally destructive

tumor, usually seen in patients over 20 years of age, a

borderline lesion between benign and malignant

tumours, with a good prognosis, despite of recurrences

and, more rarely, pulmonary metastases. The most

frequent genetic finding is telomeric associations.

Genetics

Note

In a study by Scotlandi et al., 2000, was found that a

minor population of cells from giant cell tumour of

bone samples had an EWSR1/FLI1 transcript, but this

was found in a high percentage of samples (13/15).


FLI1

Location

11q24

Protein

From N-term to C-term: a 5' ETS domain, a Fli-1-

specific transcriptional activation domain, and a 3' ETS

transcriptional activation domain. Member of ETS

transcription factor gene family. FLI1 binds to DNA in

a sequence-specific manner.

EWSR1

Location

22q12

Protein

From N-term to C-term: a transactivation domain

(TAD) containing multiple degenerate hexapeptide

repeats, 3 arginine/glycine rich domains (RGG

regions), a RNA recognition motif, and a RanBP2 type

Zinc finger. Role in transcriptional regulation for

specific genes and in mRNA splicing.

Result of the chromosomal anomaly

Hybrid Gene

Description

5' EWSR1 - 3' FLI1. EWSR1 exon 7 is fused in frame

to FLI1 exon 6 and/or 5 (type 1 and type 2 fusions

respectively), indicating, when both transcripts were

produced in a given sample, genetic heterogeneity

within the tumour.

Fusion Protein

Description

Fusion of the N terminal transactivation domain of

EWSR1 to the ETS type DNA binding domain of FLI1.

References Scotlandi K, Chano T, Benini S, Serra M, Manara MC, Cerisano V, Picci P, Baldini N. Identification of EWS/FLI-1 transcripts in giant-cell tumor of bone. Int J Cancer. 2000 Aug 1;87(3):328-35


Huret JL. t(11;22)(q24;q12) in giant cell tumour of bone. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):299.





t(11;22)(q24;q12) in rhabdomyosarcomas (RMS) Jean-Loup Huret


(JLH)


Online updated version : http://AtlasGeneticsOncology.org/Tumors/t1122q24q12RhabdoID6285.html DOI: 10.4267/2042/44991



Disease

Rhabdomyosarcomas, the most common pediatric soft

tissue sarcomas, are tumours related to the skeletal

muscle lineage. The 2 major subtypes are alveolar

rhabdomyosarcoma (ARMS) and embryonal

rhabdomyosarcoma (ERMS). Other subtypes are

botryoid, spindle cell, anaplastic, pleomorphic, and

undifferentiated RMS.

Note

Most ARMS cases are characterised by either a

t(2;13)(q35;q14), resulting in a PAX3/FOXO1 hybrid

gene, or a t(1;13)(p36;q14) resulting in a

PAX7/FOXO1 hybrid gene. Most ERMS are

characterized by chromosome gains and a loss of

heterozygocity in 11p15.

Epidemiology

Three cases of RMS with t(11;22)(q24;q12) have been

described to date, including a two-years-old girl with a

mixed embryonal and alveolar RMS, who died 14

months after diagnosis, a 4.5-year-old girl, also with a

mixed embryonal and alveolar RMS, who was alive

and well 9 months after diagnosis (Sorensen et al.,

1993; Thorner et al., 1996).

Genetics

Note

A t(2;13) hybrid transcript was excluded in the two

cases described by Thorner et al., 1996. In the 4.5-year-

old girl case, a highly abnormal karyotype was found,

with 85 to 200 chromosomes per mitosis, and MDM2

was amplified more than a hundred times.


FLI1

Location

11q24

Protein






EWSR1

Location

22q12

Protein








Hybrid Gene

Description


to FLI1 exon 6.

Fusion Protein

Description



t(11;22)(q24;q12) in rhabdomyosarcomas (RMS) Huret JL


References Sorensen PH, Liu XF, Delattre O, Rowland JM, Biggs CA, Thomas G, Triche TJ. Reverse transcriptase PCR amplification of EWS/FLI-1 fusion transcripts as a diagnostic test for peripheral primitive neuroectodermal tumors of childhood. Diagn Mol Pathol. 1993 Sep;2(3):147-57

Thorner P, Squire J, Chilton-MacNeil S, Marrano P, Bayani J, Malkin D, Greenberg M, Lorenzana A, Zielenska M. Is the EWS/FLI-1 fusion transcript specific for Ewing sarcoma and peripheral primitive neuroectodermal tumor? A report of four cases showing this transcript in a wider range of tumor types. Am J Pathol. 1996 Apr;148(4):1125-38


Huret JL. t(11;22)(q24;q12) in rhabdomyosarcomas (RMS). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):300-301.





t(11;22)(q24;q12) in solid pseudopapillary tumour of the pancreas Jean-Loup Huret


(JLH)


Online updated version : http://AtlasGeneticsOncology.org/Tumors/t1122q24q12PseudopapID6284.html DOI: 10.4267/2042/44990



Disease

Solid pseudopapillary tumour of the pancreas is a rare

epithelial tumour of low malignancy, typically occuring

in young female patients (mean age 27 years, sex ratio

is 1 male to 8 or 9 female patients). It accounts for 1%

of all pancreatic tumours. It is a encapsulated lesion

with well-defined borders, with about 15% of cases

demonstrating malignant behaviour with recurrence

and metastasis (Yu et al., 2010).

Cytogenetics

Cytogenetics Morphological

A t(11;22)(q24;q12), accompanied with +8, was found

in one case (Maitra et al., 2000).


FLI1

Location

11q24

Protein






EWSR1

Location

22q12

Protein








Hybrid Gene

Description


to FLI1 exon 6 (type 1 fusion).

Fusion Protein

Description



References Maitra A, Weinberg AG, Schneider N, Patterson K. Detection of t(11;22)(q24;q12) translocation and EWS-FLI-1 fusion transcript in a case of solid pseudopapillary tumor of the pancreas. Pediatr Dev Pathol. 2000 Nov-Dec;3(6):603-5

Yu PF, Hu ZH, Wang XB, Guo JM, Cheng XD, Zhang YL, Xu Q. Solid pseudopapillary tumor of the pancreas: a review of 553 cases in Chinese literature. World J Gastroenterol. 2010 Mar 14;16(10):1209-14


Huret JL. t(11;22)(q24;q12) in solid pseudopapillary tumour of the pancreas. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):302.





MTA1 of the MTA (metastasis-associated) gene family and its encoded proteins: molecular and regulatory functions and role in human cancer progression Yasushi Toh, Garth L Nicolson

Department of Gastroenterological Surgery, National Kyushu Cancer Center, Fukuoka, 811-1395, Japan

(YT), Department of Molecular Pathology, The Institute for Molecular Medicine, Huntington Beach, CA

92647, USA (GLN)


Online updated version : http://AtlasGeneticsOncology.org/Deep/MTAinCancerID20088.html DOI: 10.4267/2042/44992


Abstract MTA1(a metastasis-associated gene) is a newly discovered human gene (residing on chromsome 14q32.3) that belongs

to a family of cancer progression-related genes (MTA). The mRNA product of MTA1along with its protein product

MTA1 have been reported to be over-expressed in a wide variety of animal and human tumors. For example, the

expression of MTA1 and its encoded protein MTA1 correlates with the malignant properties of many human cancers,

including cancers of the breast, colon, stomach, liver, prostate and others. The MTA proteins have been shown to be

ubiquitinated transcriptional co-repressors that function in histone deacetylation and are part of the NuRD complex, a

nucleosome remodeling and histone deacetylating complex whose stability appears to be regulated by ubiquitinated

MTA1 binding to E3 ubiquitin ligase constitutive photomorphogenesis protein-1 (COP1). The MTA1 protein plays an

essential role in c-MYC-mediated cell transformation, and its expression correlates with mammary gland tumor

formation. In the latter, MTA1 helps convert mammary cells to more aggressive phenotypes by repression of the

estrogen receptor (ER) via trans-activation through deacetylation of chromatin in the ER-responsive element of ER-

responsive genes. Another member of the MTA family, MTA3, is induced by estrogen and represses the expression of

the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transformation, resulting in the

expression of the cell adhesion molecule E-cadherinand maintenance of a differentiated, normal epithelial phenotype in

mammary cells. An important activity mediated by both MTA1 and MTA2 is deacylation and inactivation of tumor

suppressor p53protein, in part by controlling its stability by inhibiting ubiquitination, leading to inhibition of growth

arrest and apoptosis. Another factor deacetylated and stabilized by MTA1 NuRD complex is hypoxia-inducible factor-

1α (HIF-1α), which is involved in angiogenesis. Therefore, the MTA proteins represent a possible set of master co-

regulatory molecules involved in the carcinogenesis and progression of various malignant tumors. As such, they could

be important new tools for cancer diagnosis and treatment.

1. Introduction - The MTA gene family An important advance in cancer research has been the

discovery of a wide variety of new molecules involved

in carcinogenesis and cancer progression. Although

additional cancer-related molecules will be identified in

the future, these molecules must fulfill two major

requirements in order to be clinically useful as

molecular targets useful for the diagnosis and treatment

of human cancers (Toh and Nicolson, 2009).

The first is that abnormalities must occur in the

expression or structure of the molecules of interest, and

their clinical relevance must be definitely demonstrated

in independent studies on human cancers. The second

is that the underlying molecular mechanisms necessary

http://atlasgeneticsoncology.org/Genes/MTA1ID41443ch14q32.html

http://atlasgeneticsoncology.org/Tumors/breastID5018.html

http://atlasgeneticsoncology.org/Tumors/colon5006.html

http://atlasgeneticsoncology.org/Tumors/GastricTumOverviewID5410.html

http://atlasgeneticsoncology.org/Tumors/LiverOverviewID5273.html

http://atlasgeneticsoncology.org/Genes/CDH1ID166ch16q22.html

http://atlasgeneticsoncology.org/Genes/P53ID88.html

MTA1 of the MTA (metastasis-associated) gene family and its encoded proteins: Toh Y, Nicolson GL molecular and regulatory functions and role in human cancer progression


for the molecules to exert their functions in

carcinogenesis or cancer progression must be

determined and confirmed in animal tumor models and

clinical specimens.

There have been a number of cancer-related genes and

molecules that have been discovered in the last few

years. In our laboratory we identified a candidate

metastasis-associated gene by the use of a differential

cDNA screening method. Using this approach we

identified a gene that was differentially over-expressed

in highly metastatic rat mammary adenocarcinoma cell

lines compared to poorly metastatic lines (Toh et al.,

1994; Toh et al., 1995). When this gene was sequenced,

it was shown to be a completely new, novel gene

without any homologous or related genes in the

database at the time. This gene was named mta1

(metastasis-associated gene-1). A homologous gene

was also expressed in human cancer cell lines, and its

human cDNA counterpart, MTA1, was subsequently

cloned (Nawa et al., 2000) and found to reside on

chromosome 14q32.3 (Cui et al., 2001).

Using surgically removed human tissues we showed

that high levels of MTA1mRNA expression were

correlated to the invasive and growth properties of

gastrointestinal cancers, including esophageal, gastric

and colorectal cancers (Toh et al., 1997; Toh et al.,

1999). After these studies, several reports from other

groups found similar correlations between MTA1

expression and the malignant potentials of human

cancers (reviewed in Toh and Nicolson, 2009).

In addition to MTA1, other genes related to MTA1 have

now been identified. This gene family, which we

termed the MTAfamily, now has several members plus

some splice variants (Toh and Nicolson, 2003;

Manavathi and Kumar, 2007; Toh and Nicolson, 2009).

Furthermore, studies on the molecular biological and

biochemical properties of the MTA family have shown

that the gene products of the main members of the

family (proteins MTA1, MTA2 and MTA3) are tightly

associated in a protein complex called NuRD

(nucleosome remodeling and histone deacetylation),

which has transcriptional regulatory functions via

histone deacetylation and chromatin remodeling (Toh

et al., 2000; Bowen et al., 2004). Interestingly, histone

deacetylase activities correlate with squamous cell

carcinoma invasion (Toh et al., 2003). At the moment,

the MTA family has attracted widespread attention as

one of several key molecules that play indispensable

roles in the pathogenesis and progression of a wide

variety of cancers (Toh and Nicolson, 2003; Kumar et

al., 2003; Manavathi et al., 2007b; Toh and Nicolson,

2009). We will examine the significance of the

expression of MTA family members in human cancers

and the important molecular mechanisms that are

currently known by which MTA proteins exert their

cellular actions as well as discuss the potential clinical

applications of this protein family for the diagnosis and

treatment of human cancers.

2. The MTA family of proteins, their structures and cell location The MTA proteins represent a family of gene products

encoded by three distinct genes (MTA1, MTA2 and

MTA3), and six reported isoforms (MTA1, MTA1s,

MTA1-ZG29p, MTA2, MTA3, and MTA3L). The

molecular masses of the gene products of MTA1,

MTA2 and MTA3 are approximately 80 kDa, 70 kDa

and 65 kDa, respectively (Manavathi and Kumar, 2007;

Toh and Nicolson, 2009). The nucleotide and protein

alignment homologies and the phylogenetic

comparative analyses have been discussed previously

(Bowen et al., 2004; Manavathi and Kumar, 2007; Toh

and Nicolson, 2009).

The MTA gene family sequences, with the exception of

ZG-29p, contain several common domain structures

(Singh and Kumar, 2007; Toh and Nicolson, 2009).

One of these, the BAH (bromo-adjacent homology)

domain is involved in protein-protein interactions.

Another, the SANT (SWI, ADA2, N-CoR, TFIIIB-B)

domain shares a high degree of homology with the

DNA-binding domain of the Myb-related proteins,

suggesting that this domain may be involved in DNA-

binding. The ELM (egl-27 and MTA1 homology)

domain has an unknown function but could be involved

in embryonic patterning (Solari et al., 1999).

The MTA family members contain a highly conserved

GATA-type zinc finger motif, suggesting direct

interactions with DNA (Nawa et al., 2000). The MTA1

protein has two src-homology (SH)-binding motifs at

its C-terminal end-such binding domains are known to

be important in signal transduction involving kinase,

adaptor and scaffolding proteins (Toh et al., 1994; Toh

et al., 1995; Singh and Kumar, 2007). Similarly, SH2-

and SH3-binding domains are also found in MTA2 and

MTA3 protein sequences (Toh et al., 1994; Toh et al.,

1995; Singh and Kumar, 2007). These common domain

structures demonstrate that the MTA family is involved

in protein-protein and protein-DNA interactions,

indicating the anticipated roles of the MTA family of

proteins in signal transduction and transcriptional

regulation (Toh and Nicolson, 2003; Singh and Kumar,

2007; Toh and Nicolson, 2009).

In addition to protein-protein and protein-DNA

binding, MTA proteins contain basic nuclear

localization signals (Toh et al., 1994; Toh et al., 1995;

Singh and Kumar, 2007). They also localize in the

nucleus in many human cancer cells (Toh et al.,1997;

Toh et al., 1999); however, MTA1 localizes to both the

cytoplasm and nucleus in some tumors (Moon et al.,

2004; Balasenthil et al., 2006; Bagheri-Yarmand et al.,

2007). MTA3 also localizes to the nucleus, but without

any apparent nuclear localization signal (Fujita et al.,

2003). A short splice-variant of MTA1, called MTA1s,

is predominantly localized in the cytoplasm (Kumar et

al., 2002).



3. MTA protein expression in various cancers and its possible clinical relevance Since the report by Toh et al. (1997) that the over-

expression of MTA1was significantly correlated to the

malignant properties of human gastric and colorectal

cancers, there have been several reports on the

expression levels of MTA family members, especially

of MTA1, in various human cancers (reviewed in Toh

and Nicolson, 2009). These studies revealed that the

expression levels of MTA family members correlate

with pathogenic significance and prognosis (Toh and

Nicolson, 2009). The biological relevance of MTA

proteins to carcinogenesis and cancer progression has

been investigated in a few cancer models, such as

breast and gastrointestinal cancers, and these will be

discussed in more detail below.

3.1 MTA1 protein and breast cancer

MTA1 protein was originally identified as a candidate

cancer progression molecule that was associated with

breast cancer metastasis (Toh et al., 1994; Toh et al.,

1995). Subsequently, using antisense RNA of MTA1 a

role for MTA1 in the growth properties of metastatic

breast cancer cells was investigated. Using

MTA1antisense RNA we found that the growth rates of

highly metastatic breast cancer cell lines were inhibited

significantly in a dose-dependent manner (Nawa et al.,

2000). More direct evidence to demonstrate an

association of MTA1 expression levels with breast

cancer malignant properties was obtained by

Mazumdar et al. (2001). They demonstrated that forced

expression of the MTA1 protein in the human breast

cancer cell line MCF-7 was accompanied by an

enhancement in the ability of MCF-7 cells to invade an

artificial matrix and ability to grow in an anchorage-

independent manner. They also showed that the

enhancement was associated with the interaction

between MTA1 protein and histone deacetylase,

resulting in a repression of estrogen receptor (ER)

mediated transcription (Mazumdar et al., 2001).

Using an animal model the Mazumdar et al. (2001)

study was extended by further experiments

demonstrating a direct in vivo effect of MTA1 on the

carcinogenesis of breast cancer cells (Bagheri-Yarmand

et al., 2004; Singh and Kumar, 2007). This group

established a transgenic mice system that over-

expressed the MTA1 protein, and these MTA1-

transgenic mice revealed inappropriate development of

their mammary glands. The MTA1-transgenic mice

eventually developed hyperplastic nodules and

mammary tumors, including mammary

adenocarcinomas and lymphomas.

The involvement of MTA1 in the carcinogenesis and

progression of breast cancers was also shown by Martin

et al. (2001; 2006). First, they mapped the

chromosomal locus in 14q that might be responsible for

axillary lymph node metastases in human breast

cancers by comparing the rate of loss of heterozygosity

between node-positive and node-negative breast

cancers. The MTA1 gene was found to be contained in

the same gene locus, suggesting that MTA1is a

candidate for a breast cancer metastasis-promoting

gene. Next, using immunohistochemistry they

examined MTA1 protein expression in primary human

breast cancer samples and demonstrated that node-

negative breast cancers with over-expression of MTA1

protein had a higher risk of disease relapse similar to

node-positive tumors. Therefore, the over-expression of

MTA1 was proposed as a potential predictor of early

disease relapse independent of node status (Martin et

al,. 2006).

Using surgically resected human breast cancer

specimens Jang et al. (2006) showed that MTA1 over-

expression was closely associated with higher tumor

grade and high intratumoral microvessel density. This

suggested that MTA1 could be a useful predictor of an

aggressive phenotype, and the MTA1 molecule could

be considered as a possible angiogenesis-promoting

molecule in breast cancers (Jang et al., 2006).

3.2 MTA1 protein and gastrointestinal cancers

MTA1 over-expression has been shown to be

pathogenically significant in human gastrointestinal

cancers. Using a reverse-transcription polymerase chain

reaction (RT-PCR) method on surgically resected

human gastric and colorectal cancer specimens were

compared to paired normal counterpart tissues, and we

found that the higher expression of MTA1 mRNA was

significantly correlated to the depth of cancer invasion

and extent of lymph node metastasis (Toh et al., 1997).

This study was the first to demonstrate the clinical

relevance of MTA1 expression to the malignant

potentials of human cancers. Over-expression of MTA1

mRNA was also shown in colorectal cancers compared

to the normal counterpart tissues (Giannini et al.,

2005).

Esophageal cancers have also been investigated for

MTA1/MTA1 over-expression. Using a RT-PCR

method we found that human esophageal squamous cell

cancers over-express MTA1mRNA (Toh et al., 1999).

The over-expressing cancer cells showed significantly

higher frequencies of adventitial invasion and lymph

node metastasis and tended to have higher rates of

lymphatic involvement (Toh et al., 1999). Using

immunohistochemistry we further examined the

expression levels of MTA1 protein in human

esophageal squamous cell cancers and reconfirmed the

results obtained by RT-PCR (Toh et al., 2004). In the

same study we also demonstrated that MTA1 protein

was a predictor of poor prognosis after surgery (Toh et

al., 2004).

The roles of MTA1/MTA1 in small intestinal cancers

have also been evaluated. Kidd et al. (2006a; 2007) and

Modlin et al. (2006b) showed that it was useful to

examine the expression of MTA1mRNA and MTA1

protein in order to determine the malignant potential



and the propensity to metastasize of enterochromaffin

cell cancers (small intestinal carcinoid tumors). When

compared to nonmetastatic primary tumors, the

expression of MTA1 was increased in malignant,

invasive small intestinal carcinoid tumors and in

metastases to liver and lymph nodes. In these cells loss

of TGFβ expression modified expression, including

increased MTA1 expression, of the genes involved in

malignant behavior (Kidd et al., 2007). It was further

reported that MTA1was a good candidate genetic

molecular marker to discriminate between gastric

carcinoids and other gastric neoplasms (Kidd et al.,

2006b) as well as malignant appendiceal carcinoids

from benign tissue (Modlin et al., 2006a). In these

studies, MTA1 and MTA1 expression were thought to

be good markers of the malignant potential of carcinoid

tumors.

Other gastrointestinal-linked cancers, such as a

pancreatic cancers and hepatocellular carcinomas, have

also been examined for the involvement of

MTA1/MTA1 over-expression in carcinogenesis and

cancer progression. Iguchi et al. (2000) examined

MTA1 mRNA expression in pancreatic cancer cell lines

and resected pancreatic cancer tissues and found that

increased levels of MTA1 mRNA expression in the

more progressed pancreatic cancers. Direct evidence on

the role of MTA1/MTA1 in the progression of

pancreatic cancer was provided by Hofer et al. (2004).

Using a pancreatic cell line (PANC-1) they transfected

MTA1cDNA into the cells and found that enhanced

expression of MTA1 promoted the acquisition of an

invasive and metastatic phenotype and enhanced the

malignant potentials of the transformed cells

(pancreatic adenocarcinomas) by modulation of the

cytoskeleton via IQGAP1. In addition, Miyake et al.

(2008) showed the expression level of the MTA1

protein correlated with poorer prognosis of pancreatic

cancer patients.

An association between MTA1/MTA1 expression and

the malignant properties of hepatocellular carcinomas

(HCC) was first reported by Hamatsu et al. (2003). In

this study, MTA1 mRNA level was assessed by RT-

PCR in resected human HCC tissues, and its high

expression predicted a lower disease-free survival rate

after curative HCC hepatectomy. Using

immunohistochemistry Moon et al. (2004) examined

MTA1 protein expression in resected human HCC

specimens and found that over-expression of MTA1

was associated with HCC growth and vascular invasion

and that nuclear localization of ERa inversely

correlated with MTA1 expression. This suggested that

MTA1 was involved in negative regulatory

mechanisms.

Recently, Yoo et al. (2008) demonstrated that hepatitis

B virus (HBV) X (HBx) protein strongly induced the

expression of MTA1 and histone deacetylase 1

(HDAC1). This suggests that positive crosstalk

between HBx and MTA1/HDAC1 complex may occur,

and this could be important in stabilizing hypoxia-

inducible factor-1α (HIF1-1α), which appears to play a

critical role in angiogenesis and metastasis of HBV-

associated HCC (Yoo et al., 2008). Interestingly, it was

reported that MTA1 was closely associated with

microvascular invasion, frequent postoperative

recurrence, and poor prognosis in patients with HCC,

especially in those with HBV-associated HCC (Ryu et

al., 2008).

3.3 MTA1 protein and other cancers

The reports on MTA1/MTA1 over-expression in human

cancers have been reinforced by the experimental over-

expression or under-expression of MTA1 in human

cells. For example, Mahoney et al. (2002) transfected

MTA1 cDNA into immortalized human keratinocytes

and demonstrated that forced over-expression of

MTA1contributed to some metastatic cell properties,

such as increased cell migration, invasion and survival

in an anchorage independent medium. Nawa et al.

(2000) used antisense MTA1 to suppress MTA1 levels

and inhibit the growth of breast cancer cells in vitro.

These authors subsequently showed that in vitro

invasion of human MBA-MB-231 cells could be

inhibited by antisense MTA1 (Nicolson et al., 2003).

Similarly, using a human esophageal squamous cell

carcinoma cell line, Qian et al. (2005) inhibited MTA1

expression by RNA interference and found significant

inhibition of the cells' in vitro invasion and migration

properties.

Possible relationships between MTA1/MTA1

expression and malignant cell properties, such as

invasion and metastasis, have been investigated in other

carcinoma and sarcoma systems. Using human non-

small cell lung cancer cells high expression of MTA1

mRNA was correlated with lymph node metastasis

(Sasaki et al., 2002). This has also been found to be the

case in human ovarian cancers (Yi et al., 2003).

Additionally, in thymomas advanced stage and

invasiveness was related to MTA1 expression (Sasaki et

al., 2001).

Using various techniques the relationship between

MTA1 expression and malignancy has been

investigated in various cancers. For example, a

potential role for MTA1 protein over-expression in the

progression of human endometrial carcinomashas been

found by Balasenthil et al. (2006). Whereas in prostate

cancers Hofer et al. (2004) showed that metastatic

prostate tumors had significantly higher levels of

MTA1 protein expression and higher percentages of

tissue cores staining positive for MTA1 protein over-

expression than in clinically localized prostate cancers

or benign prostate lesions. Most interestingly, using

transgenic mice Kumar's group showed that MTA1

over-expression was accompanied by a high incidence

of spontaneous B cell lymphomas, including diffuse

large B cell lymphomas (Bagheri-Yarmand et al., 2007;

Balasenthil et al., 2007). The high expression of MTA1

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in human diffuse B-cell lymphomashas been reported

(Hofer et al., 2006). In the transgene model, mammary

adenocarcinomas also developed (Bagheri-Yarmand et

al., 2004).

Microarrays have also been used to follow MTA1and

other genes' expression. Using DNA microarray

analysis Roepman et al. (2006) investigated gene

expression patterns in lymph node metastases of head

and neck squamous cell carcinomas. They found that

the MTA1 gene was the only single gene that showed

consistent over-expression between large numbers of

matched paired samples of primary tumor and lymph

node metastases.

4. Biological significance of the MTA proteins It has been demonstrated by different laboratories (see

Section 3) that MTA1/MTA1 over-expression is closely

correlated with cancer progression (and in some cases

with carcinogenesis) in a wide range of different

cancers. This strongly indicates that the MTA1 protein

may be an important functional molecule in

malignancy. Thus, it is necessary to clarify the

molecular mechanisms by which the MTA protein

family members exert their functions.

Only then can MTA proteins be utilized for diagnosis

or treatment of human cancers. There are several

important cellular functions of MTA proteins that have

been recently clarified, such as those that are related to

carcinogenesis and cancer progression.

4.1 MTA proteins and the nucleosome remodeling-

histone deacetylation (NuRD) complex and

transcriptional regulation

The molecular and biochemical functions of the MTA1

protein were first investigated by four independent

groups. In these studies, two different chromatin-

modifying activities, ATP-dependent nucleosome

remodeling activity and histone deacetylation, were

functionally and physically linked in the same protein

complex. This complex has been named the NuRD

(Nucleosome Remodeling and Histone Deacetylation).

The NuRD complex contains HDAC1, HDAC2, the

histone binding proteins RbAp46/48 and the

dermatomyositis-specific autoantigen Mi-2, which has

been shown to have transcription repressing activity

(Tong et al., 1998; Xue et al., 1998; Wade et al., 1999;

Zhang et al., 1999; Bowen et al., 2004).

The MTA1 protein was found in the NuRD complex by

Xue et al. (1998), and this complex also possessed

strong transcription repressing activity. Subsequently,

Zhang et al. (1999) reported that a protein similar to

MTA1 (named the MTA2 protein) was also a

component of the NuRD complex, and they found that

MTA2 protein was highly expressed in rapidly dividing

cells. Later, MTA3 protein was identified as an

estrogen-inducible gene product that is present in a

distinct NuRD complex (Fujita et al., 2003). We also

reported the physical interaction between MTA1

protein and HDAC1 (Toh et al., 2000).

Figure 1. MTA proteins in a chromatin remodeling and histone deacetylation complex (NuRD). This complex has transcription repression properties. The NuRD complex also contains histone deacetylases (HDAC1 and 2), major DNA binding protein 3 (MDB3), histone binding proteins RbAp46/48 and the dermatomyositis-specific autoantigen Mi-2 (from Toh and Nicolson, 2009 with permission).

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The basic functions of the MTA protein family

members appear to be exerted through NuRD

complexes as chromatin remodeling and histone

deacetylating activities (Figure 1). Although there are

also non-histone deacetylating proteins in NuRD

complexes, MTA proteins appear to be among the

principal components (Figure 1). In addition, the MTA-

NuRD complexes show transcriptional repression

activities (Feron, 2003; Kumar et al., 2003; Manavathi

et al., 2007b; Singh and Kumar, 2007). Although all

MTA protein family members are found in NuRD

complexes, each MTA protein may form a distinct

NuRD complex that targets different sets of promoters

(Bowen et al., 2004).

4.2 MTA protein repression of the trans-activating

activity of estrogen receptor-alpha

The involvement of MTA proteins in NuRD complexes

suggested that such complexes might function in

chromatin remodeling and histone deacetylation, but a

direct target of a MTA protein had to first be identified

(Mazumdar et al., 2001). MTA1 protein was identified

as a molecule induced by heregulin-beta1 (HRG), a

growth factor that is a natural ligand of the human

epidermal growth factor receptors HER3 and HER4. It

can also trans-activate HER2 (c-erbB-2) in human

breast cancer cell lines. Mazumdar et al. (2001) showed

that MTA1 protein directly interacted with the ligand-

binding domain of the estrogen receptor ERα and that

HRG stimulated the association of MTA1 and HDAC2

on the chromatin site of an ER-responsive

element (ERE) in the promoter regions of estrogen

responsive genes, such as pS2 and c-myc. This could

explain the activation of the HRG/HER2 pathway in

ER-positive breast cancers and the suppression of ERα

functions, which could result in the more invasive and

aggressive phenotypes observed in ER-negative breast

cancers (Cui et al., 2006).

The repressive function of MTA1 protein on ERα is

mediated via histone deacetylation by HDAC1 and

HDAC2, suggesting that MTA1 protien has a potent

co-repressor function during the trans-activation of

ERα through histone deacetylation (Figures 2 and 3).

MTA2 protein has also been shown to physically

interact with ERα and to repress its trans-activating

function. This could explain the over-expression of

MTA2 protein in cells that were unresponsive to

estrogen as well as suppression of estrogen-induced

colony formation in breast cancer cells (Cui et al.,

2006).

MTA proteins also have other activities. For example,

Khaleque et al. (2008) showed that MTA1 protien

binds to a heat shock factor 1 (HSF1), the

transcriptional activator of the heat shock genes, in

vitro and in human breast carcinoma samples. They

demonstrated that HSF1-MTA1 complex formation

was strongly induced by HRG and that the complex

was incorporated into a NuRD complex that

participated in the repression of estrogen-dependent

transcription in breast cancer cells treated with HRG

(Khaleque et al., 2008).

Figure 2. A possible role for MTA proteins in carcinogenesis and cancer progression. In this scheme the main functions of the MTA family of proteins are presented. (A) MTA1 protein is included in a NuRD complex that represses the transactivation function of estrogen receptor (ER), rendering breast cancer cells more phenotypically aggressive. MTA1 proteins in NuRD complexes are proposed to be one of the first downstream targets of c-MYC function, and it is essential for the transformation potential of c-MYC. MTA1s is a splice-variant of MTA1 that localizes in the cytoplasm where it sequesters ERα, preventing the ligand-induced nuclear translocation of Erα, thus stimulating the development of the malignant phenotype of breast cancer cells. (B) MTA3 protein induced by estrogen represses the expression of the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transitions, resulting in the expression of the cell adhesion molecule E-cadherin and maintenance of a differentiated, normal epithelial status in breast cells (from Toh and Nicolson, 2009 with permission).



There are apparently several molecules, such as

ménage-à-trois 1 (MAT1), MTA1-interacting co-

activator (MICoA) and nuclear receptor interacting

factor 3 (NRIF3), that can interact with MTA1 protein

and repress the trans-activation function of ERα

(Manavathi et al., 2007b). These three MTA1-binding

proteins themselves have co-activator properties upon

ERα trans-activation. MAT1, an assembly and

targeting ring finger factor for cyclin-dependent kinase-

activating kinase (CAK), has been identified by

Talukder et al. (2003) as a MTA1-binding protein. The

interactions between CAK and MTA1 protein

apparently regulate the trans-activation activity of ERα

in a CAK-dependent manner in breast cancer cells. In

contrast, MICoA-mediated ERα trans-activation

functions are opposed by MTA1 protein through the

recruitment of HDACs (Mishra et al., 2003). In

addition, the interactions between MTA1 protein and

NRIF3 (an estrogen-inducible gene) may be important

in modulating the sensitivity of breast cancer cells to

estrogen (Talukder et al., 2004).

Another MTA1-binding protein partner, Lim-only

protein 4 (LMO4), has been identified by Singh et al.

(2005). LMO4 was found to be a component of the

MTA1 co-repressor complex, and its over-expression

repressed ERα trans-activation in a HDAC-dependent

manner. This has been proposed to result in the

acquisition of an ERα-negative phenotype with its

known increased aggressiveness in breast cancer cells

(Singh et al., 2005).

Variants of MTA1 protein have also been found. For

example, a truncated form of MTA1 protein has been

identified and named MTA1s (Balasenthil et al., 2006).

MTA1s is a splice-variant of MTA1, and it contains an

ER-binding motif (nuclear binding motif) without any

nuclear localization signals at its C-terminus. This

truncated MTA protein localizes in the cytoplasm

where it sequesters ERα, resulting in the blockage of

ERα ligand-induced nuclear translocation and

stimulation of acquisition of the malignant phenotype

of breast cancer cells. This suggests that the regulation

of the cellular localization of ERα by MTA1s protein

may represent a mechanism for redirecting nuclear

receptor signaling by nuclear exclusion. MTA1s

protein has also been shown to associate with casein

kinase I-gamma2, which is an estrogen-responsive

kinase (Mishra et al., 2004).

MTA3 protein is the newest addition to the MTA

family. It was identified as an estrogen-dependent

component of the Mi-2/NuRD transcriptional co-

repressor complex in breast epithelial cells (Fujita et

al., 2003).

The absence of MTA3 protein as well as the absence of

ER results in an aberrantly increased expression of the

transcriptional repressor Snail, a master regulator of

epithelial-to-mesenchymal transition (EMT). This

increased expression of Snail results in reduction in

expression of the cell adhesion molecule E-cadherin,

which subsequently modifies epithelial cell architecture

and enhances invasive growth. MTA3 protein is a

transcriptional target of ERα, and in the presence of

estrogen ERα directly binds to the MTA3 promoter at

the SP1 site in close proximity to the ERE half-site,

resulting in stimulation of MTA3 transcription (Fujita et

al., 2004; Mishra et al., 2004). Thus, MTA3 protein

may function to maintain a well-differentiated, normal

epithelial phenotype in breast cells. This is in stark

contrast to MTA1 or MTA1s protein, where up-

regulation of MTA1 or MTA1s protein in breast cancer

cells may repress MTA3 expression through repression

of the ERα function, resulting in up-regulation of Snail,

down-regulation of E-cadherin, promotion of an EMT

phenotype and potentially an increase in metastatic

potential.

Forced expression of MTA3 protein inhibits ductal

branching in virgin and pregnant mammary glands in

MTA3-transgenic mice (Zhang et al., 2006). This

property is in marked contrast to MTA1-transgenic

mice, where there is inappropriate development of

mammary glands, resulting in the development of

hyperplastic nodules and mammary tumors, including

adenocarcinomas and lymphomas (Bagheri-Yarmand et

al., 2004; Manavathi and Kumar, 2007). MTA3 protein

also represses Wnt4 transcription and secretion by

inhibiting Wnt-target genes in mammary epithelial

cells. This repression of Wnt4 transcription was found

to be mediated through a MTA3-NuRD complex,

which interacts with the Wnt4-containing chromatin in

an HDAC-dependent process (Zhang et al., 2006).

The fundamental actions of the MTA proteins are

exerted via transcriptional repression by histone

deacetylation; however, a transcriptional activating

function has also been described for MTA complexes.

Gururaj et al. (2006a, 2006b) showed that breast cancer

amplified sequence 3 (BCAS3), a gene amplified and

over-expressed in breast cancers, was a chromatin

target of MTA1 protein, and the transcription of

BCAS3 was stimulated by MTA1 protein. This

suggested that MTA1 protein has a transcriptional co-

activator function in addition to its co-repressor

function. A similar property has been also been

suggested for mouse Mta2 protein (Matsusue et al.,

2001).



Figure 3. Deacetylation of non-histone proteins by MTA protein family complexes. (A) Tumor suppressor p53 protein is deacetylated and inactivated by both MTA1 and MTA2 proteins in NuRD complexes, resulting in inhibition of growth arrest and apoptosis. (B) Hypoxiainducible factor-1α (HIF-1α) is also deacetylated and stabilized by MTA1 protein, leading to angiogenesis (from Toh and Nicolson, 2009 with permission).

4.3 MTA-NuRD protein complexes and

deacetylation of non-histone proteins

Chromatin histones and non-histone proteins are the

protein targets for deacetylation by HDAC via NuRD

complexes containing MTA proteins. The tumor

suppressor gene p53 protein was the first non-histone

protein that was reported to be deacetylated by MTA

protein-containing NuRD complexes. Luo et al. (2000)

reported that the deacetylation of p53 was mediated by

an HDAC1 complex containing MTA2 protein. A

MTA2-associated NuRD complex was involved, and

this HDAC1/MTA2 complex interacted with p53 in

vitro and in vivo and reduced significantly the steady-

state levels of acetylated p53. Deacetylation of p53

causes an increase in its own degradation through

MDM2 and a reduction in p53-dependent

transcriptional activation. Eventually this results the

repression of the normal p53 function that mediates cell

growth arrest and apoptosis (Figure 3). The same

phenomenon was observed between p53 and MTA1

complexes. HDAC1/MTA1 complexes possessed

deacetylation activity against p53 protein in human

non-small cell carcinoma and human hepatoma cells,

and the complexes were found to inhibit p53-induced

apoptosis by attenuating the trans-activation function of

p53 (Moon et al., 2007). More recently the stability of

p53 was determined to be affected by MTA1 inhibiting

p53 ubiquitination by E3 ubiquitin ligases double

minute 2 (Mdm2) and constitutive photomorphogenic

protein 1 (COP1). MTA1 competes with COP1 to bind

to p53 and/or destabilize COP1 and Mdm2 (Li et al.,

2009b). MTA1 stability and degradation itself is

controlled by ubiquitination, and degradation of MTA1

is promoted by COP1-mediated hydrolysis (Li et al.,

2009b).

HIF-1α (hypoxia-inducible factor-1α) is another

important non-histone protein that is deacetylated by

HDAC1/MTA1 complexes (Figure 3). HIF-1α is a key

regulator of angiogenic factors (Yoo et al., 2006). The

expression of MTA1 was strongly induced under

hypoxic conditions in breast cancer cell lines, and

MTA1 protein over-expression enhanced the

transcriptional activity and stability of HIF-1α protein.

MTA1 protein physically bound to HIF-1α and

deacetylated it by increasing the expression of HDAC1,

leading to the stabilization of HIF-1α (Yoo et al.,

2006). These results indicated possible positive cross-

talk between MTA1 and HIF-1α, mediated by HDAC1

recruitment.

Moon et al. (2006) found a close connection between

MTA1-associated metastasis and HIF-1α-induced

tumor angiogenesis. They showed that MTA1 protein

increased the transcriptional activity of HIF-1α and a

target molecule of HIF-1α, vascular endothelial growth

factor (VEGF). Conditioned medium collected from

MTA1-transfectants increased angiogenesis in vitro and

in vivo (Moon et al., 2006). Functional links between

HIF-1α and MTA1 protein have been demonstrated in

clinical samples of pancreatic carcinoma. Using

immunohistochemistry and surgically resected

pancreatic carcinomas Miyake et al. (2008) examined

the expression of HIF-1α, HDAC1 and MTA1 proteins

and suggested that HIF-1α expression, which is

associated with a poor prognosis in patients with

pancreatic cancers, might be regulated by

HDAC1/MTA1 complexes. The contribution of MTA1

protein to tumor angiogenesis was also demonstrated in

human breast cancers. Using immunohistochemistry

Jang et al. (2006) examined MTA1 protein expression

and intra-tumoral microvessel density (MVD) in

clinical samples of breast cancer and showed that

MTA1 protein expression was significantly correlated

with higher tumor grade and higher tumor MVD.

The relationship between MTA1 protein expression and

MVD was also observed in hepatitis B-associated HCC

(Ryu et al., 2008). In this tumor system hepatitis B



virus X protein (Hbx) induces the expression of MTA1

protein and its HDAC1 complex, which enhances

hypoxia signaling in HCC (Yoo et al., 2006). This

suggests that the HDAC1 complex containing MTA1

protein may be important in stabilizing HIF-1α, and

thus play a role in angiogenesis and metastasis.

The relationship between the protein members of

NuRD complexes, including MTA1 and MTA2

proteins, and the ataxia teleangiectasia mutated (ATM)-

and Rad3-related protein (ATR) has been shown by co-

immunoprocipitation of these proteins (Schmidt and

Schreiber, 1999). ATR is a phosphatidylinositol-

kinase-related kinase that has been implicated in the

response of human cells to multiple forms of DNA

damage and may play a role in the DNA replication

checkpoint. This suggests that MTA proteins may

contribute to the regulation of DNA checkpoints (Toh

and Nicolson, 2009).

4.4 MTA proteins: other possible functions in

cancer cells

Other reports have been forthcoming suggesting some

possible roles of MTA proteins in carcinogenesis and

cancer progression. The most important of these may

be the relationship of MTA1 protein with c-MYC

oncoprotein (Figure 2). Using expression profiling,

Zhang et al. (2005) identified the MTA1 protein as a

target of the c-MYC protein in primary human cancer

cells. They showed that c-MYC binds to the genomic

MTA1 locus and recruits transcriptional co-activators.

They also found that the MTA1 proteins in NuRD

complexes were one of the first downstream targets of

c-MYC function, and this was essential for the

transformation potential of c-MYC. Indeed, reduction

of MTA1 expression by a short hairpin RNA blocked

the ability of c-MYC to transform mammalian cells

(Zhang et al., 2005).

Another milestone was the establishment of a

transgenic mice model that over-expressed MTA1

protein. Kumar and his collaborators found that the

MTA1-transgenic mice showed inappropriate

development of mammary glands. These mice also

developed hyperplastic nodules and mammary tumors

(Bagheri-Yarmand et al., 2004; Singh and Kumar,

2007). In this study, the underlying molecular

mechanisms of MTA1 protein action and its regulation

were also examined, and the results suggested that

MTA1 protein dysregulation in mammary epithelium

and cancer cells triggered down-regulation of the

progesterone receptor-B isoform and up-regulation of

the progesterone receptor-A isoform, resulting in the

up-regulation of the progesterone receptor-A target

genes Bcl-XL and cyclin D1 in mammary glands of

virgin mice. These authors also found that spontaneous

B-cell lymphomas were induced in the MTA1-

transgenic mice (Bagheri-Yarmand et al., 2007).

Recently, Molli et al. (2008) reported that

MTA1/NuRD complexes negatively regulated BRCA1

transcription by physically associating with ERE of the

BRCA1 promoter in an ERα-dependent manner. This

repressive effect of MTA1 on BRCA1 expression

resulted in the acquisition of abnormal centrosomes and

chromosomal instability (Molli et al., 2008).

The expression of MTA1 and HDAC1 proteins can also

be increased by the interaction of hepatitis B virus X

(HBx) protein at the transcriptional level (Yoo et al.,

2008). Since MTA1 and HDAC1/2 proteins are

physically associated with HIF-1α in vivo in the

presence of HBx protein, HBx-induced deacetylation

stabilizes HIF-1α by inhibiting proteosomal

degradation. These results indicated the existence of

positive cross-talk between HBx and the MTA1/HDAC

complex, and it further suggests that such cross-talk

may play a role in angiogenesis and metastasis of

HBV-associated hepatocellular carcinomas.

Direct interactions between MTA1 protein and

endophilin 3 have also been reported by Aramaki et al.

(2005). This suggests that MTA1 protein might be

involved in the regulation of endocytosis mediated by

endophilin 3.

An important treatment modality in cancer is the use of

ionizing radiation. MTA1 protein has been implicated

in ionizing radiation-induced DNA damage response by

regulating p53-dependent DNA repair (Li et al.,

2009a).

5. MTA/MTA genes and proteins as new clinical targets This review and others (Nicolson et al., 2003;

Manavathi and Kumar, 2007; Toh and Nicolson, 2009)

have discussed the available data on the likelyhood that

MTA proteins have important and critical roles in the

genesis and progression of a wide variety of cancers.

MTA1 protein can be thought of as a master co-

regulatory molecule (Manavathi and Kumar, 2007; Toh

and Nicolson, 2009). This clearly suggests the

possibility that MTA1 protein (or the MTA1 gene or its

RNA product) could be an excellent molecular target

for cancer therapy as well as its use in cancer

diagnosis/prognosis.

The first studies that suggested the possibility of

targeting MTA1 RNA were reported by Nawa et al.

(2000) and Nicolson et al. (2003). Using antisense

phosphorothioate oligonucleotides against MTA1

mRNA, these authors found growth inhibitory effects

and inhibition of invasion of human metastatic breast

cancer cell lines.

Different techniques have been used to regulate

MTA1/MTA1 expression in order to determine the

effects of MTA1 protein on cellular functions. Using

RNA interference (RNAi) Qian et al. (2007) inhibited

MTA1 expression in a human esophageal squamous

cell carcinoma cell line and demonstrated significant

inhibition of in vitro invasion and migration properties

of the cancer cells (Qian et al., 2005). In a metastasis

model based on murine melanoma Qian et al. (2007)

examined the therapeutic use of lowering MTA1



protein levels in the melanoma cells and demonstrated

that down-regulation of MTA1 protein by RNAi

successfully suppressed growth in vitro and

experimental metastasis in vivo. Using microRNAs

against MTA1 Reddy et al. (2009) were able to inhibit

the expression of MTA1 protein in human breast cancer

cells, resulting in decreased cell mobility, invasiveness,

anchorage-dependent growth and tumorigenicity.

Results such as these suggest a potential role of the

MTA1 gene as a target for cancer gene therapy.

Other MTA/MTA genes and proteins may also be

useful targets. For example, MTA1s may be a useful

target in the treatment of breast cancer. MTA1s

functions as a repressor of ERα transcriptional activity

by binding and sequestering the ERα in the cytoplasm

(Kumar et al., 2002). MTA1s has a unique C-terminal

33-amino acid region containing a nuclear receptor-box

motif that mediates the interaction of MTA1s protein

with ERα. Singh et al. (2006) showed that the MTA1s

peptide containing this motif could effectively repress

the ERα transactivation function, measured by

estrogen-induced proliferation and anchorage-

independent growth of the human breast cancer cell

line MCF-7. Using an animal model they also showed

the effect of MTA1s peptide in blocking tumor

progression of MCF-7 breast cancer cells that over-

expressed ERα (Singh et al., 2006).

The use of MTA1 protein as a target of immunotherapy

has also been considered. MTA1 protein is a promising

antigen for tumor rejection, because it is over-

expressed in many different tumors and is only

expressed at lower levels in normal tissues (Toh and

Nicolson, 2009). In reviewing a model for

immunotherapy Assudani et al. (2006) proposed using

a vector that contained disabled infectious single cycle-

herpes simplex virus (DISC-HSV). Their initial studies

demonstrated the presence of immunogenic MHC class

I-restricted peptides of MTA1 protein. Next, MTA1

protein was identified as a SEREX antigen, and hence

it is likely to be capable of inducing a T-cell response

in cancer patients (Chen and Han, 2001).

6. MTA/MTA genes and proteins: future directions This and previous reviews (Toh and Nicolson, 2003;

Manavathi and Kumar, 2007; Toh and Nicolson, 2009)

have focused on the clinical and biological significance

of the newly emerging gene family named MTA. The

family of MTA proteins is made up of transcriptional

co-repressors that function via NuRD complexes

containing chromatin remodeling and histone

deacetylating molecules. These actions clearly have a

role in tumor formation and progression. For example,

the repression of ERα trans-activation function by

MTA1 protein through deacetylation of ERE chromatin

of the ER-responsive genes has been the most

extensively investigated, and the data clearly

demonstrated that MTA1 expression results in tumor

formation in mammary glands and renders breast

cancer cells phenotypically more aggressive (reviewed

in Manavathi and Kumar, 2007).

In addition to chromatin histones, MTA proteins also

deacetylate non-histone proteins. For example, the

tumor suppressor p53 protein is deacetylated and

inactivated by both MTA1 and MTA2 proteins,

resulting in inhibition of growth arrest and apoptosis.

HIF-1α is also deacetylated and stabilized by MTA1,

leading to angiogenesis. Thus, it has been proposed that

MTA proteins, especially MTA1 protein, represent

master co-regulatory molecules involved in the

carcinogenesis and progression of various malignant

tumors (Manavathi and Kumar, 2007; Toh and

Nicolson, 2009). Since, it is assumed that these

properties are important to the survival and progression

of cancer cells, ultimately this could lead to novel

clinical applications of MTA genes or MTA proteins as

new molecular targets for cancer therapy.

There are other examples of the potential use of MTA

proteins as therapeutic targets. Inhibition of MTA1

protein expression or function may enhance the

chemosensitivity of cancer cells by restoring tumor

suppressor function of p53, or it may inhibit tumor

angiogenesis by destabilizing the angiogenesis

promoting function of HIF-1α. Moreover, MTA

proteins may cooperate with HDAC inhibitors, which

are now expected to be the target of a new class of

anticancer agents (Toh and Nicolson, 2009).

MTA1 will also be clinically useful for the prognosis or

prediction of the malignant potentials of various human

cancers, such as esophageal, gastric and colorectal

cancers (Toh and Nicolson, 2009). Thus, evaluating the

expression levels of MTA proteins in individual cases

of various cancers may provide us with important

clues.

Finally, the MTA proteins are clearly present in

completely normal cells to provide them with certain

necessary functions. Thus, it will be important to

understand their physiological functions and underlying

mechanisms in normal cells. For example, C. elegans

has MTA1 homologues, egl-27 and egr-1, that function

in embryonic patterning and development (Solari et al.,

1999; Chen and Han, 2001), suggesting that MTA1

protein may have an embryonic developmental

function. MTA1 protein is also thought to play a crucial

role in postnatal testis development and

spermatogenesis (Li et al., 2007a; Li et al., 2007b), and

MTA1 protein is a direct stimulator of rhodopsin

expression (Manavathi et al., 2007a). These are only a

few of the known physiological functions of MTA1

protein (Toh and Nicolson, 2009), and it is expected

that other MTA proteins have important roles in normal

physiology and development. Thus, determining the

normal physiological functions of MTA proteins will

be absolutely necessary in understanding the

pathological functions of MTA proteins in human

cancers.



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Role of p38α in apoptosis: implication in cancer development and therapy Almudena Porras, Carmen Guerrero

Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, UCM, Ciudad Universitaria,

28040 Madrid, Spain (AP); Centro de Investigación del Cáncer, IBMCC, Universidad de Salamanca-CSIC,

37007 Salamanca, Spain (CG)


Online updated version : http://AtlasGeneticsOncology.org/Deep/MAPK14-p38ainCancerID20089.html DOI: 10.4267/2042/44993


1- Introduction The family of p38 mitogen-activated protein kinases

(MAPKs) belongs to the MAPK superfamily. They are

strongly activated by different stress signals and

inflammatory cytokines, although non-stressful stimuli

also activate p38 MAPKs leading to the regulation of

cellular functions such as proliferation, differentiation

and survival. Four different p38 MAPK family

members have been identified so far: p38α, β, γ and δ,

also known as stress-activated kinase 2a (SAPK2a),

SAPK2b, SAPK3 and SAPK4, respectively, which may

have both overlapping and specific functions (reviewed

by Cuenda and Rousseau, 2007; Kyriakis and Avruch,

2001; Nebreda and Porras, 2000; Ono and Han, 2000).

p38α was initially identified as a 38 KDa protein,

which became phosphorylated in Tyr in response to the

bacterial endotoxin LPS in macrophages (Han et al.,

1994). In parallel, other two groups identified p38α as a

kinase activated by stress and IL1, able to

phosphorylate and activate MAPKAP-K2 (MAPK-

activated protein kinase 2) (Freshney et al., 1994;

Rouse et al., 1994). p38α (encoded by MAPK14gene) is

broadly expressed and is also the most abundant p38

isoform present in most cell types. Targeted

inactivation of the mouse p38α gene results in

embryonic death due to a placental defect (Adams et

al., 2000; Mudgett et al., 2000; Tamura et al., 2000).

p38 MAPKs are mainly activated by MKK3 and

MKK6 through dual phoshorylation in Tyr and Thr,

although MKK4 can sometimes activate p38α (Cuenda

and Rousseau, 2007).

MKK3 and MKK6 are in turn activated by

phosphorylation by a MAPK kinase kinase (MKKK)

such as MLKs, ASK1, TAK1 or MEKKs, which are

activated by small and heterotrimeric G proteins

(Cuenda and Rousseau, 2007; Wagner and Nebreda,

2009).

p38α can be also activated through MKK-independent

pathways such as that involving p38α

autophospohorylation upon interaction with TAB1

(Cuenda and Rousseau, 2007).

Once p38 MAPKs are activated, they phoshorylate

different transcription factors such as p53, ATF2,

MEF2 or C/EBPb and protein kinases, including

MAPKAP-K2 and MAPKAP-K3 (also known as MK-2

and MK-3), MSK-1 (mitogen- and stress-activated

protein kinase 1) and MNK-1 and MNK-2 (MAP

kinase-interacting serine/threonine kinase 1 and 2)

(Cuenda and Rousseau, 2007; Wagner and Nebreda,

2009).

The analysis of the function of p38α has been initially

based on the effect of chemical inhibitors such as

SB203580 and SB202190, which inhibit p38α but can

also inhibit p38β at a higher dose (reviewed by

Nebreda and Porras, 2000). Therefore, the main effects

observed upon treatment with those inhibitors are a

consequence of p38α inhibition, sometimes are also

due to p38β inhibition. The generation of mice with

specific genetic inactivation of the different p38

isoforms or the use of interference RNA technology

during the last few years has allowed a more precise

study of the role played by each isoform and they have

been essential to establish the role of p38α in different

cellular processes.

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Role of p38α in apoptosis: implication in cancer development and therapy Porras A, Guerrero C


Figure 1. p38 MAPK signaling pathway. A variety of stimuli, mainly stress stimuli, activate p38 MAPK through complex kinase cascades including a MAP3K that phosphorylates a MAPK2 that, in turn, phosphorylates p38MAPKs. Cellular intermediates, like

GTPases, receptor adapter proteins or cell cycle checkpoint proteins, among others, transmit the stimulus to the kinase cascades. Once activated, the different p38MAPKs either phosphorylate cytoplasmic targets or translocate into the nucleus leading to the regulation of transcription factors involved in cellular responses. MKK6 is the major activator of all p38 MAPK isoforms, while MKK3 and MKK4 are

more specific and can only activate some isoforms.

2- Dual role of p38 MAPK in cell death: function of p38α in apoptosis p38 MAPK can play a dual role as a regulator of cell

death, thus it can either mediate cell survival or cell

death through different mechanisms, including

apoptosis. Therefore, the specific function of p38

MAPKs in apoptosis appears to depend on the cell

type, the stimuli and/or the isoform (reviewed by

Nebreda and Porras, 2000; Wagner and Nebreda,

2009).

As indicated above, some studies have been based on

the using of chemical inhibitors such as SB203580

which is able to inhibit p38α and p38β, thus the

participation of the p38β isoform in apoptosis can not

be excluded. In fact, there is a good evidence for a role

of p38α and/or p38β MAPKs as mediators of apoptosis

in several cell types such as neurons (Ciesielski-Treska

et al., 2001; De Zutter and Davis, 2001; Ghatan et al.,

2000; Le-Niculescu et al., 1999) or cardiac cells

(Mackay and Mochly-Rosen, 1999; Saurin et al., 2000;

Wang et al., 1998). Moreover, the induction of

apoptosis by many types of stimuli such TNF-

α(Valladares et al., 2000), TGF-β (Edlund et al., 2003)

or oxidative stress (Zhuang et al., 2000) involves one of

these p38MAPKs.

p38α is a mediator of apoptosis in response to a number

of cellular stresses through transcriptional and

posttranscriptional mechanisms, which can involve the

regulation of apoptotic and/or survival pathways

(reviewed by Wagner and Nebreda, 2009).

For example, p38α sensitizes cardiomyocytes and

MEFs-derived cell lines to apoptosis induced by

different stimuli through both, up-regulation of the pro-

apoptotic proteins Fas and Baxand down-regulation of

the activity of ERKs and Akt survival pathways (Porras

et al., 2004; Zuluaga et al., 2007a). According to this,

overexpression of p38α enhances apoptosis induced by

the constitutive active mutant MKK3bE in

cardiomyocytes, while overexpression of p38β

promotes cell survival (Nebreda and Porras, 2000).

p38α is also a negative regulator of survival in

embryonic stem (ES) (Guo and Yang, 2006). In

addition, p38α is able to suppress tumor initiation

induced by H-rasoncogene through induction of

apoptosis (Dolado et al., 2007). Upon expression of

oncogenic H-Ras, ROS are generated leading to p38α

activation and apoptotic cell death.

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Moreover, p38α MAPK plays an important role in the

apoptosis induced by some chemotherapeutical drugs.

For example, it is essential for cisplatin-induced

apoptosis in the colon carcinoma derived cell line,

HCT116 (Bragado et al., 2007).

On the other hand, anti-apoptotic roles of p38 MAPKs

have been described in DNA-damaged fibroblasts

(Héron-Milhavet and LeRoith, 2002), differentiating

neurons (Okamoto et al., 2000) and activated

macrophages (Park et al., 2002). In some cases, p38α

has been identified as the isoform responsible for this

survival effect. For example, in pulmonary arterial

endothelial cells exposed to anoxia-reoxygenation

during ischemia-reperfusion, carbon monoxide (CO)

protects from apoptosis through p38α activation (Zhang

et al., 2003; Zhang et al., 2005). In response to H2O2,

p38α also mediates cell survival in MEFs, while p38α

mediates apoptosis triggered by serum deprivation

(Gutiérrez-Uzquiza et al., 2010). In both situations,

C3G acts as a negative regulator of p38α. This

crosstalk between the C3G and the p38α pathways has

been also observed in CML cell line K562, where p38α

mediates STI-571-induced apoptosis (Maia et al.,

2009).

In contrast to the apoptotic role of p38 in CML cells

treated with STI-571, p38 MAPK pathway exerts an

antiapoptotic role in CML cells, as well as acute

promyelocytic leukemia cells (APL), treated with

arsenic trioxide (AT) (Verma et al., 2002b).

In some tumor cells p38α can also induce a pro-

survival effect known as tumor dormancy, which

maintains cells in a quiescent state related to drug

resistance (reviewed by Aguirre-Ghiso, 2007). In

addition, p38α can also induce cell survival of

colorectal cancer cells through inhibition of autophagy

(Comes et al., 2007).

3- Pathways regulating p38α apoptotic function There are different signaling pathways that are

involved in the regulation of p38 apoptotic function.

Some of them can be considered classical and/or

canonical, while others such as C3G-Rap1 or PKC are

novel or less known.

Classically, two small GTPases from the Rho family,

Rac1 and Cdc42, have been described to be activators

of p38α MAPK (Nobes and Hall, 1995). The existence

of a functional crosstalk between Rac and p38 MAPK

has been largely reported in relation with different

cellular functions. p38 can act either as an effector or as

an activator of Rac. In particular, in cardiomyocytes

p38α acts as a positive or a negative regulator of Rac1

depending on the presence of growth factors (Zuluaga

et al., 2007b). On the other hand, Rac-GTP induces p38

activation in many systems (Nobes and Hall, 1995). For

example, Rac-1 is an upstream regulator of p38 in

retinoic acid-induced differentiation and apoptosis of

malignant NB-4 (acute pro-myelocytic leukemia) and

MCF-7 (breast carcinoma) cell lines (Alsayed et al.,

2001).

Recent novel findings support the participation of

another member of the Ras family, the GTPase Rap1,

in the regulation of p38α function in apoptosis

(Gutiérrez-Uzquiza et al., 2010; Maia et al., 2009).

Rap1 can either activate or inhibit p38α depending on

the cell context. In CML cells, the C3G-Rap1 pathway

downregulates the pro-apoptotic activity of

p38αMAPK as a mediator of STI effects (Maia et al.,

2009). In contrast, in MEFs C3G is a negative regulator

of p38α MAPK activity and Rap1 a positive one,

leading to pro- or anti-apoptotic effects depending on

the stress stimulus (Gutiérrez-Uzquiza et al., 2010).

There are also evidences of a cross-talk between PKCs

and p38 MAPK in order to regulate apoptosis. For

example, in LNCaP prostate cancer cells, PMA induces

apoptosis through a mechanism, which involves PKCα

and d-mediated p38α/β activation (Tanaka et al., 2003).

An autocrine pro-apoptotic loop is also generated in

these cells by PKCδ, which is mediated by death

receptor ligands through a mechanism dependent on

caspase-8, FADD, p38α/β MAPK and JNK (Gonzalez-

Guerrico and Kazanietz, 2005). In addition, in vascular

smooth muscle cells activation of PKCδ leads to

apoptotic cell death involving p53 induction through a

mechanism partially dependent on p38α/β MAPK

(Ryer et al., 2005).

4- Targets of p38α in the regulation of apoptosis The mechanisms by which p38α can mediate apoptosis

or cell survival, include those involving the regulation

of the expression and/or activity of different members

of the Bcl-2 family.

Bax is a pro-apoptotic member of the Bcl-2 family,

which can be regulated by p38α through different

mechanisms. In cardiomyocytes derived cell lines,

p38α mediates an up-regulation of Bax mRNA and

protein, which sensitizes cells to apoptosis induced by

different stimuli (Porras et al., 2004). In addition, in

primary neonatal cardiomyocytes p38 (α/β) mediates

Bax translocation to mitochondria upon simulated

ischemia (Capano and Crompton, 2006). This p38

(α/β)-induced Bax translocation was also observed in a

human hepatoma cell line (HepG2) treated with

different pro-apoptotic stimuli (Kim et al., 2006). In

contrast, in the anoikis-induced apoptosis in mammary

epithelial cells, p38α is not required for Bax

mitochondrial translocation, but it acts as a part of a

mitochondrial complex allowing Bax activation and

cytochrome-c release (Owens et al., 2009).

Bimlevels (Cai and Xia, 2008) and activity (Cai et al.,

2006) are also positively regulated by p38α, which

contributes to induce apoptosis. Thus, in PC12 cells

treated with sodium arsenite, p38α induces

FOXO3anuclear translocation, which stimulates Bim

transcription, leading to an increase in BimEL protein

levels (Cai and Xia, 2008). In addition, p38α

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contributes to BimEL phosphorylation in Ser65 (Cai et

al., 2006).

Bcl-2 has been shown to be phosphorylated by p38α

upon NGF withdrawal in memory B lymphocytes,

which leads to inhibition of its anti-apoptotic activity

and to induction of cytochrome-c release (Torcia et al.,

2001). Similarly, H2O2 induces p38α-mediated Bcl-2

phoshorylation in adult rat cardiac myocytes

contributing to apoptosis (Markou et al., 2009).

p38 can also mediate survival through regulation of the

expression and/or activity of proteins from the Bcl-2

family. For example, carbon monoxide protects from

ischemia-reperfusion lung injury through a p38α-

dependent up-regulation of Bcl-2 and Bcl-xLprotein

levels (Zhang et al., 2003). According to this, UVA-

induced p38 (α/β) activation in a human keratinocyte

cell line (HaCaT cells) results in an increase in Bcl-xL

protein levels through post-transcriptional mechanisms

mediated by the 3'-unstranslated region (UTR)

(Bachelor and Bowden, 2004). In addition, in primary

human trophoblasts p38 appears to mediate EGF-

mediated Bad phosphorylation in Ser 112, which

protects from hypoxia-induced apoptosis (Humphrey et

al., 2008).

p38α is also involved in the stimulation of Fas and

FasL expression, which can contribute to apoptosis in a

number of cellular systems. p38α was shown to

participate in the anti-CD3-induced upregulation of Fas

and FasL expression in T cells, although p38α alone

was unable to increase Fas expression (Hsu et al.,

1999). Similarly, p38 MAPK is a mediator of hepatitis

B virus X protein-induced Fas and FasL expression

(Wang et al., 2004). p38α activation also mediates Fas

expression through phosphorylation of STAT1 in Ser-

727 in cardiac myocytes exposed to

ischemia/reperfusion (Stephanou et al., 2001).

According to this, Fas expression is down-regulated in

p38α-deficient embryonic cardiomyocytes under basal

conditions which contributes to sensitize cells to

apoptosis (Porras et al., 2004). In contrast, p38

downregulates Fas expression through inhibition of

NF-kB in human melanoma cells (Ivanov and Ronai,

2000).

Apart from the role of p38α in the regulation of

Fas/FasL expression, p38α mediates caspase 8

activation in Fas-activated Jurkat cells by inhibiting the

phoshorylation and presence of c-FLIPs in the DISC

(Tourian et al., 2004). p38 also mediates TGF-β-

induced activation of caspase 8 (Schrantz et al., 2001)

as well as the regulation of membrane blebbing and

nuclear condensation (Deschesnes et al., 2001).

The tumor suppressor protein p53, which is a relevant

mediator of apoptosis in response to a great variety of

stimuli, is also an important p38 target. In fact, there

are a number of evidences supporting a role for p38α in

the regulation of p53. For example, in response to

different chemotherapeutic drugs such as cisplatin p38α

phoshorylates and/or activates p53 leading to onset of

apoptosis (Bragado et al., 2007; Sánchez-Prieto et al.,

2000). p38α can also induce apoptosis through p53 in

response to other stimuli,. Thus, p53 phosphorylation

by p38α is essential in the apoptosis induced by the

HIV-1 envelope (Perfettini et al., 2005). In addition to

this role of p38α as a direct regulator of p53, p38α also

acts through phosphorylation and stabilization of the

p53 coactivator, p18Hamlet (Cuadrado et al., 2007). In

response to DNA damage p18Hamlet is accumulated,

which contributes to induce apoptosis through the

stimulation of some p53-regulated genes, such as Noxa.

Other alternative mechanisms are involved in p38α-

mediated apoptosis. For example, phosphorylation of

H2AX by p38α has been shown to be required for

serum starvation-induced apoptosis (Lu et al., 2008).

In addition to the above referred effects of p38α

through direct regulation of pro- and/or antiapoptotic

proteins, p38α is also a negative regulator of PI3K/Akt

and ERKs survival pathways. Thus, in p38α-deficient

derived cardiomyocytes or MEFs cell lines ERKs and

Akt activities are increased, which contributes to the

enhanced survival of these cells (Porras et al., 2004;

Zuluaga et al., 2007a). In particular, in cardiomyocytes

p38α can negatively modulate Akt activity,

independently of PI3K, favoring the interaction

between caveolin-1 and PP2A and the activation of

PP2A through a mechanism dependent on cell

attachment (Zuluaga et al., 2007a).

There are additional mechanisms that can be involved

in the p38α-induced survival, such as the activation of

the ATF6α-Rheb-mTOR survival pathway (Schewe

and Aguirre-Ghiso, 2008). Other mechanisms include

the p38α mediated induction of antioxidant enzymes

expression in response to oxidative stress (unpublished

data from our group). In addition, the increase in BNIP-

3 levels and the low Bim/Bcl-xL ratio induced by p38α

would contribute to this survival effect upon H2O2

treatment (Gutiérrez-Uzquiza et al., 2010).

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Figure 2. Mechanisms involved in p38α-mediated apoptosis. p38Î± induces the expression of pro-apoptotic proteins such as the

death receptor, Fas and its ligand, FasL, as well as Bax, Bim or Noxa from the Bcl-2 family. This expression is induced by the phosphorylation of transcription factors, such as p53 and/or coactivators such as p18Hamlet. This would lead to the activation of the Fas

death receptor pathway and the mitochondrial pathway. p38α can also activate some of these pro-apoptotic proteins, such as Bim, through phosphorylation or through alternative mechanisms as it happens for Bax. Alternatively, p38α can inhibit c-FLIP phosphorylation

impairing its function as an inhibitor of caspase 8 activation. p38α can also inactivate the anti-apoptotic protein Bcl-2 through phosphorylation. In addition, p38α negatively regulates ERKs and Akt survival pathways, being PP2A the mediator of Akt inactivation.

5- Disregulation of p38α function in apoptosis: involvement in human diseases Different evidences from the literature indicate that the

pro-apoptotic function of p38α is deregulated in some

human diseases such as cancer, although recent

findings have also linked p38 signaling with

neurodegenerative diseases such as Alzheimer's disease

(AD), Parkinson disease (PD) and amyolotrophic

lateral sclerosis (ALS) (reviewed by Kim and Choi,

2010).

5.1- Role of p38α in cancer: implication in cancer

development and therapy

5.1.1- p38α as a tumor suppressor and/or mediator

Several data indicate that p38α can act as a tumour

suppressor (Review by Wagner and Nebreda, 2009). In

particular, p38α negatively regulates malignant

transformation induced by Ras through different

mechanisms including apoptosis induction. For

example, in MEFs expressing H-RasV12, sustained

activation of p38α MAPK inhibits transformation

through a mechanism involving ROS-induced

apoptosis (Dolado et al., 2007). H-RasV12-induced

ROS is only able to trigger apoptosis in MEFs

expressing p38α, but not in those deficient in this

protein. As a consequence, p38α-deficient MEFs

transformed by Ras accumulate high levels of ROS

which contributes to tumour progression. This type of

response is produced in the transformation induced by

different oncogenes able to generate ROS. In fact,

many human cancer cell lines have developed

mechanisms to uncouple ROS generation to p38α

activation and therefore induction of apoptosis.

However, as referred above, p38α can also induce a

survival state known as tumor dormancy in some

cancer cells such as those from squamous carcinoma,

which maintains cells in a quiescent state related to

drug resistance through mechanisms including

activation of the p38α-ATF6α-Rheb-mTOR survival

pathway (reviewed by Aguirre-Ghiso, 2007; Schewe

and Aguirre-Ghiso, 2008).

5.1.2- Role of p38 in chronic myeloid leukemia and

other hematopoietic malignancies

p38 MAPK, mainly the p38α isoform, is a key player in

the maintenance of hematopoiesis homeostasis, as it

balances both proliferative and growth inhibitory

signals triggered by the growth factors and cytokines

that regulate normal hematopoiesis (Feng et al., 2009;

Uddin et al., 2004). Alterations in this p38 MAPK-

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controlled balance may result in either overproduction

or depletion of myelosuppressive cytokines leading to

the development of certain bone marrow failure

syndromes. For example, p38α is responsible for the

enhanced stem cell apoptosis characteristic of low

grade myeolodysplastic syndromes (MDSs) (Navas et

al., 2006; Zhou et al., 2007). On the other hand,

imbalance toward the proliferative side may conduct to

the development of myeloproliferative syndromes

(MPSs), such as leukemia, lymphomas and myelomas.

The role played by different members of the p38 kinase

family in the regulation of acute leukemia blasts growth

(myeloid and lymphoid), is not well defined, although

they seem to participate in the generarion of resistance

to chemotherapeutic agents (Feng et al., 2009;

Platanias, 2003) In contrast, the participation of the p38

MAPK pathways in the pathogenesis and

pathophysiology of chronic leukemias have been

extensively studied, although its role is contradictory.

On one side, p38 is activated by the Rho-GEF domain

of Bcr, contributing to transformation through the

regulation of NF-κB activation (Korus et al., 2002). On

the other side, p38 MAPK is selectively activated by

IFNα and mediates the growth suppressive effects of

IFNα in CML cells (Mayer et al., 2001) as well as in

normal hematopoiesis (Verma et al., 2002a).

p38 MAPK also seems to play a role in chronic

lymphocytic leukemia(CLL). p38 and its effector MK-

2 (MAP kinase-activated protein kinase-2) are activated

by rituximab in cultures of CLL cells and contributes to

the generation of the antileukemic effects of rituximab

in CLL (Pedersen et al., 2002). Finally, there are

evidences supporting the participation of p38 in cell

proliferation and adhesion of lymphomas and multiple

myelomas in response to growth factors (Feng et al.,

2009; Platanias, 2003).

Another important function of p38α MAPK in

hematopoiesis is to promote cell differentiation.

Curiously, while GTP-induced erythroid differentiation

of K562 cells is mediated through p38-dependent

caspase activation (Moosavi et al., 2007), caspases do

not participate in STI-571-induced erythroid

differentiation in CML cells, where STI-571-mediated

apoptosis and differentiation are independent events

(Jacquel et al., 2007). The differentiation-inducing

therapy may be a good therapeutical alternative for

CML patients that develop resistance to STI-571.

5.2- Role of p38α in neurodegenerative diseases

Recent findings support the involvement of p38 MAPK

signaling pathway in neurodegenerative diseases. Thus,

persistent activation of the p38 MAPK signaling has

been suggested to contribute to neuronal apoptosis in

Alzheimer's disease (AD), Parkinson disease(PD) and

Amyotrophic lateral sclerosis (ALS).

Alzheimer's disease is an incurable, neurodegenerative

disease characterized by a progressive deterioration of

the congnitive, memory and learning ability including

lost of motor coordination at advanced stages. AD is

the result of the accumulation of plaques containing

amyloidogenic Aβ proteins and tangles containing the

microtubule-associated protein tau in a

hiperphosphorylated state (Giacobini and Becker,

2007). The ASK1-MKK6-p38 signaling pathway

participates in amyeloid precursor protein (APP) and

tau phosphorylation in response to oxidative stress and

contributes to the expression of the β-secretase gene

(Galvan et al., 2007) and the induction of neuronal

apoptosis triggered by ROS (D'Amico et al., 2000; Puig

et al., 2004; Tamagno et al., 2003).

Parkinson disease is a degenerative disorder of the

central nervous system characterized by muscle

rigidity, tremor and loss of physical movement caused

by a progressive loss of dopaminergic neurons. A

number of specific genetic mutations causing

Parkinson's disease have been discovered, being the

mutations in α-Synuclein (a major constituent of Lewy

bodies) among the best characterized (Gandhi and

Wood, 2005). α-Synuclein activates p38 MAPK in

human microglia promoting a potent inflammatory

stimulation of microglial cells (Klegeris et al., 2008).

The p38 MAPK is also activated in PD cellular and

animal models and plays a role in dopaminergic neural

apoptosis through the phosphorylation of p53 and

expression of the pro-apoptotic protein Bax

(Karunakaran et al., 2008; Mathiasen et al., 2004; Silva

et al., 2005).

Amyotrophic lateral sclerosis is a progressive, lethal,

degenerative disorder of motor neurons in the brain and

spinal cord, leading to paralysis of voluntary muscles.

Aberrant chemistry and oxidative stress have been

described to be involved in ALS development, being

SOD1 (superoxide dismutase 1) the most frequently

mutated gene in the inherited cases of ALS. Numerous

evidences point to a role of p38 MAPK in the

development and progression of ALS induced by

mutations in SOD1 gene (Bendotti et al., 2004;

Bendotti et al., 2005; Dewil et al., 2007; Holasek et al.,

2005; Tortarolo et al., 2003). Mutant SOD1 provokes

aberrant oxyradical reactions that increase the

activation of p38 MAPK in motor neurons and glial

cells. This increase in active p38 MAPK may

phosphorylate cytoskeletal proteins and activate

cytokines and nitric oxide, thus contributing to

neurodegeneration through different mechanisms

including apoptosis (Bendotti et al., 2005; Tortarolo et

al., 2003).

5.3- p38α in cancer therapy

p38α can act as a tumor suppressor in the initial phases

of malignant transformation, which involves apoptosis

induction.

Similarly, p38α is also a mediator of apoptosis upon

treatment with many chemotherapeutical drugs such as

cisplatin, arsenic trioxide or others. Therefore, p38α

can be considered as a target in the treatment of cancer,

although sometimes is also responsible for the

resistance to some of these treatments.

It is well established that p38 MAPK mediates the

responses to several DNA-damaging agents, such as

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UV radiation, cisplatin (CDDP) and Ara-C through

activation of p53-dependent and independent responses

(Bulavin et al., 1999; Huang et al., 1997; Sánchez-

Prieto et al., 2000). Cisplatin is an anti-cancer drug that

induces apoptosis in a number of cancer cell lines

through mechanisms that in many cases are p38

dependent. In the colon carcinoma cell line HCT116,

the activation of p38α was shown to be necessary for

CDDP-induced apoptosis, upon activation by p53-

mediated ROS production (Bragado et al., 2007). Once

p38α MAPK is activated, it contributes to further

activation of p53, which leads to a positive feedback

loop. So, a p53/ROS/p38α MAPK cascade is essential

for cisplatin-induced cell death and the subsequent

p38α/p53 positive feedback loop strongly enhances the

initial p53 activation in HCT116 cells. In other cancer

cell lines, c-Abl is required for CDDP-induced p38

activation and for its antitumoral effect. Thus, CDDP-

induced cell death appears to be dependent on c-Abl

expression, rather than on its tyrosine kinase activity,

and on stabilization of MKK6 protein levels (Galán-

Moya et al., 2008). This pro-apoptotic effect of CDDP

is not selective for tumoral cells. Hence, in NIH3T3

cells, which are non-transformed cells, an involvement

of p38(α/β) MAPK was described as mediator of

CDDP-induced apoptosis through a mechanism

dependent on the phosphorylation of p53 in Ser33 and

the subsequent p53 transcriptional activation (Sánchez-

Prieto et al., 2000).

Similarly to CDDP, other anticancer drugs also lead to

the generation of ROS as an important mechanism to

induce apoptosis in cancer cells, being p38α a relevant

mediator. Thus, diamine, a thiol-oxidizing compound,

induces apoptosis in a gastric human

adenocarcinomacell line (AGS) through a mechanism

dependent on Trx1/p38α/p53 pathway, while CaCo2

cells are resistant to this compound due to the absence

of a functional p53 (Piccirillo et al., 2009).

As previously mentioned, activation of p38 MAPK

plays a key role in some hematopoietic malignancies by

different mechanisms including increasing resistance to

chemotherapeutic agents (Feng et al., 2009). Ara C is a

classic anti-neoplasic agent widely used in the

treatment of CML blast crisis, which provides a typical

example of genotoxic stress mediated through c-Abl-

p38 MAPK pathway (Huang et al., 1997; Pandey et al.,

1996; Stadheim et al., 2000). Although p38 MAPK is

activated by c-Abl in response to CDDP in some cancer

cell lines (Galán-Moya et al., 2008) and by

overexpression of Bcr-Abl, Ara-C (1-β-D-

arabinofuranosylcytosine) is unable to activate p38

MAPK in cells with constitutive expression of Bcr-Abl

such as the CML derived cell line K562 (Sánchez-

Arévalo Lobo et al., 2005). In those cells, Bcr-Abl

induces a sustained activation of the p38 MAPK

pathway (not related to increased apoptosis), rendering

them insensitive to further activation in response to

Ara-C. Therefore, Bcr-Abl mediated p38 MAPK

activation is a key mechanism to explain resistance to

Ara-C. In fact, lack of p38 MAPK activation is a

general mechanism for Ara-C resistance, as it had been

previously proposed (Stadheim et al., 2000). This could

provide a clue for new therapeutic approaches based on

the combined use of specific Abl and p38 MAPK

inhibitors.

In contrast, p38 MAPK signaling cascade mediates the

antiproliferative effects of STI-571 (or imatinib, the

current drug used in CML treatment) and cisplatin on

Bcr-Abl expressing cells (Galán-Moya et al., 2008;

Parmar et al., 2004). Moreover, we have recently found

a functional relationship between C3G and p38 in

CML, so that the pro-apoptotic activity of p38α

MAPK, as a mediator of STI-571 effects, is

downregulated by the C3G-Rap1 pathway and the

antitumoral effects of STI-571 could be improved by

C3G gene silencing as it enhances p38α activation and

pro-apoptotic activity (Maia et al., 2009).

In contrast, p38 appears to be a negative regulator of

the antitumoral effects of all-trans-retinoic acid (RA),

which induces differentiation and growth arrest in

several malignant cell types, including acute

promyelocytic leukemia (APL) and breast carcinoma.

The Rac-p38 MAPK-MK2 pathway is activated by RA

and negatively regulates RA-effects in NB-4 APL and

MCF-7 breast cancer cell lines, as p38α/β inhibitor

SB203580 enhances RA-dependent cell differentiation

and apoptosis. Therefore, pharmacological inhibition of

p38α/β may prevent resistance to RA that is developed

in nearly all cases (Alsayed et al., 2001).

Arsenic trioxide (AT), which also induces

differentiation and apoptosis of leukemia cells in vitro

and in vivo, activates the Rac-p38 MAPK-MK2

pathway, which can act as a negative regulator of AT

functions, suggesting a possible role of p38 MAPK

pathways in AT-induced resistance (Verma et al.,

2002b). However, AT can sensitize human

promonocytic cells U937 to TNF-α-induced apoptosis,

which is dependent on p38α/β activation (Amran et al.,

2007). AT increases the levels of TNF-α receptor

leading to an enhancement of TNF-α-induced

apoptosis.

The role of p38 MAPK in resistance to

chemotherapeutic agents is also observed in an

experimental model of head and neck cancer, where

lower activation or lack of activation of p38 MAPK

correlates with a more resistant phenotype (Losa et al.,

2003).

Therefore, based on all these data described here and

other published data, p38α would play a dual role in

cancer treatment. Depending on the type of tumour and

the chemotherapeutic agent, p38α can either induce

apoptosis or survival, leading to the disappearance of

the tumour or making it resistant to chemotherapy.

Concluding remarks p38α MAPK (encoded by MAPK14 gene) is broadly

expressed and is also the most abundant p38 isoform

present in most cell types. It can be activated by several



http://atlasgeneticsoncology.org/Genes/ABL.html



extracellular signals, including stress stimuli, leading to

the regulation of different cellular processes, which

includes cell death. Although in many cases p38α is a

mediator of apoptosis, recent data indicate that it can

also have a pro-survival role. Therefore, p38α would

play a dual role in apoptosis, which is dependent on the

cell context and/or stimulus.

p38α regulates apoptosis through transcriptional and

posttranscriptional mechanisms, being of particular

relevance the modulation of proteins from the Bcl-2

family and proteins involved in the death receptor

pathway such as Fas and FasL.

p38α-mediated apoptosis or survival is deregulated in a

number of pathologies. For example, some

neurodegenerative diseases are linked to an enhanced

apoptosis mediated by p38α. p38α can also act as a

tumour suppressor in the initial stages of malignant

transformation through activation of apoptosis. In

addition, the antitumoral effects of a number of

chemotherapeutical drugs are based on the activation of

apoptosis through p38α. However, it should be notice

that in some tumours p38α MAPK is involved in

chemotherapeutical resistance.

We apologize to the authors whose original work is not

included in the references due to extension constrains.

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Porras A, Guerrero C. Role of p38α in apoptosis: implication in cancer development and therapy. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(3):316-326.



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volume 15 - number 3 march...

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