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The PDF version of the Atlas of Genetics and Cytogenetics in Oncology and Haematology is a reissue of the original articles published in collaboration with the Institute for Scientific and Technical Information (INstitut de l’Information Scientifique et Technique - INIST) of the French National Center for Scientific Research (CNRS) on its electronic publishing platform I-Revues. Online and PDF versions of the Atlas of Genetics and Cytogenetics in Oncology and Haematology are hosted by INIST-CNRS.

Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Volume 15 - Number 8 August 2011

The PDF version of the Atlas of Genetics and Cytogenetics in Oncology and Haematology is a reissue of the original articles published in collaboration with the Institute for Scientific and Technical Information (INstitut de l’Information Scientifique et Technique - INIST) of the French National Center for Scientific Research (CNRS) on its electronic publishing platform I-Revues. Online and PDF versions of the Atlas of Genetics and Cytogenetics in Oncology and Haematology are hosted by INIST-CNRS.



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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Jean-Loup Huret Genetics, Department of Medical Information, University Hospital F-86021 Poitiers, France tel +33 5 49 44 45 46 or +33 5 49 45 47 67 [email protected] or [email protected]

Staff Mohammad Ahmad, Mélanie Arsaban, Marie-Christine Jacquemot-Perbal, Maureen Labarussias, Vanessa Le Berre, Anne Malo, Catherine Morel-Pair, Laurent Rassinoux, Alain Zasadzinski. Philippe Dessen is the Database Director, and Alain Bernheim the Chairman of the on-line version (Gustave Roussy Institute – Villejuif – France).

The Atlas of Genetics and Cytogenetics in Oncology and Haematology (ISSN 1768-3262) is published 12 times a year by ARMGHM, a non profit organisation, and by the INstitute for Scientific and Technical Information of the French National Center for Scientific Research (INIST-CNRS) since 2008. The Atlas is hosted by INIST-CNRS (http://www.inist.fr)

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© ATLAS - ISSN 1768-3262

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8)







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 8, August 2011

Table of contents

Gene Section

ING4 (inhibitor of growth family, member 4) 620 Angela Greco, Claudia Miranda

LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) 625 Dongsheng Guo, Baofeng Wang

MAPK14 (mitogen-activated protein kinase 14) 628 Almudena Porras, Carmen Guerrero

RAD51L3 (RAD51-like 3 (S. cerevisiae)) 632 Mary K Taylor, Michael K Bendenbaugh, Susan M Brown, Brian D Yard, Douglas L Pittman

SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) 637 Wendy S McDonough, Michael E Berens

USP15 (ubiquitin specific peptidase 15) 645 Monica Faronato, Sylvie Urbé, Judy M Coulson

CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) 652 Joanna Fares, Linda Wolff, Juraj Bies

DLX4 (distal-less homeobox 4) 658 Patricia E Berg, Saurabh Kirolikar

IL17A (interleukin 17A) 662 Norimitsu Inoue, Takashi Akazawa

MYBBP1A (MYB binding protein (P160) 1a) 667 Claudia Perrera, Riccardo Colombo

PLCD1 (phospholipase C, delta 1) 670 Xiaotong Hu

PYY (peptide YY) 674 Maria Braoudaki, Fotini Tzortzatou-Stathopoulou

SIAH2 (seven in absentia homolog 2 (Drosophila)) 677 Jianfei Qi, Ze'ev Ronai

TP53BP2 (tumor protein p53 binding protein, 2) 681 Kathryn Van Hook, Zhiping Wang, Charles Lopez

Leukaemia Section

8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome) 686 Paula Aranaz, José Luis Vizmanos

i(5)(p10) in acute myeloid leukemia 695 Nathalie Douet-Guilbert, Angèle Herry, Audrey Basinko, Marie-Josée Le Bris, Nadia Guéganic, Clément Bovo, Frédéric Morel, Marc De Braekeleer

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




+20 or trisomy 20 (solely) 697 Jean-Loup Huret

Deep Insight Section

TMPRSS2:ETS gene fusions in prostate cancer 699 Julia L Williams, Maisa Yoshimoto, Alexander H Boag, Jeremy A Squire, Paul C Park

Gene Section Review

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 620



ING4 (inhibitor of growth family, member 4) Angela Greco, Claudia Miranda

Dept Experimental Oncology, Molecular Mechanisms Unit, Istituto Nazionale Tumori IRCCS Foundation - via Venezian 1 - 20133 Milan Italy (AG, CM)

Published in Atlas Database: December 2010

Online updated version : http://AtlasGeneticsOncology.org/Genes/ING4ID40978ch12p13.html DOI : 10.4267/2042/45999

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: MGC12557; my036; p29ING4

HGNC (Hugo): ING4

Location: 12p13.31

DNA/RNA Description ING4 belongs to family of highly homologous five members containing PHD domain and has been identified through a computational sequence

homology search for expressed tag clones with a PHD finger motif (Shiseki et al., 2003). ING4 gene is located on chromosome 12p13.31 and consists of eight exons encoding a 29-kDa protein expressed in multiple human tissues.

Transcription Multiple alternatively spliced transcript variants have been observed using different splice sites in the coding region; transcript variants span from 1461 bp to 1313 bp. Wobble splicing events have been described at exon 4 and 5 boundary.

Figure adapted from Atlas of Genetics and Cytogenetics in Oncology and Haematology.

ING4 (inhibitor of growth family, member 4) Greco A, Miranda C


Different splicing variants have been identified (among them -v1, -v2, -v3 and -v4/∆4AA) involving 12 bp (379-390) and resulting in in frame deletions of one to four aminoacids in NLS (Tsai and Lin, 2006). Several splicing variants have been described lacking exon 2, 3 and 6 (entirely or in part), and named ING4-∆Ex2, -∆Ex3, -∆Ex6A and -∆Ex6B, respectively (Raho et al., 2007). Splicing variants have been detected in all tissues analysed, indicating that are not tissue specific (Tsai and Lin, 2006; Raho et al., 2007). More recently five novel spliced variants of ING4-v1 and -v2 were identified, causing codon frame shift and eventually deletion of NLS or PHD domains. Increased expression of these variants was found in gastric adenocarcinomas compared to normal tissue (Li M et al., 2009).

Protein Note 249 aminoacids, 29 kDa protein.

Description ING4 protein contains several conserved regions: i) a leucine zipper-like (LZL) domain, probably involved in protein interactions, located at the N-terminus; ii) a functional bipartite nuclear localization signal (NLS1); iii) a C-terminal plant homeo-domain (PHD), a Cys4-His-Cys3 zinc finger motif spanning 50-80 residues, found in many nuclear proteins, such as transcription factors and proteins regulating chromatin structure; iv) a non functional NLS located at the C-terminal end.

Expression Ubiquitous.

Localisation p29ING4 is a nuclear protein. It possesses a bipartite nuclear localization signal. ING4 splicing variants have been described involving the NLS1 domain; most/all of them retains nuclear localization. Furthermore ING4-v1 is translocated to the nucleolus and such subcellular localization is modulated by two wobble-splicing events at the exon 4-5 boundary, causing displacement from the nucleolus to the nucleus.

Function ING4 was also isolated through a screening for genes able to suppress loss of contact inhibition, thus suggesting its tumor suppressor role. ING4 is a nuclear protein participating to a variety of cellular functions, such as apoptosis, cell-cycle

regulation, chromatin remodeling, and regulation of gene expression. Several ING4 partners have been described. Similarly to the other ING members, ING4 was described to interact with p53 and to modulate p53 transcriptional activity (Shiseki et al., 2003). The interaction of ING4 with p53 is mediated by the bipartite ING4 nuclear localization signal (NLS) (Zhang et al., 2005) and drives an increase of p53 acetylation at lysine 382 (Shiseki et al., 2003). ING4 is a critical regulator of chromatin acetylation required for gene expression. In particular, ING4 associates with the HAT complex HBO1 and it is required for the majority of histone 4 acetylation and for normal progression through S phase (Doyon et al., 2006; Shi et al., 2006). Recently a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions has been shown (Hung et al., 2009). ING4 can also function as repressor of factors mediating angiogenesis. It was demonstrated that ING4 plays an inhibitory role on NF-kappaB activity by interaction with p65NF-kappaB and that the lack of inhibition of the NF-kappaB pathway by ING4 results in increased angiogenesis in glioblastomas (Garkavtsev et al., 2004). More recently, it has been described that physiologic levels of ING4 govern innate immunity in mice by regulating the levels of IkappaB and NF-kappaB proteins and the activation of select cytokine promoters (Coles et al., 2010). ING4 was also described to repress the ability of hypoxia inducible factor (HIF)-1 to activate transcription of its downstream target genes by interacting with the HPH-2 prolyl hydroxylase. Under hypoxic conditions, ING4 may act as an adapter protein recruiting transcriptional repressors to mediate HIF activity (Ozer et al., 2005). Involvement of ING4 in regulation of apoptosis has been demonstrated in several cellular systems. Its overexpression can induce apoptosis through the downregulation of Bcl-2 and the upregulation of p21 and Bax expression (Shiseki et al., 2003; Yu et al., 2007; Li X et al., 2009b; Cai et al., 2009).

Homology ING4 protein shares homology with other ING family members with respect to the following regions: i) a leucine zipper-like (LZL) domain, probably involved in interaction with proteins, located at the N-terminus of all the ING proteins except for ING1; ii) a nuclear localization signal (NLS); iii) a C-terminal plant homeo-domain (PHD) involved in chromatin.

LZL: leucine zipper-like; NLS1: nuclear localization signal 1; PHD: plant homology domain; NLS2: nuclear localization signal 2.



Mutations Note The following ING4 point mutations have been found in lung adenocarcinoma and small cell lung carcinoma (H23 and H28, respectively) human cancer cell lines. N214D: it alters ING4 capability of inhibition of proliferation, anchorage independent cell migration reducing protein stability by proteasome mediated degradation. Y121N: it does not alter ING4 functions (Moreno et al., 2010).

Implicated in Breast cancer Cytogenetics Analysis of CGH data revealed that 10-20% of primary breast tumors present deletions in 12p13. The deletions appear to affect only one copy of the gene; no genomic mutations were found in the remaining allele of ING4 (Kim et al., 2004).

Head and neck squamous cell carcinoma (HNSCC) Cytogenetics LOH of 12p13.

Oncogenesis Loss of heterozygosity at 12p12-13 region was found in 66% (33/50) of head and neck squamous cell carcinomas by using six highly polymorphic microsatellite markers. No mutations of the ING4 gene were found. Quantitative real-time RT-PCR analysis demonstrated decreased expression of ING4 mRNA in 76% of primary tumors compared to matched normal samples (Gunduz et al., 2005).

Glioma Oncogenesis Expression of ING4 is significantly reduced in gliomas as compared with normal human brain tissue, and the extent of reduction correlates with the progression from lower to higher grades of tumours. ING4 regulates brain tumour angiogenesis through transcriptional repression of NF-kB-responsive genes (Garkavtsev et al., 2004).

Astrocytoma Prognosis A potential of role of ING4 as a biomarker for the prediction of the grade of astrocytic neoplasms has been suggested (Klironomos et al., 2010).

Oncogenesis Significantly reduced levels of ING4 were observed in human astrocytomas compared to normal brain tissue, suggesting that down-regulation of this protein might be involved in the pathogenesis of human astrocytic

tumors. Decreased ING4 expression correlated significantly with tumor progression, with lower expression levels of ING4 observed in cases of high-grade neoplasms. A statistical significant negative correlation between expression of ING4 and expression of nuclear p65 was noticed (Klironomos et al., 2010).

Hepatocellular carcinoma (HCC) Prognosis Survival and metastasis analysis indicated that HCC patients with lower ING4 expression had poorer overall and disease-free survival than those with high expression. Multivariable Cox regression analysis revealed that the ING4 expression level was an independent factor for prognosis (Fang et al., 2009).

Oncogenesis The ING4 mRNA and protein levels were significantly lower in HCC than paracarcinomatous liver tissue. ING4 expression level correlates with prognosis and metastatic potential, suggesting that ING4 as a candidate prognostic marker of HCC (Fang et al., 2009).

Multiple myeloma (MM) Prognosis MM patients with high IL-8 production and microvascular density (MVD) have significantly lower ING4 levels compared with those with low IL-8 and MVD.

Oncogenesis ING4 suppression in MM cells up-regulated IL-8 and OPN under hypoxic conditions, increasing the hypoxia inducible factor-1alpha (HIF-1alpha) activity and its target gene NIP-3 expression. ING4 suppression in MM cells significantly increased vessel formation in vitro, blunted by blocking IL-8 or OPN (Colla et al., 2007).

Lung cancer Oncogenesis Reduced ING4 nuclear and cytoplasmic expression were both revealed in lung cancer and associated with tumour grade. ING4 expression in the cytoplasm was found higher than in the nucleus in a high percentage of tumors. Nuclear ING4 inhibition correlated with the tumour stage and lymph node metastasis, thus suggesting that ING4 is involved in the initiation and progression of lung cancers (Wang et al., 2010).

Gastric cancer Oncogenesis ING4 RNA and protein were drastically reduced in stomach adenocarcinoma cell lines and tissues, significantly less in female than male patients. Novel spliced forms of ING4-v1 and -v2 were identified in both normal and tumor tissue; increased expression of the novel spliced variants was observed in tumors; however no correlation with clinical parameters was observed (Li M et al., 2009).



References Shiseki M, Nagashima M, Pedeux RM, Kitahama-Shiseki M, Miura K, Okamura S, Onogi H, Higashimoto Y, Appella E, Yokota J, Harris CC. p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity. Cancer Res. 2003 May 15;63(10):2373-8

Garkavtsev I, Kozin SV, Chernova O, Xu L, Winkler F, Brown E, Barnett GH, Jain RK. The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis. Nature. 2004 Mar 18;428(6980):328-32

Kim S, Chin K, Gray JW, Bishop JM. A screen for genes that suppress loss of contact inhibition: identification of ING4 as a candidate tumor suppressor gene in human cancer. Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16251-6

Gunduz M, Nagatsuka H, Demircan K, Gunduz E, Cengiz B, Ouchida M, Tsujigiwa H, Yamachika E, Fukushima K, Beder L, Hirohata S, Ninomiya Y, Nishizaki K, Shimizu K, Nagai N. Frequent deletion and down-regulation of ING4, a candidate tumor suppressor gene at 12p13, in head and neck squamous cell carcinomas. Gene. 2005 Aug 15;356:109-17

Ozer A, Bruick RK. Regulation of HIF by prolyl hydroxylases: recruitment of the candidate tumor suppressor protein ING4. Cell Cycle. 2005 Sep;4(9):1153-6

Ozer A, Wu LC, Bruick RK. The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF). Proc Natl Acad Sci U S A. 2005 May 24;102(21):7481-6

Zhang X, Wang KS, Wang ZQ, Xu LS, Wang QW, Chen F, Wei DZ, Han ZG. Nuclear localization signal of ING4 plays a key role in its binding to p53. Biochem Biophys Res Commun. 2005 Jun 17;331(4):1032-8

Doyon Y, Cayrou C, Ullah M, Landry AJ, Côté V, Selleck W, Lane WS, Tan S, Yang XJ, Côté J. ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation. Mol Cell. 2006 Jan 6;21(1):51-64

Shi X, Hong T, Walter KL, Ewalt M, Michishita E, Hung T, Carney D, Peña P, Lan F, Kaadige MR, Lacoste N, Cayrou C, Davrazou F, Saha A, Cairns BR, Ayer DE, Kutateladze TG, Shi Y, Côté J, Chua KF, Gozani O. ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature. 2006 Jul 6;442(7098):96-9

Tsai KW, Lin WC. Quantitative analysis of wobble splicing indicates that it is not tissue specific. Genomics. 2006 Dec;88(6):855-64

Unoki M, Shen JC, Zheng ZM, Harris CC. Novel splice variants of ING4 and their possible roles in the regulation of cell growth and motility. J Biol Chem. 2006 Nov 10;281(45):34677-86

Colla S, Tagliaferri S, Morandi F, Lunghi P, Donofrio G, Martorana D, Mancini C, Lazzaretti M, Mazzera L, Ravanetti L, Bonomini S, Ferrari L, Miranda C, Ladetto M, Neri TM, Neri A, Greco A, Mangoni M, Bonati A, Rizzoli V, Giuliani N. The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) regulates the production of proangiogenic molecules by myeloma cells and suppresses hypoxia-inducible factor-1 alpha (HIF-1alpha) activity: involvement in myeloma-induced angiogenesis. Blood. 2007 Dec 15;110(13):4464-75

Raho G, Miranda C, Tamborini E, Pierotti MA, Greco A. Detection of novel mRNA splice variants of human ING4 tumor suppressor gene. Oncogene. 2007 Aug 9;26(36):5247-57

Yu X, Zhang HF, Wang JZ, Xie YF, Yang JC, Miao JC. [Ad-ING4 inhibits K562 cell growth]. Zhonghua Xue Ye Xue Za Zhi. 2007 Jun;28(6):396-400

Li X, Cai L, Liang M, Wang Y, Yang J, Zhao Y. ING4 induces cell growth inhibition in human lung adenocarcinoma A549 cells by means of Wnt-1/beta-catenin signaling pathway. Anat Rec (Hoboken). 2008 May;291(5):593-600

Nozell S, Laver T, Moseley D, Nowoslawski L, De Vos M, Atkinson GP, Harrison K, Nabors LB, Benveniste EN. The ING4 tumor suppressor attenuates NF-kappaB activity at the promoters of target genes. Mol Cell Biol. 2008 Nov;28(21):6632-45

Palacios A, Muñoz IG, Pantoja-Uceda D, Marcaida MJ, Torres D, Martín-García JM, Luque I, Montoya G, Blanco FJ. Molecular basis of histone H3K4me3 recognition by ING4. J Biol Chem. 2008 Jun 6;283(23):15956-64

Tsai KW, Tseng HC, Lin WC. Two wobble-splicing events affect ING4 protein subnuclear localization and degradation. Exp Cell Res. 2008 Oct 15;314(17):3130-41

Cai L, Li X, Zheng S, Wang Y, Wang Y, Li H, Yang J, Sun J. Inhibitor of growth 4 is involved in melanomagenesis and induces growth suppression and apoptosis in melanoma cell line M14. Melanoma Res. 2009 Feb;19(1):1-7

Fang F, Luo LB, Tao YM, Wu F, Yang LY. Decreased expression of inhibitor of growth 4 correlated with poor prognosis of hepatocellular carcinoma. Cancer Epidemiol Biomarkers Prev. 2009 Feb;18(2):409-16

Hung T, Binda O, Champagne KS, Kuo AJ, Johnson K, Chang HY, Simon MD, Kutateladze TG, Gozani O. ING4 mediates crosstalk between histone H3 K4 trimethylation and H3 acetylation to attenuate cellular transformation. Mol Cell. 2009 Jan 30;33(2):248-56

Li M, Jin Y, Sun WJ, Yu Y, Bai J, Tong DD, Qi JP, Du JR, Geng JS, Huang Q, Huang XY, Huang Y, Han FF, Meng XN, Rosales JL, Lee KY, Fu SB. Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma. J Pathol. 2009 Sep;219(1):87-95

Li X, Cai L, Chen H, Zhang Q, Zhang S, Wang Y, Dong Y, Cheng H, Qi J. Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG. Pathobiology. 2009a;76(4):181-92

Li X, Zhang Q, Cai L, Wang Y, Wang Q, Huang X, Fu S, Bai J, Liu J, Zhang G, Qi J. Inhibitor of growth 4 induces apoptosis in human lung adenocarcinoma cell line A549 via Bcl-2 family proteins and mitochondria apoptosis pathway. J Cancer Res Clin Oncol. 2009b Jun;135(6):829-35

Tzouvelekis A, Aidinis V, Harokopos V, Karameris A, Zacharis G, Mikroulis D, Konstantinou F, Steiropoulos P, Sotiriou I, Froudarakis M, Pneumatikos I, Tringidou R, Bouros D. Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis. Respir Res. 2009 Feb 27;10:14

Coles AH, Gannon H, Cerny A, Kurt-Jones E, Jones SN. Inhibitor of growth-4 promotes IkappaB promoter activation to suppress NF-kappaB signaling and innate immunity. Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11423-8

Kim S, Welm AL, Bishop JM. A dominant mutant allele of the ING4 tumor suppressor found in human cancer cells exacerbates MYC-initiated mouse mammary tumorigenesis. Cancer Res. 2010 Jun 15;70(12):5155-62

Klironomos G, Bravou V, Papachristou DJ, Gatzounis G, Varakis J, Parassi E, Repanti M, Papadaki H. Loss of inhibitor of growth (ING-4) is implicated in the pathogenesis and progression of human astrocytomas. Brain Pathol. 2010 Mar;20(2):490-7



Moreno A, Palacios A, Orgaz JL, Jimenez B, Blanco FJ, Palmero I. Functional impact of cancer-associated mutations in the tumor suppressor protein ING4. Carcinogenesis. 2010 Nov;31(11):1932-8

Palacios A, Moreno A, Oliveira BL, Rivera T, Prieto J, García P, Fernández-Fernández MR, Bernadó P, Palmero I, Blanco FJ. The dimeric structure and the bivalent recognition of H3K4me3 by the tumor suppressor ING4 suggests a mechanism for enhanced targeting of the HBO1 complex to chromatin. J Mol Biol. 2010 Mar 5;396(4):1117-27

Wang QS, Li M, Zhang LY, Jin Y, Tong DD, Yu Y, Bai J, Huang Q, Liu FL, Liu A, Lee KY, Fu SB. Down-regulation of ING4 is associated with initiation and progression of lung cancer. Histopathology. 2010 Aug;57(2):271-81

This article should be referenced as such:

Greco A, Miranda C. ING4 (inhibitor of growth family, member 4). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):620-624.

Gene Section Mini Review




LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) Dongsheng Guo, Baofeng Wang

Dept of Neurosurgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, People's Republic of China (DG, BW)


Online updated version : http://AtlasGeneticsOncology.org/Genes/LRIG1ID41198ch3p14.html DOI: 10.4267/2042/46009


Identity Other names: DKFZp586O1624; LIG-1; LIG1

HGNC (Hugo): LRIG1

Location: 3p14.1

DNA/RNA Description Genomic DNA encoding LRIG1 spans a region of 122.89 kilobases on chromosome 3, at 3p14. LRIG1 gene is encoded on the reverse strand.

Transcription The pre-mRNA comprises 19 exons. Coding sequence: 4812 bp.

Protein Description LRIG1 is a transmembrane cell-surface protein consisting of 1093 amino acids. LRIG1 contains extracellular part containing 15 leucine-rich repeats (LRR) and three C2-type immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail. LRIG1 can be cut into soluble LRIG1 ectodomain

by proteolytic processing, which also is a functional molecule. Expression LRIG1 is expressed ubiquitously in various epithelial cells, endothelial cells, heart, smooth and striated muscles, and in large neurons.

Localisation Differential subcellular distribution in a cell type-specific manner.

Function LRIG1 acts as a suppressor of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) family, MET (hepatocyte growth factor receptor), and RET. The interaction of the extracellular LRR domain and immunoglobulin-like domains of LRIG1 with the extracellular parts of the human EGFR results in recruitment of c-Cbl to the cytoplasmic domains, and induction of EGFR degradation. LRIG1 is involved in signal transduction, cell proliferation, cell apopotosis, cell cycle, cell migration, and cell invasion. LRIG1 as a putative tumor suppressor gene often be down-regulated in various human tumors. Soluble ectodomain of LRIG1 protein can modulate EGFR signaling and its growth-promoting activity in a paracrine fashion.

LRIG1 gene. Exons are represented by red boxes (in scale). Exons 1 to 19 are from the 5' to 3' direction.

LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) Guo D, Wang B


LRIG1 protein. SP: signal peptide; NF: N-terminal cysteine-rich flanking domain; LRR: leucine-rich repeat; CF: C-terminal cysteine-rich flanking domain; Ig C2: C2-type immunoglobulin-like domains; TM: transmembrane domain; Cyto: cytoplasmic domain.

Implicated in Ependymoma Note Higher cytoplasmic immunoreactivity of LRIG1 correlates with older patient age and higher LRIG1 nuclear immunoreactivity with lower WHO grade.

Prostate cancer Note High LRIG1 expression is significantly associated with short overall and prostate cancer-specific survival for 256 Swedish patients analysed. In contrast, in the U.S. series, high LRIG1 expression is significantly associated with longer overall survival.

Renal cell carcinoma (RCC) Note LRIG1 expression is generally downregulated in conventional and papillary RCC but not in chromophobic RCC.

Cutaneous squamous cell carcinoma (SCC) Note LRIG-1 expression is highest in well-differentiated lesions of cutaneous SCC. LRIG-1 expression intensity of tumor cells is significantly correlated with histologic differentiation of SCC. The SCC patients have significant survival benefits in the high LRIG-1 expression groups compared with low LRIG-1 expression groups.

Breast tumor Note LRIG1 protein levels are significantly suppressed in the majority of human breast tumors expressing ErbB2.

Cervical squamous cell carcinoma Note LRIG1 appears to be a significant prognosis predictor in early-stage cervical cancer, independent of the other tumor markers.

Astrocytic tumor Note Perinuclear staining of LRIG1 is associated with low WHO grade and better survival of the patients.

Psoriasis Note In psoriasis, LRIG1 is mostly absent from the cell

surfaces in the spinous layers.

References Nilsson J, Vallbo C, Guo D, Golovleva I, Hallberg B, Henriksson R, Hedman H. Cloning, characterization, and expression of human LIG1. Biochem Biophys Res Commun. 2001 Jun 29;284(5):1155-61

Nilsson J, Starefeldt A, Henriksson R, Hedman H. LRIG1 protein in human cells and tissues. Cell Tissue Res. 2003 Apr;312(1):65-71

Thomasson M, Hedman H, Guo D, Ljungberg B, Henriksson R. LRIG1 and epidermal growth factor receptor in renal cell carcinoma: a quantitative RT--PCR and immunohistochemical analysis. Br J Cancer. 2003 Oct 6;89(7):1285-9

Guo D, Holmlund C, Henriksson R, Hedman H. The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in Ascidiacea. Genomics. 2004 Jul;84(1):157-65

Gur G, Rubin C, Katz M, Amit I, Citri A, Nilsson J, Amariglio N, Henriksson R, Rechavi G, Hedman H, Wides R, Yarden Y. LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation. EMBO J. 2004 Aug 18;23(16):3270-81

Laederich MB, Funes-Duran M, Yen L, Ingalla E, Wu X, Carraway KL 3rd, Sweeney C. The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases. J Biol Chem. 2004 Nov 5;279(45):47050-6

Ye F, Guo DS, Niu HQ, Tao SZ, Ou YB, Lu YP, Lei T. [Molecular mechanism of LRIG1 cDNA-induced apoptosis in human glioma cell line H4]. Ai Zheng. 2004 Oct;23(10):1149-54

Tanemura A, Nagasawa T, Inui S, Itami S. LRIG-1 provides a novel prognostic predictor in squamous cell carcinoma of the skin: immunohistochemical analysis for 38 cases. Dermatol Surg. 2005 Apr;31(4):423-30

Guo D, Nilsson J, Haapasalo H, Raheem O, Bergenheim T, Hedman H, Henriksson R. Perinuclear leucine-rich repeats and immunoglobulin-like domain proteins (LRIG1-3) as prognostic indicators in astrocytic tumors. Acta Neuropathol. 2006 Mar;111(3):238-46

Jensen KB, Watt FM. Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence. Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):11958-63

Yang WM, Yan ZJ, Ye ZQ, Guo DS. LRIG1, a candidate tumour-suppressor gene in human bladder cancer cell line BIU87. BJU Int. 2006 Oct;98(4):898-902

Goldoni S, Iozzo RA, Kay P, Campbell S, McQuillan A, Agnew C, Zhu JX, Keene DR, Reed CC, Iozzo RV. A soluble ectodomain of LRIG1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity. Oncogene. 2007 Jan 18;26(3):368-81

Guo D, Han L, Shu K, Chen J, Lei T. Down-regulation of leucine-rich repeats and immunoglobulin-like domain proteins

LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) Guo D, Wang B


(LRIG1-3) in HP75 pituitary adenoma cell line. J Huazhong Univ Sci Technolog Med Sci. 2007 Feb;27(1):91-4

Hedman H, Henriksson R. LRIG inhibitors of growth factor signalling - double-edged swords in human cancer? Eur J Cancer. 2007 Mar;43(4):676-82

Ljuslinder I, Golovleva I, Palmqvist R, Oberg A, Stenling R, Jonsson Y, Hedman H, Henriksson R, Malmer B. LRIG1 expression in colorectal cancer. Acta Oncol. 2007;46(8):1118-22

Shattuck DL, Miller JK, Laederich M, Funes M, Petersen H, Carraway KL 3rd, Sweeney C. LRIG1 is a novel negative regulator of the Met receptor and opposes Met and Her2 synergy. Mol Cell Biol. 2007 Mar;27(5):1934-46

Karlsson T, Mark EB, Henriksson R, Hedman H. Redistribution of LRIG proteins in psoriasis. J Invest Dermatol. 2008 May;128(5):1192-5

Ledda F, Bieraugel O, Fard SS, Vilar M, Paratcha G. Lrig1 is an endogenous inhibitor of Ret receptor tyrosine kinase activation, downstream signaling, and biological responses to GDNF. J Neurosci. 2008 Jan 2;28(1):39-49

Lindström AK, Ekman K, Stendahl U, Tot T, Henriksson R, Hedman H, Hellberg D. LRIG1 and squamous epithelial uterine cervical cancer: correlation to prognosis, other tumor markers, sex steroid hormones, and smoking. Int J Gynecol Cancer. 2008 Mar-Apr;18(2):312-7

Miller JK, Shattuck DL, Ingalla EQ, Yen L, Borowsky AD, Young LJ, Cardiff RD, Carraway KL 3rd, Sweeney C.

Suppression of the negative regulator LRIG1 contributes to ErbB2 overexpression in breast cancer. Cancer Res. 2008 Oct 15;68(20):8286-94

Stutz MA, Shattuck DL, Laederich MB, Carraway KL 3rd, Sweeney C. LRIG1 negatively regulates the oncogenic EGF receptor mutant EGFRvIII. Oncogene. 2008 Sep 25;27(43):5741-52

Yi W, Haapasalo H, Holmlund C, Järvelä S, Raheem O, Bergenheim AT, Hedman H, Henriksson R. Expression of leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins in human ependymoma relates to tumor location, WHO grade, and patient age. Clin Neuropathol. 2009 Jan-Feb;28(1):21-7

Thomasson M, Wang B, Hammarsten P, Dahlman A, Persson JL, Josefsson A, Stattin P, Granfors T, Egevad L, Henriksson R, Bergh A, Hedman H. LRIG1 and the liar paradox in prostate cancer: a study of the expression and clinical significance of LRIG1 in prostate cancer. Int J Cancer. 2011 Jun 15;128(12):2843-52

Yi W, Holmlund C, Nilsson J, Inui S, Lei T, Itami S, Henriksson R, Hedman H. Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1. Exp Cell Res. 2011 Feb 15;317(4):504-12


Guo D, Wang B. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):625-627.





MAPK14 (mitogen-activated protein kinase 14) Almudena Porras, Carmen Guerrero

Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, UCM, Ciudad Universitaria, 28040 Madrid, Spain (AP), Centro de Investigacion del Cancer, IBMCC, Universidad de Salamanca-CSIC, 37007 Salamanca, Spain (CG)


Online updated version : http://AtlasGeneticsOncology.org/Genes/MAPK14ID41292ch6p21.html DOI: 10.4267/2042/46010


Identity Other names: CSBP1; CSBP2; CSPB1; EXIP; Mxi2; PRKM14; PRKM15; RK; SAPK2A; p38; p38ALPHA

HGNC (Hugo): MAPK14

Location: 6p21.31

DNA/RNA Description The gene spans a region of 83.53 kb and the coding part is divided into 41 different exons.

Larger transcripts contain 12 or 13 exons. (GATExplorer).

Transcription 9 types of transcripts have been described, although only 5 are protein coding transcripts. The larger 4319-nucleotide transcript encodes a protein of 360 amino acid residues. The first and last exons are partially untranslated.

Pseudogene None described so far.

Schematic representation of human chromosome 6 indicating the position of MAPK14 locus (p21.31) (red bar).

MAPK14 gene locus. Representation of the MAPK14 gene organization indicating the position of the exons (coding region) and untranslated regions.

MAPK14 (mitogen-activated protein kinase 14) Porras A, Guerrero C


MAPK14 protein domains. Schematic representation of MAPK14 protein indicating the position of its functional domains. 30-54: protein kinase ATP signature, ATP-binding region; 59-162: MAPK signature; 24-308: protein kinase domain.

Protein Description MAPK14 is a Ser/Thr kinase composed of 90 to 360 residues depending on the transcript variant.

Crystal structure of MAPK14 at 2.3 A resolution. From PDB (access number: 1WFC).

Expression p38alpha MAPK is ubiquitously expressed, being the p38 most abundant isoform.

Localisation p38alpha is mainly present in the cytosol, but it can translocate to the nucleus. In addition, it can be localized in the mitochondria or in other subcellular compartments.

Function p38alpha is mainly activated by various environmental stresses and proinflammatory cytokines, but many other extracellular signals, including growth factors, also lead to p38alpha activation. The canonical activation requires its phosphorylation in threonine and tyrosine residues by dual-specificity MAP kinase kinases (MKKs), MKK3, MKK6 and MKK4. Substrates of this kinase include transcription factors, such as ATF1, ATF2, ATF6, p53, MEF2 or C/EBPbeta and protein kinases, such as MAPKAP-K2 and MAPKAP-K3 (also known as MK-2 and MK-3), MSK-1, MNK-1/MNK-2 and other proteins. p38alpha MAPK is essential for embryonic

development and it regulates different cellular functions such as proliferation, differentiation, cell death, adhesion, migration, as well as the response to stress and many metabolic pathways, among others. It does so through regulation of transcription, mRNA stability, chromatin remodelling, protein synthesis, etc. Concerning cell death, although p38alpha plays an important role as a pro-apoptotic signal, it can play a dual role, acting as either a mediator of cell survival or of cell death, depending on the cell type and the stimuli. Related with its function as a negative regulator of proliferation and a mediator of apoptosis, p38alpha acts as a tumor suppressor in the initial stages of a tumorigenic process, while at later stages it can promote metastasis.

Signaling through p38alphaMAPK. Signaling through MAPK14 cascade and its role in the regulation of cellular functions. MAPK14 is involved in signaling pathways triggered by a variety of stimuli such as growth factors, oxidative stress, UV, cytokines and DNA damage. Depending on the stimulus, different receptors and intermediates (adaptors, GTPases or kinases) are activated leading to the activation of the p38alpha MAPK cascade. This cascade is initiated by activation of MAPKKKs, which phosphorylate and activate MAPKKs (MKK3/6/4), which in turn lead to activation of MAPK14 through dual phosphorylation in Tyr and Thr. Once phosphorylated, MAPK14 phosphorylates a number of cytosolic and nuclear substrates, including transcription factors, which lead to the control of many cellular responses.



Mutations Somatic 4 somatic mutations according to Ensembl: COSM21366; COSM20563; COSM35409; COSM12875.

Implicated in Hematopoietic malignancies Disease p38 MAPK, mainly the p38alpha 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. Alterations in this p38 MAPK-controlled balance may result in either overproduction or depletion of myelosuppressive cytokines leading to the development of certain bone marrow failure syndromes. For example, p38alpha is responsible for the enhanced stem cell apoptosis characteristic of low grade myeolodysplastic syndromes (MDSs). On the other hand, imbalance toward the proliferative side may conduct to the development of myeloproliferative syndromes (MPSs), such as leukemia, lymphomas and myelomas. In particular, p38alpha MAPK plays a pro-apoptotic role in chronic myeloid leukemia (CML). In fact, p38alpha MAP kinase pathway mediates the growth inhibitory effects of IFNalpha and STI-571, two drugs used in the CML treatment, which underscores the importance of this pathway in the generation of antileukemic responses.

Alzheimer's disease Disease Alzheimer is an incurable, neurodegenerative disease characterized by a progressive deterioration of the cognitive, memory and learning ability due to the accumulation of plaques containing amyloidogenic Abeta proteins and tangles containing hyperphosphorylated tau protein. 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 beta-secretase gene and the induction of neuronal apoptosis triggered by ROS.

Parkinson disease Disease Parkinson 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. Mutations in alpha-synuclein are one of the main causes of Parkinson. alpha-synuclein activates p38alpha MAPK in human microglia promoting a potent inflammatory stimulation of microglial cells. Additionally, the

p38alpha MAPK plays a role in dopaminergic neural apoptosis through the phosphorylation of p53 and expression of the pro-apoptotic protein Bax.

Amyotrophic lateral sclerosis Disease ALS is a progressive, lethal, degenerative disorder of motor neurons leading to paralysis of voluntary muscles. Numerous evidences point to a role of p38 MAPK in the development and progression of ALS induced by mutations in SOD1 (superoxide dismutase 1) gene. 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.

To be noted Note See also the Deep Insight: "Role of p38alpha in apoptosis: implication in cancer development and therapy".

References Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, Nebreda AR. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell. 1994 Sep 23;78(6):1027-37

Wilson KP, Fitzgibbon MJ, Caron PR, Griffith JP, Chen W, McCaffrey PG, Chambers SP, Su MS. Crystal structure of p38 mitogen-activated protein kinase. J Biol Chem. 1996 Nov 1;271(44):27696-700

Huang Y, Yuan ZM, Ishiko T, Nakada S, Utsugisawa T, Kato T, Kharbanda S, Kufe DW. Pro-apoptotic effect of the c-Abl tyrosine kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine. Oncogene. 1997 Oct 16;15(16):1947-52

Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, Fornace AJ Jr. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J. 1999 Dec 1;18(23):6845-54

Adams RH, Porras A, Alonso G, Jones M, Vintersten K, Panelli S, Valladares A, Perez L, Klein R, Nebreda AR. Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development. Mol Cell. 2000 Jul;6(1):109-16

D'Amico M, Hulit J, Amanatullah DF, Zafonte BT, Albanese C, Bouzahzah B, Fu M, Augenlicht LH, Donehower LA, Takemaru K, Moon RT, Davis R, Lisanti MP, Shtutman M, Zhurinsky J, Ben-Ze'ev A, Troussard AA, Dedhar S, Pestell RG. The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways. J Biol Chem. 2000 Oct 20;275(42):32649-57

Sanchez-Prieto R, Rojas JM, Taya Y, Gutkind JS. A role for the p38 mitogen-acitvated protein kinase pathway in the transcriptional activation of p53 on genotoxic stress by chemotherapeutic agents. Cancer Res. 2000 May 1;60(9):2464-72



Park JI, Choi HS, Jeong JS, Han JY, Kim IH. Involvement of p38 kinase in hydroxyurea-induced differentiation of K562 cells. Cell Growth Differ. 2001 Sep;12(9):481-6

Platanias LC. The p38 mitogen-activated protein kinase pathway and its role in interferon signaling. Pharmacol Ther. 2003 May;98(2):129-42

Tamagno E, Robino G, Obbili A, Bardini P, Aragno M, Parola M, Danni O. H2O2 and 4-hydroxynonenal mediate amyloid beta-induced neuronal apoptosis by activating JNKs and p38MAPK. Exp Neurol. 2003 Apr;180(2):144-55

Tortarolo M, Veglianese P, Calvaresi N, Botturi A, Rossi C, Giorgini A, Migheli A, Bendotti C. Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression. Mol Cell Neurosci. 2003 Jun;23(2):180-92

Mathiasen JR, McKenna BA, Saporito MS, Ghadge GD, Roos RP, Holskin BP, Wu ZL, Trusko SP, Connors TC, Maroney AC, Thomas BA, Thomas JC, Bozyczko-Coyne D. Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells. Brain Res. 2004 Apr 2;1003(1-2):86-97

Porras A, Zuluaga S, Black E, Valladares A, Alvarez AM, Ambrosino C, Benito M, Nebreda AR. P38 alpha mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli. Mol Biol Cell. 2004 Feb;15(2):922-33

Puig B, Gómez-Isla T, Ribé E, Cuadrado M, Torrejón-Escribano B, Dalfó E, Ferrer I. Expression of stress-activated kinases c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), and tau hyperphosphorylation in neurites surrounding betaA plaques in APP Tg2576 mice. Neuropathol Appl Neurobiol. 2004 Oct;30(5):491-502

Bendotti C, Bao Cutrona M, Cheroni C, Grignaschi G, Lo Coco D, Peviani M, Tortarolo M, Veglianese P, Zennaro E. Inter- and intracellular signaling in amyotrophic lateral sclerosis: role of

p38 mitogen-activated protein kinase. Neurodegener Dis. 2005;2(3-4):128-34

Silva RM, Kuan CY, Rakic P, Burke RE. Mixed lineage kinase-c-jun N-terminal kinase signaling pathway: a new therapeutic target in Parkinson's disease. Mov Disord. 2005 Jun;20(6):653-64

Cuenda A, Rousseau S. p38 MAP-kinases pathway regulation, function and role in human diseases. Biochim Biophys Acta. 2007 Aug;1773(8):1358-75

Zhou L, Opalinska J, Verma A. p38 MAP kinase regulates stem cell apoptosis in human hematopoietic failure. Cell Cycle. 2007 Mar 1;6(5):534-7

Zuluaga S, Alvarez-Barrientos A, Gutiérrez-Uzquiza A, Benito M, Nebreda AR, Porras A. Negative regulation of Akt activity by p38alpha MAP kinase in cardiomyocytes involves membrane localization of PP2A through interaction with caveolin-1. Cell Signal. 2007 Jan;19(1):62-74

Karunakaran S, Saeed U, Mishra M, Valli RK, Joshi SD, Meka DP, Seth P, Ravindranath V. Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of substantia nigra leads to nuclear translocation of p53 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. J Neurosci. 2008 Nov 19;28(47):12500-9

Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer. 2009 Aug;9(8):537-49

Cuadrado A, Nebreda AR. Mechanisms and functions of p38 MAPK signalling. Biochem J. 2010 Aug 1;429(3):403-17


Porras A, Guerrero C. MAPK14 (mitogen-activated protein kinase 14). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):628-631.

Gene Section Review




RAD51L3 (RAD51-like 3 (S. cerevisiae)) Mary K Taylor, Michael K Bendenbaugh, Susan M Brown, Brian D Yard, Douglas L Pittman

South Carolina College of Pharmacy, University of South Carolina, Coker Life Sciences Building, 715 Sumter Street, Columbia, SC 29208, USA (MKT, MKB, SMB, BDY, DLP)


Online updated version : http://AtlasGeneticsOncology.org/Genes/RAD51L3ID347ch17q12.html DOI: 10.4267/2042/46011


Identity Other names: HsTRAD; R51H3; RAD51D; Trad

HGNC (Hugo): RAD51D

Location: 17q12

Note Of the five RAD51 paralog proteins, four come together to form the BCDX2 complex, which includes RAD51L1 (RAD51B; chromosome 14), RAD51L2 (RAD51C; chromosome 17), RAD51L3 (RAD51D; chromosome 17), and XRCC2 (chromosome 14). The protein complex is involved in homologous recombination repair of double-stranded breaks that

result during DNA replication or from DNA-damaging agents, e.g., cisplatin (Masson et al., 2001). The RAD51L3 protein directly interacts with RAD51L2 (RAD51C) and XRCC2. It does not directly interact with RAD51L1 (RAD51B) (Schild et al., 2000).

DNA/RNA Note The human gene is composed of 10 exons. The study by Kawabata and Saeki (1999) describes alternative splicing of the human gene using a numbering scheme of 12 alternatively spliced exons. The exon alignment is illustrated below.

Human RAD51D alternative splicing. A. Exons 4 and 8 of the Kawabata and Saeki numbering scheme are considered "alternative exons" and not included in the reference sequence. B. Summary of splice variants and predicted translation products (for further details see the annexed document below).

RAD51L3 (RAD51-like 3 (S. cerevisiae)) Taylor MK, et al.


Further descriptions of mouse alternatively spliced variants are described in Gruver et al., 2009 and Kawabata et al., 2004.

Transcription The HsTRAD transcript is the predominant variant. It is the full-length transcript and is made up of 2418 base pairs. This transcript will be used as the reference for the information that follows. There are multiple splice variants for the RAD51L3 gene that translate into one of seven putative protein isoforms.

Protein Note The Saccharomyces cerevisiae Rad51 protein is homologous to the RecA protein of Escherichia coli. The RecA protein is known to promote repair via ATP-dependent mechanisms and is responsible for pairing and strand transfer between homologous DNA sequences. This is similar to the actions of the RAD51 protein in repair pathways. There are 5 members of the RAD51 family that share similar roles in recombination and DNA repair. RAD51D is one of these RecA-like genes (Pittman et al., 1998; Cartwright et al., 1998). The RAD51D gene is predicted to encode seven different protein isoforms through alternative splicing. Isoform 1 is the predominant protein and is translated from the HsTRAD transcript mentioned previously (Kawabata and Saeki, 1999). The diagram below is based on this predominant form.

Description The RAD51D protein contains regions necessary for interactions with other RAD51 paralogs as well as those that are required for proper function of the protein. RAD51D contains an ATP binding domain with highly conserved Walker A and B motifs (Pittman et al., 1998; Cartwright et al., 1998). Mutations targeting the conserved residues of glycine and lysine within the Walker A motif region resulted in a reduction in RAD51C binding ability and were shown to be required for DNA repair (Gruver et al., 2005). The Walker B motif contains a "GGQRE" sequence between residues 219-223 that is also required for DNA repair (Wiese et al., 2006). Furthermore, RAD51D-XRCC2 complex formation is significantly reduced with mutations targeting a highly conserved aspartate residue within the Walker B motif (Wiese et al., 2006). A carboxyl terminal domain spanning amino acids 77-329 has been identified to be required for RAD51D to interact with RAD51C. In addition, the "linker region" located between

residues 54-77 in the amino terminus is required for proper interactions with XRCC2. Together, these interactions aid in the repair of DNA damage (Miller et al., 2004; Gruver et al., 2009).

Expression According to the study by Kawabata and Saeki (1999), RAD51L3 transcripts are expressed to varying degrees in the colon, prostate, spleen, testis, ovaries, thymus, small intestine and leukocytes.

Localisation Located in the nucleus. Specifically, RAD51L3 localizes to the telomeres during both mitosis and meiosis (Tarsounas et al., 2004). There is evidence that RAD51L3 is found in the cytoplasm as well (Gruver et al., 2005).

Function RAD51D is one of five members of the RAD51 gene family that is known to participate in repair of double stranded DNA breaks via homologous recombination. Without repair, the DNA damage can result in cell death or chromosomal aberrations that can ultimately lead to cancer (Thacker, 2005). Knockout studies with mice have shown a dramatic increase in levels of chromosomal aberrations, most notably, chromatid and chromosome breaks that occur through unrepaired replication forks (Smiraldo et al., 2005; Hinz et al., 2006). Proteomic studies have identified an interaction between RAD51D with the SFPQ protein (Rajesh et al., 2011). Exposure of mouse RAD51D-deficient cells to a strong alkylating agent results in G2/M cell cycle arrest and ultimately apoptosis (Rajesh et al., 2010). RAD51D has recently been shown to play a diverse role in cellular processes through its interaction with proteins involved in cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cellular structure (Rajesh et al., 2009). RAD51L3 is directly associated with telomeres prevents their dysfunction (Tarsounas et al., 2004). In mouse studies, RAD51L3 foci were present at telomeres in both meiosis and mitosis. Knockout studies showed that "RAD51D-deficient" mice exhibited an increase in end-to-end fusion and telomere attrition (Smiraldo et al., 2005). In addition, human studies using RAD51D-deficient cells have shown repeated shortening of the telomeric DNA, leading to chromosomal instability. This suggests a role for "RAD51D" in telomere capping. Failure to provide this function can lead to chromosomal aberrations (Tarsounas and West, 2005).

RAD51L3 protein structure. Isoform 1 (from full-length transcript).



Homology

Canis lupus familiaris [Dog]

Official gene name: RAD51-like 3 (S. cerevisiae) Genomic location: chromosome 9

Reference material: no primary references found

Pan troglodytes [Chimpanzee]



Bos taurus [Cow]


Reference material: Zimin et al., 2009

Gallus gallus [Chicken]



Rattus norvegicus [Rat]


Reference material: Strausberg et al., 2002

Mus musculus [Mouse]


Reference material: Pittman et al., 1998 Cartwright et al., 1998

Arabidopsis thaliana [Thale cress]

Official gene name: RAD51D (ARABIDOPSIS HOMOLOG OF RAD51D) Genomic location: chromosome 1

Reference material: Durrant et al., 2007

Oryza sativa [Rice]

Official gene name: Os09g0104200 Genomic location: chromosome 9 **Hypothetical protein**


Danio rerio [Zebrafish]

Official gene name: zgc:77165 Genomic location: chromosome 5


** Protein alignments and protein sequences are available at the HomoloGene database.

Mutations Note Single nucleotide polymorphisms have been identified in RAD51L3. However, only a small number of the major mutations occur in coding regions. The majority of the other mutations are present in various locations within the introns. Of the mutations affecting the gene, only one has an observed clinical association. It is observed that a mutation of the mRNA position 954

(SNP ID: rs28363284) results in an allele change to GGG (from the wild type GAG). This point mutation affects the 233rd amino acid as a glycine residue is observed in this particular mutation rather than the natural glutamic acid. This particular variation in amino acid sequence has been implicated as a precursor to breast cancer (see "Implicated In" section below). Another mutation observed in the coding region is at mRNA position 188 (SNP ID: rs1871892), resulting in a change in the sequence to TCA (from the wild type



CCA). This particular substitution results in the insertion of proline at the 36th protein position rather than a serine. A third mutation observed is noted to occur at mRNA positions 810 (SNP ID: rs4796033). A mutation at this location results in a sequence of CAG (from the natural CGG). The effect of this substitution is the insertion of a glutamine residue at the 185th amino acid position rather than the arginine observed in the wild type gene. It is noted, that this particular mutation also occurs in 2 additional transcripts of the gene at the mRNA positions 750 and 414 affecting the 165th and 53rd amino acid residues respectively. Other mutations in the coding region include E237K (SNP ID: rs115031549), R252Q (SNP ID: rs28363283), A245T (SNP ID: rs28363282), A210T (SNP ID: rs80116829), E177D (SNP ID: rs55942401), and R24S (SNP ID: rs28363257).

Implicated in Cancer Disease Cancer arises in part due to the accumulation of genetic damage. Furthermore, such damage has a greater tendency to be found in significant levels when genetic repair pathways such as DNA mismatch repair and homologous recombination (HR) are defective. Involved in the pathway of HR are numerous proteins that are known as the RAD51 paralogs (RAD51L1, RAD51L2, RAD51L3, XRCC2 and XRCC3). It is believed that the lack of genetic stability created from the loss of this pathway, HR, is significant in initiation and potentially the progression of cancer. In particular, defects in the HR pathway have been noted to be associated with breast and ovarian cancer (Thacker, 2005); however, it is plausible that such a defect could potentially lead to multiple forms of cancer due to the accumulation of genetic mutations (although it takes significant damage accumulation to lead to tumor formation). A RAD51L3 variant does have an association with increased familial breast cancer risk (Rodríguez-López et al., 2004).

Breast cancer Note Although conflicting data exist, the RAD51D-E233G variant allele has been identified as a potential precursor to breast cancers in women with high familial risk but do not possess a BRCA1/BRCA2 mutation (Rodríguez-López et al., 2004; Dowty et al., 2008).

Disease In an initial study that screened for possible breast cancer alleles, it was determined that the exon 8 mutation led to an increased frequency of breast cancer in a specific group of cases (familial cancer cases) versus the control group (Rodríguez-López et al., 2004). Additionally, individuals expressing the

RAD51D-E233G variant have been shown to have higher proliferative indices and a less favorable clinical immunohistochemical pattern (Rodríguez-López et al., 2004). However, another study found no statistically significant evidence that this variant is associated with breast cancer risk. Yet, this study did find that it was plausible that the variable could lead to a small increase in the risk of breast cancer and that a small, yet insignificant, effect was made by the variant on the risk of breast cancer (approximately 30%) (Dowty et al., 2008).

Prognosis It has been noted that the RAD51D-E223G variant confers increased resistance to DNA damaging agents such as: mitomycin C, cisplatin, ultraviolet light, and methyl methane sulfonate, and taxol. This presents clinical implications as these are commonly utilized therapies. Furthermore, the variant has increased cellular proliferation and telomere maintenance compared to the wild-type and exhibits reduced interaction with the binding partner RAD51C but does not affect binding to XRCC2 (Nadkarni et al., 2009b).

Bloom's syndrome Disease Bloom's syndrome is an autosomal recessive disorder of rare occurrence. Characteristics include short stature, immunodeficiency, fertility defects, and increased risk for the development of various types of cancer. Cells associated with this disorder are noted for their genomic instability. They exhibit an increase in sister chromatid and homologous chromosome exchanges. In normal, healthy cells, BLM, a helicase of the RecQ family, interacts with the RAD51L3 portion of the RAD51L3-XRCC2 heteromeric complex. Upon joining with the complex, BLM disrupts synthetic 4-way junctions that resemble Holliday junctions suggesting an important role for the protein-protein interaction in DNA repair. The mutated form of the gene encoding for this protein, which occurs in Bloom's syndrome, results in the inability for BLM to bind to RAD51L3. Absence of normal BLM function leads to the characteristic elevation in recombination events seen in Bloom's syndrome (Braybrooke et al., 2003).

References Cartwright R, Dunn AM, Simpson PJ, Tambini CE, Thacker J. Isolation of novel human and mouse genes of the recA/RAD51 recombination-repair gene family. Nucleic Acids Res. 1998 Apr 1;26(7):1653-9

Pittman DL, Weinberg LR, Schimenti JC. Identification, characterization, and genetic mapping of Rad51d, a new mouse and human RAD51/RecA-related gene. Genomics. 1998 Apr 1;49(1):103-11

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Gene Section Review




SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) Wendy S McDonough, Michael E Berens

The Translational Genomics Research Institute, 445 N Fifth Street, Phoenix, Arizona 85004, USA (WSM, MEB)


Online updated version : http://AtlasGeneticsOncology.org/Genes/SLC9A3R1ID46023ch17q25.html DOI: 10.4267/2042/46012


Identity Other names: EBP50; NHERF; NHERF1; NPHLOP2

HGNC (Hugo): SLC9A3R1

Location: 17q25.1

DNA/RNA Description The SLC9A3R1 gene is comprised of 6 exons and spans approximately 20.7 kb of genomic DNA.

Transcription The SLC9A3R1 gene encodes a 1978 bp mRNA transcript. Reported regulatory transcription factor binding sites upstream of the SLC9A3R1 promoter region include: NF-kappaB1, HNF-4alpha2, COUP-TF1, NF-kappaB, NRSF form 2, NRSF form 1, FOXD1, PPAR-gamma2, PPAR-gamma1, GATA-1.

SLC9A3R1 Physical Map.

DNA size 20.71 Kb; mRNA size 1978 bp; 6 exons.

SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) McDonough WS, Berens ME


NHERF (human)-358 aminos acids.

Protein Description The SLC9A3R1 protein is composed of 353 amino acids (389 kDa). Post-transcriptional regulation of SLC9A3R1 occurs via Serines S77-p, S162-p, S339-p, S340-p. SLC9A3R1 is also phosphroylated on T95-p. SLC9A3R1 has two PDZ (DHR) domains. SLC9A3R1 can exist as a homodimer or heterodimer with SLC9A3R2.

Expression SLC9A3R1 is expressed in liver, salivary glands, kidney, pancreas, trachea, small intestine, stomach, prostate and brain.

Localisation SLC9A3R1 is a cytoplasmic protein. SLC9A3R1 translocates from the cytoplasm to the apical cell membrane in a PODXL-dependent manner. SLC9A3R1 colocalizes with actin in microvilli-rich apical regions of the syncytiotrophoblast. SLC9A3R1 has been found in microvilli, ruffling membrane and filopodia of HeLa cells. SLC9A3R1 is also been discovered in lipid rafts of T-cells. Subcellular localization is present in cells with apical specialized structure such as micorvili and cilia. SLC9A3R1 also has a membranous expression in cells of non-epithelial origin (astrocytes) and hematopoietic stem cells and has been found in membrane rafts in lymphocytes and at the rear edge of neutrophils.

Function SLC9A3R1 is a scaffold protein that connects plasma membrane proteins with members of the ezrin/moesin/radixin family and thereby helps to link them to the actin cytoskeleton to regulate their surface expression. SLC9A3R1 has been shown to be necessary for recycling of internalized ADRB2. SLC9A3R1 regulates SLC9A3 as well as its subcellular location. SLC9A3R1 is required for cAMP-mediated phosphorylation and inhibition of SLC9A3R1. SLC9A3R1 interacts with MCC. SLC9A3R1 may participate in HTR4 targeting to microvilli. SLC9A3R1 has been shown to play a role in the WNT signaling pathway.

Induction: SLC9A3R1 can be induced by estrogen. SLC9A3R1 has been show to have binary interaction with the following proteins: CFTR, CLCN3, MSN, NF2, RDX.

Mutations Note SLC9A3R1 has been shown to have three natural variants. Natural variant 110L --> V in NPHLOP2; the mutant expressed in cultured renal cells increases the generation of cyclic AMP (cAMP) by parathyroid hormone (PTH) and inhibits phosphate transport. Natural variant 153R --> Q in NPHLOP2; the mutant expressed in cultured renal cells increases the generation of cAMP by PTH and inhibits phosphate transport. Natural variant 225E --> K in NPHLOP2; the mutant expressed in cultured renal cells increases the generation of cAMP by PTH and inhibits phosphate transport.

Implicated in Cancer progression Note There is growing evidence SLC9A3R1 plays an important role in cancer progression. SLC9A3R1 functions as an adaptor protein to control cell transformation. In addition, recent evidence suggests that SLC9A3R1 has a dual role either acting as a tumor suppressor when it is localized as the cell membrane or as an oncogenic protein when it is localized in the cytoplasm (Georgescu et al., 2008).

Glioblastoma Note The invasive nature of glioblastoma multiforme presents a clinical problem rendering tumors incurable by conventional treatment modalities such as surgery, ionizing radiation, and temozolomide. SLC9A3R1 has been implicated to play a role in sustaining glioma cell migration and invasion. SLC9A3R1 has been shown to be over-expressed in invading glioma cells as compared to the tumor core (Kislin et al., 2009).



Breast cancer Note Increased cytoplasmic expression of SLC9A3R1 in breast tumors suggests a key role of its localization and compartmentalization in defining cancerogenesis, progression, and invasion. SLC9A3R1 overexpression has been associated with increasing tumor cytohistological grade, aggressive clinical behavior, unfavorable prognosis, and increased tumor hypoxia. Moreover, SLC9A3R1 co-localizes with the oncogenic receptor HER2/neu in HER2/neu-overexpressing carcinoma and in distant metastases (Mangia et al., 2009). The switch from apical membranous to cytoplasmic expression is compatible with a dual role for NHERF1 as a tumour suppressor or tumour promoter dependent on its subcellular localization (Georgescu et al., 2008).

Cystic fibrosis Note The inherited disease cystic fibrosis is one of the most common chronic lung diseases in children and young adults and may lead to an early death. Cystic fibrosis transmembrane regulator (CFTR) functions as a cAMP-regulated chloride channel, and mutations in CFTR are contributory in cystic fibrosis. CFTR contains a C-terminal SLC9A3R1 consensus sequence affording the two proteins to bind with high affinity. Recent experiments have postulated two roles for SLC9A3R1 in CFTR function. Guggino, Stanton, and coworkers have proposed that NHERF functions as a membrane retention signal for CFTR (Moyer et al., 1999). Raghuram et al. suggest that SLC9A3R1 facilitates the dimerization of CFTR leading to its full expression of chloride channel activity (Raghuram et al., 2001). Lastly, ss2-adrenoceptors have been shown to physically interact with CFTR Na+/H+ Exchanger Regulatory Factor 1 SLC9A3R1 protein. This function of SLC9A3R1 could be a new therapeutic target in CF patients to facilitate the trafficking of mutated CFTR to plasma membrane (Bossard et al., 2011).

Hypophosphatemia and nephrolithiasis Note SLC9A3R1 plays an important role in tumor phosphorous transport. Inactivating missense mutations in SLC9AR1 have been identified in patients with hypercalciuria and neprolithiasis (Karim et al., 2008).

To be noted Note We acknowledge the support of Michael Northrop for scientific illustrations.

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Pan Y, Wang L, Dai JL. Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1). Breast Cancer Res. 2006;8(6):R63

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Takahashi Y, Morales FC, Kreimann EL, Georgescu MM. PTEN tumor suppressor associates with NHERF proteins to attenuate PDGF receptor signaling. EMBO J. 2006 Feb 22;25(4):910-20

Terawaki S, Maesaki R, Hakoshima T. Structural basis for NHERF recognition by ERM proteins. Structure. 2006 Apr;14(4):777-89

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Kwon SH, Pollard H, Guggino WB. Knockdown of NHERF1 enhances degradation of temperature rescued DeltaF508 CFTR from the cell surface of human airway cells. Cell Physiol Biochem. 2007;20(6):763-72

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Gene Section Review




USP15 (ubiquitin specific peptidase 15) Monica Faronato, Sylvie Urbé, Judy M Coulson

Physiology Department, School of Biomedical Sciences, Faculty of Health and Life Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK (MF, SU, JMC)


Online updated version : http://AtlasGeneticsOncology.org/Genes/USP15ID44585ch12q14.html DOI: 10.4267/2042/46013


Identity Other names: KIAA0529; UNPH4; Unph-2

HGNC (Hugo): USP15

Location: 12q14.1

DNA/RNA Note USP15 is a member of the ubiquitin-specific protease (USP) family; these cysteine proteases comprise the largest sub-group of deubiquitinase enzymes (DUBs). USP15 cleaves the isopeptide bonds of polyubiquitin chains, and can cleave linear ubiquitin fusion proteins (Baker et al., 1999).

Description The USP15 gene spans 145 kb of genomic DNA.

Transcription Four transcripts of the human USP15 gene are described by Ensembl and are summarized in the accompanying diagram and table. According to Entrezgene, USP15 encodes a single reference sequence mRNA of 4611bp (NM_006313.1) composed of 21 exons, which corresponds to USP15-203. However, three other USP15 splice variants utilise several alternative-splicing sites between exon 5 and exon 7 of this reference sequence. USP15-201, a 4698 bp mRNA comprised of 22 exons, is expressed at similar levels to the reference sequence (Angelats et al., 2003). Expression of the remaining variants, USP15-204 and the truncated USP15-202, is less well studied.

Schematic illustrating four human USP15 transcripts. The USP15 reference sequence mRNA (USP15-203) and three alternative splice variants are illustrated. The approximate position and size of exons within the USP15 gene, according to Ensembl, is shown for each splice variant.

USP15 (ubiquitin specific peptidase 15) Faronato M, et al.


Summary table of USP15 transcripts.

Name Ensembl Entrez Aceview Size (bp) Exons

USP15-203 ENST00000353364 NM_006313.1 Variant b 4611 21

USP15-201 ENST00000280377 Variant a 4698 22

USP15-204 ENST00000393654 4626 23

USP15-202 ENST00000312635 Variant e 717 7

Schematic illustrating four human USP15 isoforms. The domain structure is shown for the reference sequence protein (USP15-203) and three alternative isoforms according to Ensembl. DUSP, domain present in ubiquitin-specific proteases; UBL, ubiquitin-like fold; UCH, ubiquitin carboxyl-terminal hydrolase. The cysteine motifs that form the zinc-binding site are shown in purple and the amino acids comprising the catalytic triad are shown in red. The approximate location of nuclear export sequences (triangles) and a putative nuclear localisation signal (inverted triangle) are shown above isoform USP15-203. Differences in amino acid sequence between isoforms are shown in light blue. The UCH is absent in isoform USP15-202, but USP15-201, USP15-203 and USP15-204 are predicted to be catalytically active. Summary table of USP15 protein isoforms.

Name Ensembl Entrez Size (aa) MW (kDa)

USP15-203 ENSP00000258123 NP_006304.1 952 109

USP15-201 ENSP00000280377 981 112

USP15-204 ENSP00000377264 957 109

USP15-202 ENSP00000309240 235 40



Protein Description All USP15 isoforms encompass a single N-terminal DUSP (domain in USPs) characterised by a novel tripod-like fold with a conserved hydrophobic surface patch that is predicted to participate in protein-protein interaction (de Jong et al., 2006). The ubiquitin carboxyl-terminal hydrolase (UCH) catalytic core of the USPs is typically around 350 amino acids, but consists of six conserved boxes, interspersed by insertion sites for additional sequence that can confer diversity (Ye et al., 2009). The major insertion in USP15 is between boxes 3/4, and embeds an ubiquitin-like fold (UBL) domain within the catalytic domain. UBLs are commonly found in the USPs and in certain other DUB families (Zhu et al., 2007; Komander et al., 2009a). They exhibit low sequence conservation, but have high structural similarity with ubiquitin and have been proposed to play important roles in regulating DUB catalytic function or interactions (Zhu et al., 2007; Ye et al., 2009). The intercalation of a UBL between boxes 3 and 4 of the catalytic domain increases the spacing between two sets of zinc-coordinating cysteine motifs, which form a functional zinc finger that is required for activity (Hetfeld et al., 2005). In the case of USP15, a second UBL is located directly adjacent to the DUSP (Zhu et al., 2007; Ye et al., 2009). The four USP15 splice variants encode four distinct protein isoforms, which are illustrated in the diagram and summarised in the accompanying table. As a consequence of alternative splicing, isoform USP15-201 has a 29 amino acid insert within the unstructured region between the first UBL domain and the start of the UCH domain, whereas USP15-204 has a substitution of 3 amino acids for 8 residues within the first UBL. Otherwise the three isoforms that retain the catalytic domain are identical. They also retain predicted nuclear export signals (NES) (Soboleva et al., 2005) and, by homology with rat, a functional nuclear localisation signal (NLS) (Park et al., 2000).

Expression USP15 messenger RNA (mRNA) expression is prevalent throughout the tissues of the body, although its levels vary. Human USP15 is least abundant in brain, lung and kidney, consistent with observations for mouse Usp15 and the rat ortholog UBP109 (Park et al., 2000; Angelats et al., 2003). In each species, USP15 is most abundant in testes, and is variously enriched in spleen, heart, skeletal muscle or peripheral blood leukocytes.

Localisation As USP15 harbours both putative NES and NLS, its sub-cellular distribution may in part depend on the cellular context. Rat UBP109 localises to both the cytoplasm and the nuclear compartment, with the latter

dependent on a C-terminal NLS (Park et al., 2000) that is conserved across species. Using an Usp15/USP15-specific polyclonal antibody, Soboleva et al. demonstrated that in HeLa (human cervical cancer cells) endogenous USP15 localised to the cytoplasm and nucleolus, but was largely excluded from the nucleoplasm; whilst in NIH3T3 (mouse fibroblast cells), Usp15 localised in the cytoplasm and was enriched proximal to the plasma membrane (Soboleva et al., 2005). Interestingly, GFP-tagged USP15 (isoform USP15-203) adopts a largely cytoplasmic distribution in human cancer cell lines (Urbé, unpublished observation).

Function Human USP15 was cloned and characterized in 1999 (Baker et al., 1999) and belongs to the largest ubiquitin specific protease (USP) group of deubiquitinating enzymes (DUBs). Protein ubiquitination occurs at lysine residues through the concerted action of E1 activating, E2 conjugating and E3 ligase enzymes. Ubiquitin contains seven lysine residues (K6, K11, K27, K29, K33, K48 and K63), which allow poly-ubiquitin chains to assemble through alternative isopeptide bond linkages. In addition, linear ubiquitin chains may be assembled through the amino-terminus and substrate proteins may also be mono-ubiquitinated. Consequently, in addition to the classical K48-poly-ubiquitin tag that targets substrates for proteasome-mediated degradation, ubiquitination has multiple cellular functions including regulation of protein localisation and activity (Pickart and Eddins, 2004). The general role of the DUBs, in addition to processing inactive ubiquitin precursors and keeping the 26S proteasome free of inhibitory ubiquitin chains, is to reverse the ubiquitination of substrate proteins (Amerik and Hochstrasser, 2004). There are approximately 80 active human DUBs that are divided into five families (Komander et al., 2009a). These DUBs are steadily being assigned to specific substrates (Ventii and Wilkinson, 2008), which is increasingly revealing associations with signalling pathways in cancer (Sacco et al., 2010). USP15 has activity against both mono-ubiquitinated and poly-ubiquitinated substrates; the zinc-binding domain is necessary for USP15 to process poly-ubiquitin chains, but is not required for USP15 to remove ubiquitin from linear ubiquitin-GFP fusion proteins (Hetfeld et al., 2005). Although USP15 is relatively promiscuous in showing little specificity between K48- and K63-linked poly-ubiquitin chains, or between K63 and K11 di-ubiquitin linkages, it has limited activity against K11-linked poly-ubiquitin chains or linear ubiquitin (Komander et al., 2009b; Bremm et al., 2010). A recent endeavour to map protein partners of the DUBs by mass spectroscopy reported that, in common with USP4 and USP39, USP15 interacts with several proteins involved in mRNA processing and so may



play a role in ubiquitin-dependent control of splicing or mRNA decay (Sowa et al., 2009). In addition, various cancer-signalling pathways have been associated with USP15. For example, USP15 was one of twelve DUBs identified from an siRNA screen that impact on the hepatocyte growth factor (HGF)-dependent cell scattering response in non-small cell lung cancer and pancreatic cancer cells (Buus et al., 2009). A number of specific USP15 substrates have also been described, including the human papilloma virus (HPV) E6 oncoprotein (Vos et al., 2009), the RING-box protein Rbx1 (Hetfeld et al., 2005), the adenomatous polyposis coli (APC) tumour suppressor (Huang et al., 2009), and the NF-kB inhibitor IkBa (Schweitzer et al., 2007). The latter three examples are all connected with the COP9-signalosome (CSN), a conserved multi-protein complex that regulates the cullin-RING ligase (CRL) superfamily of ubiquitin E3 ligases (Wei et al., 2008). CRLs have a core complex comprised of a cullin scaffold and the RING-box protein Rbx1 that recruit alternative adapter and substrate recognition proteins to form diverse E3 complexes with different substrate specificities. The primary function of the CSN is to remove the ubiquitin-like modifier Nedd8 from the cullin component. This both terminates E3 activity and is required for the reassembly of new CRLs (Wei et al., 2008). The CSN plays a role in many cancer-associated pathways including the cell cycle and DNA damage repair, and both CSN and CRL components may be dysregulated in tumours (Richardson and Zundel, 2005). Ubp12p, an S. pombe ortholog of human USP15, was shown through a systematic mass spectrometry screen to bind the CSN (Zhou et al., 2003). This targets Ubp12p to nuclear cullins, where it is proposed to protect against auto-ubiquitination and degradation of CRL components, in particular the substrate-specific adaptors. (Zhou et al., 2003; Wee et al., 2005). Human USP15 also co-purifies with the CSN complex and was reported to stabilise the CRL core component Rbx1 (Hetfeld et al., 2005), thereby acting as a positive regulator of these E3 ligase complexes. In contrast, other studies suggest USP15 may directly oppose CRL E3 ligase activity by deubiquitinating specific substrates. For example, the CSN is involved in ubiquitin-dependent turnover of the IkBa inhibitor that retains NF-kB in the cytosol (Schweitzer et al., 2007). Phosphorylation of IkBa triggers CRL-mediated poly-ubiquitination of IkBa and subsequent proteasomal degradation, allowing NF-kB to enter the nucleus and

activate transcription (Karin and Ben-Neriah, 2000). In response to TNFalpha, IkBa has been reported to interact with the CSN leading to its deubiquitination and stabilisation by CSN-associated USP15 (Schweitzer et al., 2007). The adenomatous polyposis coli (APC) tumour suppressor and the beta-catenin oncogene are frequently mutated in cancers, particularly of the intestine, leading to constitutive wingless and Int-1 (Wnt) signalling (Clevers, 2006). The CSN is proposed to control the balance of beta-catenin and APC through formation of a regulatory super-complex. Deneddylation by the CSN promotes assembly of the beta-catenin destruction complex, whilst CSN-associated USP15 stabilises APC (Huang et al., 2009). The APC also plays a role in mitotic fidelity through interaction with the plus end-binding protein EB1 that controls microtubule growth and dynamics. In contrast to APC, EB1 is destabilised by USP15, suggesting that this is not a direct substrate, but rather that USP15 stabilises a CRL that accelerates ubiquitination and degradation of EB1 (Peth et al., 2007). It is interesting to speculate that such links with microtubule regulation may underpin recent reports that USP15 levels can influence the taxol sensitivity of cancer cells (Xu et al., 2009; Xie et al., 2010). VCP/p97 is a large AAA+-type ATPase that acts as a chaperone in many cellular processes. Its basic function is to segregate ubiquitinated proteins from macromolecular complexes, and VCP plays an important role in recognizing and handling misfolded proteins, which are then either handed over for degradation or recycled. The CSN directly interacts with VCP and USP15 can process VCP-bound poly-ubiquitinated substrates, which accumulate following USP15 depletion (Cayli et al., 2009). VCP is implicated in human neurodegenerative disorders where it co-localises with poly-glutamine aggregates and is proposed to act as both an aggregate-formase and an unfoldase (Kakizuka, 2008). Another established VCP-associated cofactor, the DUB Ataxin-3, is subject to polyglutamine repeat expansion, which causes Machado-Joseph disease (Madsen et al., 2009). Although the mechanism is as yet unclear, USP15 was recently associated with this same disorder (Menzies et al., 2010).

Homology USP15 belongs to the peptidase C19 family. The closest paralogs based on sequence homology are USP4 and USP11.



USP15 and the paralogs USP4 and USP11.The highly similar domain structure is illustrated for USP15 (NP_006304.1), USP4 (NP_003354.2) and USP11 (NP_004642.2). The degree of identity (Id) or similarity (Sim) was derived using ClustalW (EMBL-EBI); note that the indicated UCH homology includes the internal UBL domain. Overall, USP15 shares 56.9% identity (71.2% similarity) with USP4, and 42.4% identity (60.2% similarity) with USP11.

Mutations Somatic No mutations have yet been reported for USP15 according to the COSMIC database, which includes data from four studies of 595 renal carcinoma, glioblastoma, breast and colon cancers.

Implicated in Cervical cancer Note USP15 plays an oncogenic role in cervical cancer. Specific HPV strains are associated with cervical carcinoma and two HPV oncoproteins, E6 and E7, are expressed in these cancers. E6 hijacks a cellular E3 ubiquitin ligase and forms a complex with p53, whilst E7 binds the retinoblastoma (Rb) protein; in each case the viral oncoproteins facilitate degradation of the cellular tumour suppressor. It was recently found that USP15 interacts with the oncogenic HPV16 E6 protein (Vos et al., 2009). siRNA mediated depletion of USP15 led to a decrease in E6 protein, whilst overexpression of wild-type but not catalytically inactive USP15 promoted the stabilisation of E6. Interesting, another group has shown that E7 is regulated in a similar fashion by USP11 (Lin et al., 2008). Intriguingly, USP4 also has functional Rb binding motifs (Blanchette et al., 2001; DeSalle et al., 2001) that are conserved in USP11 and USP15 (Baker et al., 1999).

Ovarian cancer Note USP15 was identified from a genome-wide siRNA screen for Paclitaxel-resistance in the cervical cancer cell line HeLa and, in ovarian cancer samples, Paclitaxel-resistant cases (n=3) showed lower expression of USP15 mRNA than drug-sensitive cases (n=6) (Xu et al., 2009). Moreover, USP15 appeared to stabilise caspase-3, suggesting that reduced levels of USP15 may promote cell survival rather than apoptosis in response to drug treatment.

Gastro-intestinal cancers Note USP15 was also amongst four genes, identified by expression profiling of Docetaxel-sensitive versus resistant cells, which correlated with drug-sensitivity in a panel of gastric cell lines. However, no statistical correlation was established between elevated USP15 transcript levels and Docetaxel-sensitivity in 25 gastric cancer tissues (Xie et al., 2010). Germline mutations in APC lead to inherited colon cancer and sporadic tumours are associated with beta-catenin stabilisation. Huang et al. show a role for USP15 in stabilizing APC levels through the action of the CSN (Huang et al., 2009).

Machado-Joseph disease Note USP15 was identified from microarray analysis of a mouse model of spinocerebellar ataxia type 3. In this



study, USP15 transcript and protein levels were decreased in both the ataxin-3 model, and in a second huntingtin transgenic model of a polyglutamine disorder; although overexpression of USP15 promoted the accumulation of protein aggregates, this was independent of its activity on poly-ubiquitin chains (Menzies et al., 2010).

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Gene Section Review




CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) Joanna Fares, Linda Wolff, Juraj Bies

Lab Cell Oncology, National Cancer Institute NIH, 37 Convent Dr, Bethesda MD 20892, USA (JF, LW, JB); Biochemistry and Molecular Biology Department, Georgetown University, Washington DC 20037, USA (JF)

Published in Atlas Database: January 2011

Online updated version : http://AtlasGeneticsOncology.org/Genes/CDKN2BID187ch9p21.html DOI: 10.4267/2042/46014


Identity Other names: CDK4I; INK4B; MTS2; P15; TP15; p15INK4b

HGNC (Hugo): CDKN2B

Location: 9p21.3

DNA/RNA Description The p15INK4B gene encompasses 6.41 Kb of DNA and has 2 coding exons. It is tandemly linked to p16INK4A and p14ARF within 42 Kb of genomic locus located on chromosome 9p21. The locus is commonly referred to as INK4/ARF locus.

Transcription CDKN2B gene encodes 2 distinct transcript variants: p15 and p10. p10 arises from an alternative 5' splice donor site within intron 1 of: - p15. - p15: 3.82 Kb of mRNA. - p10: 0.86 Kb of mRNA. Regulation: A conserved DNA element with the ability to regulate the entire INK4/ARF locus has been identified in close proximity of the locus and named regulatory domain (RD). It appears to promote transcriptional repression of all three genes encoded by the locus, in a manner dependent on CDC6. In proliferating embryonic fibroblasts (MEFs), EZH2 a

member of the polycomb repressive complex 2 (PRC2) as well as BMI1 and M33 members of the polycomb repressive complex 1 (PRC1) are strongly expressed and are found to localize to the INK4/ARF RD. BMI1 has been shown to interact specifically with CDC6. These polycomb group (PcG) complexes repress the locus activity through the establishment of repressive chromatin modifications such as H3K27 trimethylation. During senescence, binding of these complexes to RD is lost and correlates with increased expression of the INK4/ARF genes (Figure 1). The p15INK4B gene is also silenced by a long non coding RNA, called antisense non-coding RNA in the INK4 locus (ANRIL), whose expression was found to be inversed to the expression of p15INK4B in leukemia cell lines. It was shown that ANRIL induces the silencing of p15INK4B in cis and trans by triggering heterochromatin formation in a Dicer-independent manner. PcG complexes are recruited to the INK4/ARF locus by ANRIL and modulate its repression (Figure 1). Additionally, a naturally occurring antisense circular ANRIL RNAs (cANRIL) has also been described. Different forms of cANRIL are produced in most INK4/ARF expressing cells, suggesting that alternative splicing events leading to different ANRIL structures can contribute to changes in PcG-mediated INK4/ARF repression. Specific transcription regulators of p15INK4B have also been reported (see Figure 2). These include TGF-b, MIZ-1, SMAD3/SMAD4 complex, SP1, c-MYC, IRF8, PU.1, SNAIL and EGR1 factors among others.

CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) Fares J, et al.


Figure 1.

p15INK4B expression is dramatically induced by TGF-b, suggesting that it is a potent downstream effector of TGF-b mediated growth arrest. MIZ-1 is a transcription factor that has been shown to bind the initiator element (Inr) in the promoter region and induce the transcription of p15INK4B in epithelial cells. However it can also recruit transcriptional co-repressors such as c-MYC and GFI-1 to the promoter region by binding to them and forming inhibitory complexes. TGF-b has been reported to re-activate the core promoter through downregulation of C-MYC and GFI-1, thereby releasing endogenous MIZ-1 from inhibition. The SMAD3/4 complex readily forms following TGF-b treatment and physical interaction between this complex, MIZ-1 and promoter-bound SP1 protein has been described. These interactions have been proposed to constitute a platform for the recruitment of co-activators, and do not seem to be affected by the suppressor activity of c-MYC. The inhibitory function of c-MYC seems to be cell-type dependent, as it was confirmed in epithelial cells but not in the hematopoietic lineage. In myeloid cells, the transcription factor c-MYB was shown to prevent the transcription and the upregulation of p15INK4B which is normally associated with the differentiation process. The mechanism by which C-MYB does this is unclear but it is not through upregulation of c-MYC, a known target of c-MYB. A tri-component transcriptional complex consisting of

SNAIL, SP1 and EGR-1 was also described for its ability to trigger the p15INK4b promoter activation upon TPA treatment. In murine myeloid cells specifically, the interferon consensus sequence-binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) in combination with PU.1 were shown to bind p15Ink4b promoter and activate the transcription of the gene in response to IFN-b treatment. In AML patients with inv(16), p15INK4B silencing was found to be caused by inv(16)-encoded core binding factor beta-smooth muscle myosin heavy chain (CBFb-SMMHC). CBFb-SMMHC was shown to displace RUNX1 from a newly determined CBF site in the promoter of p15INK4B.

Protein Description p15INK4B transcript encodes two protein isoforms p15 and p15.5 that are functionally indistinguishable. p15.5 is an N-terminally extended variant of p15 initiated from an upstream alternative in frame initiation codon. p15 protein is 138 aa long and its mass is 14.72 KDa. p10 transcript encodes the shorter variant. The protein consists of 78 aa only and its mass is 10 KDa. It shares a similar NH2 terminus to p15 but contains a different basic COOH terminus that is translated from the p15Ink4b intronic region (Figure 2).



Figure 2.

Expression p15INK4B is expressed at very low levels under normal physiological conditions. Its expression seems to be lineage restricted. In bone marrow cells the highest level of p15INK4B is mainly detected in maturing monocytes/macrophages and lymphocytes. The gene expression has also been reported to be normally up-regulated during megakaryocytic differentiation. Increased expression of p15INK4B is also detected during stress and senescence of cells.

Localisation Nucleus and cytoplasm.

Function I- Function in the cell cycle. p15INK4B belongs to the INK4 family of protein kinase inhibitors named for their high and exclusive specificity towards the catalytic activity of cyclin dependent kinases 4 (CDK4) or 6 (CDK6). Structural studies have demonstrated that the protein performs its inhibitory activity by allosteric competition with the D-type cyclins to bind CDK4/6 kinases and prevents the formation of active CDK4/6-cyclin-Ds complexes. This keeps the retinoblastoma protein (RB), which is downstream of this pathway, in its hypophosphorylated state. Hypophosporylated RB binds and inactivates the E2F transcription factors required for the transcriptional activation of genes necessary for entry into the S phase of the cell cycle and DNA synthesis. Three other members of the INK4 family of CDK inhibitors: p16INK4A, p18INK4C and p19INK4D are encoded by unique genes and share roughly 40% homology. They have similar protein structure characterized by the presence of four ankyrin-like motif tandem repeats that are predicted to be engaged in protein-protein interactions. II- Function during hematopoietic cell differentiation. Another role for p15INK4B during differentiation of

early hematopoietic progenitors has also been described. In knockout mice, loss of p15INK4B was shown to favor the differentiation of common myeloid progenitors (CMP) into granulocyte macrophage progenitors (GMP) resulting in an imbalance between the myeloid and the erythroid compartments. III- Function during cellular senescence. Cellular senescence is accompanied by hallmark features that include the up-regulation of cell cycle inhibitors like p15INK4B, p16INK4A and p21CIP. When overexpressed, p15INK4B engages the RB pathway to promote a stable senescent state which has been shown to occur in part through a process that involves alterations in heterochromatin and the stable silencing of E2F target genes. Another mechanism that has been described is the inactivation of c-MYC which results in the induction of p15INK4B expression and correlates with the global changes in heterochromatin structure known to be associated with cellular senescence.

Homology p15INK4B is highly conserved. Its sequence in homo sapiens is > 85% similar to bos taurus, mus musculus and rattus norvegicus; and > 70% similar to gallus gallus.

Mutations Note Intragenic p15INK4B mutations are highly infrequent.

Implicated in Various hematological disorders and malignancies Note p15INK4B is frequently epigenetically silenced in leukemias, myelodysplastic syndromes and



myeloproliferative diseases by mechanisms involving aberrant DNA methylation and/or histone modifications. These diseases are subcategorized by the French-American-British (FAB) co-operative group, based on the percentage of blast cells in bone marrow and peripheral blood, degree of cytopenia, and in accordance to the direction of differentiation along the myeloid or lymphoid lineages as well as the degree of maturation of the hematopoietic cells.

Myelodysplastic syndromes (MDS) Disease Myelodysplastic syndromes are heterogeneous clonal hematologic disorders characterized by dysplasia of the myeloid bone marrow cells accompanied with peripheral blood cytopenia and increased risk of transformation to acute myeloid leukemia (AML). MDS transforms into AML once the percentage of blasts in the bone marrow has exceeded 30% (FAB). MDS can arise in patients de novo (primary MDS), or following chemotherapy or exposure to toxins (secondary MDS). According to the Leukemia and Lymphoma Society reports, MDS most commonly affects males aged 70 and above, and is considered to be a disease of the elderly. About 11000 new cases are diagnosed each year, resulting in an incidence rate of 4 cases per 100000 population for both genders.

Prognosis p15INK4B is silenced by promoter hypermethylation in > 50% of MDS cases. Levels of p15INK4B methylation increase as the disease progresses and provide a marker that can predict occurrence of AML.

Chronic myelomonocytic leukemia (CMML) Disease The defining features of CMML are an absolute monocytosis in peripheral blood (> 1x109/L), increased numbers of monocytes in bone marrow, a variable degree of dysplasia and less than 5% and 20% of blasts in peripheral blood and bone marrow, respectively. There are two types of CMML: proliferative and dysplastic. Roughly half of CMML diagnosed patients have an elevated white blood cell count commonly associated with hepatomegaly and splenomegaly (myeloproliferative form of the disease). Patients lacking these features are generally considered to have the myelodysplastic form of the disease.

Prognosis Hypermethylation is found in up to 60% of CMML cases and correlates with a more aggressive form of disease. Experimentally, a LysMCre mouse model was developed in which p15INK4B gene is deleted specifically in cells of the myeloid lineage, to better mimic the loss of the gene expression the way it is observed in humans. The mice develop non-reactive monocytosis of the peripheral blood as well as increased myeloid blast progenitors in the bone

marrow. In this way the mice develop symptoms that closely resemble CMML in human patients.

Acute myeloid leukemia (AML) Disease AML is the most common type of leukemia among adults with 14000 new cases diagnosed each year, and with 9000 deaths per year in the United States. AML classification into ten different subtypes was originally defined by the FAB cooperative group according to the direction of differentiation along the different myeloid lineages as well as the degree of maturation of the cells. However, AML exemplifies a genetically heterogeneous cancer with more than a hundred genetic aberrations implicated in the disease.

Prognosis Despite the great genetic and phenotypic heterogeneity of AML, hypermethylation of the p15INK4B promoter region (CpG island) is found to occur in up to 80% of AML cases across all FAB subtypes. It correlates with a loss of p15INK4B expression, poor prognosis and shorter survival time in patients. The p15INK4B methylation status in AML patients in clinical remission is now monitored and used as a reliable prognostic marker for relapse. These findings were further experimentally confirmed in a conditional knockout mouse model where myeloid-specific gene inactivation resulted in an increased susceptibility to retrovirus-induced myeloid leukemia.

Acute lymphoblastic leukemia (ALL) Disease There are about 4000 new cases of ALL in the United States each year. It appears most often in children younger than age 10. ALL is the most common leukemia in children. However, it can appear in people of any age. About one-third of cases are adults.

Prognosis In B and T acute lymphoblastic leukemia the p15INK4B promoter methylation as well as deletion of the entire locus has been reported.

Chronic leukemia Disease Chronic leukemia can be subdivided into two subtypes, chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL). CLL is primarily an adult disease; it is very rare in children and young adults. The median age of diagnosis is 72 years, and about 60% of patients are male. In the United States, about 15000 people are diagnosed with CLL each year. This disease is also commonly referred to as B-cell chronic lymphocytic leukemia (B-CLL).

Prognosis Promoter hypermethylation has been reported in a small subset of B-CLL (11%) at all stages of the disease. In CML, silencing of p15INK4B either by



deletion or hypermethylation of its promoter was not found to be a very frequent event.

Glioblastoma multiforme (GBM) Disease GBM is the most common and very aggressive brain tumor in adults. It involves glial cells and accounts for more than 50% of parenchymal brain tumors approximately 20% of all intracranial tumors. Glioblastoma growth is characterized by a high motility of tumor cells that display broad chemoresistance leading to frequent post-surgical tumor recurrence. It is one of the most dreaded cancer diagnoses due to its poor prognosis and the limited treatment options, with the median survival duration after diagnosis varying from 6 months to 2 years.

Prognosis Homozygous deletion of the p15INK4B/p14ARF/p16INK4A locus on chromosome 9p21.3 is a signature genetic event that drives the pathogenesis of GBM. The deletion of this locus is the most common homozygous deletion present in GBM (> 75% of samples). Specific p15INK4B promoter methylation was also detected in 37% of patients diagnosed with glioblastoma and it correlated with shorter survival.

Hepatocellular carcinoma (HCC) Disease HCC is a primary malignancy of the liver that mostly arises secondary to hepatitis B or C viral infections. Outcome of the disease is poor, because only 10 - 20% of hepatocellular carcinomas can be removed completely using surgery, and the cancer is usually deadly within 3 to 6 months.

Prognosis The suppression of the C-MYC oncogene induces cellular senescence in diverse tumor types including hepatocellular carcinoma and correlates with increased p15INK4b expression. In primary HCC, p15INK4B promoter is hypermethylated in about 50% of the cases, and homozygous deletions of both p16INK4A and p15INK4B have been reported in 30% HCC patients and cell lines. This suggests that p15INK4B might be contributing to human hepatocarcinogenesis through a pathway associated with cellular senescence.

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Tsubari M, Tiihonen E, Laiho M. Cloning and characterization of p10, an alternatively spliced form of p15 cyclin-dependent kinase inhibitor. Cancer Res. 1997 Jul 15;57(14):2966-73

Drexler HG. Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukemia-lymphoma cells. Leukemia. 1998 Jun;12(6):845-59

Aoki E, Uchida T, Ohashi H, Nagai H, Murase T, Ichikawa A, Yamao K, Hotta T, Kinoshita T, Saito H, Murate T. Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes. Leukemia. 2000 Apr;14(4):586-93

Fuxe J, Raschperger E, Pettersson RF. Translation of p15.5INK4B, an N-terminally extended and fully active form of p15INK4B, is initiated from an upstream GUG codon. Oncogene. 2000 Mar 23;19(13):1724-8

Jeffrey PD, Tong L, Pavletich NP. Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. Genes Dev. 2000 Dec 15;14(24):3115-25

Latres E, Malumbres M, Sotillo R, Martín J, Ortega S, Martín-Caballero J, Flores JM, Cordón-Cardo C, Barbacid M. Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis. EMBO J. 2000 Jul 3;19(13):3496-506

Malumbres M, Ortega S, Barbacid M. Genetic analysis of mammalian cyclin-dependent kinases and their inhibitors. Biol Chem. 2000 Sep-Oct;381(9-10):827-38

Teofili L, Morosetti R, Martini M, Urbano R, Putzulu R, Rutella S, Pierelli L, Leone G, Larocca LM. Expression of cyclin-dependent kinase inhibitor p15(INK4B) during normal and leukemic myeloid differentiation. Exp Hematol. 2000 May;28(5):519-26

Amati B. Integrating Myc and TGF-beta signalling in cell-cycle control. Nat Cell Biol. 2001 May;3(5):E112-3

Schmidt M, Koller R, Haviernik P, Bies J, Maciag K, Wolff L. Deregulated c-Myb expression in murine myeloid leukemias prevents the up-regulation of p15(INK4b) normally associated with differentiation. Oncogene. 2001 Sep 27;20(43):6205-14

Seoane J, Pouponnot C, Staller P, Schader M, Eilers M, Massagué J. TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b. Nat Cell Biol. 2001 Apr;3(4):400-8

Staller P, Peukert K, Kiermaier A, Seoane J, Lukas J, Karsunky H, Möröy T, Bartek J, Massagué J, Hänel F, Eilers M. Repression of p15INK4b expression by Myc through association with Miz-1. Nat Cell Biol. 2001 Apr;3(4):392-9

Teofili L, Martini M, Di Mario A, Rutella S, Urbano R, Luongo M, Leone G, Larocca LM. Expression of p15(ink4b) gene during megakaryocytic differentiation of normal and myelodysplastic hematopoietic progenitors. Blood. 2001 Jul 15;98(2):495-7

Wolff L, Schmidt M, Koller R, Haviernik P, Watson R, Bies J, Maciag K. Three genes with different functions in transformation are regulated by c-Myb in myeloid cells. Blood Cells Mol Dis. 2001 Mar-Apr;27(2):483-8

Narita M, Nũnez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell. 2003 Jun 13;113(6):703-16

Tessema M, Länger F, Dingemann J, Ganser A, Kreipe H, Lehmann U. Aberrant methylation and impaired expression of the p15(INK4b) cell cycle regulatory gene in chronic myelomonocytic leukemia (CMML). Leukemia. 2003 May;17(5):910-8

Qin Y, Liu JY, Li B, Sun ZL, Sun ZF. Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis. World J Gastroenterol. 2004 May 1;10(9):1276-80

Schmidt M, Bies J, Tamura T, Ozato K, Wolff L. The interferon regulatory factor ICSBP/IRF-8 in combination with PU.1 up-



regulates expression of tumor suppressor p15(Ink4b) in murine myeloid cells. Blood. 2004 Jun 1;103(11):4142-9

Aggerholm A, Holm MS, Guldberg P, Olesen LH, Hokland P. Promoter hypermethylation of p15INK4B, HIC1, CDH1, and ER is frequent in myelodysplastic syndrome and predicts poor prognosis in early-stage patients. Eur J Haematol. 2006 Jan;76(1):23-32

Markus J, Garin MT, Bies J, Galili N, Raza A, Thirman MJ, Le Beau MM, Rowley JD, Liu PP, Wolff L. Methylation-independent silencing of the tumor suppressor INK4b (p15) by CBFbeta-SMMHC in acute myelogenous leukemia with inv(16). Cancer Res. 2007 Feb 1;67(3):992-1000

Papageorgiou SG, Lambropoulos S, Pappa V, Economopoulou C, Kontsioti F, Papageorgiou E, Tsirigotis P, Dervenoulas J, Economopoulos T. Hypermethylation of the p15INK4B gene promoter in B-chronic lymphocytic leukemia. Am J Hematol. 2007 Sep;82(9):824-5

Wu CH, van Riggelen J, Yetil A, Fan AC, Bachireddy P, Felsher DW. Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation. Proc Natl Acad Sci U S A. 2007 Aug 7;104(32):13028-33

Peters G. An INKlination for epigenetic control of senescence. Nat Struct Mol Biol. 2008 Nov;15(11):1133-4

Solomon DA, Kim JS, Jean W, Waldman T. Conspirators in a capital crime: co-deletion of p18INK4c and p16INK4a/p14ARF/p15INK4b in glioblastoma multiforme. Cancer Res. 2008 Nov 1;68(21):8657-60

Yu W, Gius D, Onyango P, Muldoon-Jacobs K, Karp J, Feinberg AP, Cui H. Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature. 2008 Jan 10;451(7175):202-6

Basu S, Liu Q, Qiu Y, Dong F. Gfi-1 represses CDKN2B encoding p15INK4B through interaction with Miz-1. Proc Natl Acad Sci U S A. 2009 Feb 3;106(5):1433-8

Bies J, Sramko M, Fares J, Rosu-Myles M, Zhang S, Koller R, Wolff L. Myeloid-specific inactivation of p15Ink4b results in monocytosis and predisposition to myeloid leukemia. Blood. 2010 Aug 12;116(6):979-87

Burd CE, Jeck WR, Liu Y, Sanoff HK, Wang Z, Sharpless NE. Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk. PLoS Genet. 2010 Dec 2;6(12):e1001233

Hu CT, Chang TY, Cheng CC, Liu CS, Wu JR, Li MC, Wu WS. Snail associates with EGR-1 and SP-1 to upregulate transcriptional activation of p15INK4b. FEBS J. 2010 Mar;277(5):1202-18

Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, Xiong Y. Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene. Oncogene. 2011 Apr 21;30(16):1956-62


Fares J, Wolff L, Bies J. CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):652-657.

Gene Section Review




DLX4 (distal-less homeobox 4) Patricia E Berg, Saurabh Kirolikar

The George Washington University Medical Center, Washington DC 20037, USA (PEB), The George Washington University, Department of Biochemistry and Molecular Biology, Washington DC 20037, USA (SK)


Online updated version : http://AtlasGeneticsOncology.org/Genes/DLX4ID49827ch17q21.html DOI: 10.4267/2042/46015


Identity Other names: BP1; DLX7; DLX8; DLX9

HGNC (Hugo): DLX4

Location: 17q21.33

DNA/RNA Note BP1, DLX4 and DLX7 are not interchangeable names for the same gene, as sometimes claimed. We cloned a cDNA encoding BP1 from a library made from K562 erythroleukemia cells, often used as a model for hemoglobin switching. After it was sequenced it was apparent that part of the BP1 sequence was identical to that of two other published "genes", DLX4 and DLX7; upstream of nucleotide 565 of BP1, all three had entirely different sequences, while downstream of that site the sequences were identical (Fu et al., 2001; Chase et al., 2002). This suggested that the three might be isoforms of one gene, differing only in the first exon; this can occur by alternative splicing or by use of alternative promoters. DLX7 had been mapped to

17q21-22 (Nakamura et al., 1996). We then mapped BP1, which also mapped to the same chromosomal region (Fu et al., 2001). In fact, our BAC included sequences from both BP1 and DLX7. When we published the paper showing that BP1 is a repressor of the beta-globin gene and identifying BP1, DLX4 and DLX7 as isoforms, to prevent confusion in the literature we gave the gene a single name, DLX4, based on the fact that the DLX4 DNA sequence was published first (Chase et al., 2002). Thus, the DLX4 gene encodes at least three different proteins with presumably different functions, DLX4, BP1 and DLX7. The NCBI Database is somewhat confusing in this regard - the gene is called DLX4, but BP1 is named DLX4 variant 1 and DLX7 is called DLX4 variant 2.

Description The DLX4 gene is located at 17q21.33 and is about 5761 bp in length (chr17:48,046,562-48,052,322).

Transcription Three different mRNAs are expressed by DLX4, BP1 and DLX7. BP1 mRNA is about 2012 bp.

The red lines indicate the predicted ORFs. Number 565 indicates the nucleotide of BP1 where divergence occurs among BP1, DLX4, and DLX7. HB is the homeobox region. The regions between the two vertical lines indicate the regions of DNA identity. The complete ORF is not available for DLX4.

DLX4 (distal-less homeobox 4) Berg PE, Kirolikar S


Amino acid sequence alignment between BP1 (Q92988-1), DLX7 (Q92988-2) and an unidentified isoform of DLX4 (Q92988-3). The sequences were obtained from the UniProt database which has incorrectly identified the BP1 sequence as DLX4.

Protein Description The BP1 protein is an alternatively spliced isoform derived from the DLX4 gene. The protein is about 240 aa in length with the calculated molecular weight of 26 kDa. The observed weight of the protein is about 36 kDa on Western blots using lysates from breast cancer and prostate cancer cell lines. This difference may be due to post-translational modifications of BP1 protein. BP1 protein has a 116 aa N-terminal region, 60 aa homeobox domain and a 64 aa C-terminal domain, while DLX7 is 168 aa.

Expression In normal tissue: BP1 protein is expressed in adult kidney and placenta and in low levels in normal breast and fetal liver (Chase et al., 2002).

Localisation Both nuclear and cytoplasmic immunostaining are seen in BP1 positive breast tumors and prostate tumors (Man et al., 2005; Schwartz et al., 2009).

Function Functionally, we demonstrated that BP1 is a repressor of the beta-globin gene, while DLX7 binds to the same DNA sequence upstream of the beta-globin gene but lacks the ability to repress it (Fu et al., 2001; Chase et al., 2002). Thus, the functions of BP1 and DLX7 are clearly different in this context. BP1 acts to repress embryonic and fetal globin genes during early development but is itself repressed during normal adult erythropoiesis. BP1 overexpression induces increased Bcl-2 expression and decreased apoptosis. pBP1 binds to the regulatory region of the bcl-2 gene, an anti-apoptotic gene, resulting in elevated expression of Bcl-2 protein and resistance to TNF-alpha in MCF-7 breast cancer cells (Stevenson et al., 2007). Increased BP1 is associated with decreased cleavage of caspase-7, caspase-8 and caspase-9, and increased expression of PARP. Thus,

high BP1 expression can lead to decreased cell death and, as shown below, increased proliferation. BP1 appears to be a repressor of BRCA1. Three breast cancer cell lines engineered to overexpress BP1 show decreased BRCA1 RNA and protein, while cells in which BP1 is knocked down by siRNA treatment show increased BRCA1 expression, suggesting that BP1 activity may contribute to reduced BRCA1 in some breast cancers (Kluk et al., 2010).

Implicated in Breast cancer Note BP1 is activated in about 80% of invasive ductal breast (IDC) tumors. Aberrant expression of BP1 was shown by semi-quantitative RT-PCR, where 80% of tumors were BP1 positive, and by immunostaining, where 81% of tumors were BP1 positive, a remarkable agreement between mRNA and protein expression (Fu et al., 2003; Man et al., 2005). Surprisingly, 89% of the tumors of African American women (AAW) were BP1 positive, compared with 57% of the tumors of Caucasian women (p=0.04). In addition, 100% of ER negative tumors were BP1 positive, compared with 73% of ER positive (p=0.03). Both tumors of AAW and ER negative tumors are associated with aggressiveness. A group in China quantitated BP1 mRNA in the tumors of 142 Chinese women, discovering that 65% of their tumors were BP1 positive, and confirming an association between high BP1 mRNA expression and ER negative tumors (Yu et al., 2008b). Inflammatory breast cancer (IBC) is an extremely aggressive breast cancer, with approximately half the survival seen in IDC; 100% of the forty-six cases of IBC we examined were highly BP1 immunoreactive, suggesting an association between aggressiveness, frequency of BP1 positivity, and BP1 protein (pBP1) staining intensity (Man et al., 2009). pBP1 expression correlates with breast cancer progression. The frequency of pBP1 positivity,



distribution and intensity of BP1 expression all increased with the progression of tumor development from 0% (normal) to 21% in hyperplasia, 46% in ductal carcinoma in situ, and 81% in IDC (p <0.0001) (Man et al., 2005). This suggests BP1 expression may be an important upstream factor in an oncogenic pathway and may contribute to tumor progression. Expression of BP1 is associated with larger tumor size. We have recently shown a correlation between BP1 mRNA or protein expression and tumor size in women with invasive ductal breast cancer. This correlation is also true in a mouse model (submitted). BP1 positivity correlates with increased proliferation. BP1 positive cells were significantly more frequently positive for Ki67, a proliferation marker, when 5000 BP1 positive tumor cells were compared with 5000 BP1 negative tumor cells (Man et al., 2005). BP1 appears to be associated with metastasis. Among the IBC cases, nine had metastasized. The lymph nodes corresponding to these cases were all BP1 positive, providing evidence that BP1 is expressed in metastasis (Man et al., 2009). Moreover, BP1 positive cells were observed in lymphatic ducts of patients with metastatic IBC. A correlation between high BP1 mRNA levels and metastasis in invasive ductal breast cancer was observed by Yu et al. (2008b). BP1 mRNA levels are associated with survival. Kaplan-Meier curves revealed that patients with grade III tumors expressing high BP1 mRNA levels showed decreased survival compared with patients whose grade III tumors contained lower BP1 mRNA levels (Yu et al., 2008b). BP1 is activated by DNA amplification. It is important to determine the factors that activate BP1. Approximately 33% of the tumors we examined from women with metastatic breast cancer exhibited DNA amplification of BP1. Amplification was associated with BP1 positivity by immunostaining in all cases (Cavalli et al., 2008). Overall, the data strongly suggest that BP1 may be a useful new biomarker in early detection of breast cancer and a potential therapeutic target.

Leukemia Note We examined BP1 in the bone marrow of leukemia patients by semi-quantitative RT-PCR, finding that BP1 was activated in 63% of acute myeloid leukemias (AML), including 81% of pediatric and 47% of adult patients with AML, in 32% of T-cell acute lymphocytic leukemias (ALL) but not in the pre-B ALL cases (Haga et al., 2000). Expression of BP1 occurred in primitive leukemia cells and in CD34 positive progenitors. In the same study we examined expression of DLX4 and DLX7 by designing primers specific for each isoform. Interestingly, the three isoforms were frequently co-expressed in the same cases.

Next we compared the growth-inhibitory and cyto-differentiating activities of all-trans retinoic acid (ATRA) in two acute promyelocytic leukemia (APL) cells lines, NB4 (ATRA-responsive) and R4 (ATRA-resistant) cells relative to BP1 levels (Awwad et al., 2008). NB4 cells and R4 cells both expressed BP1; BP1 was repressed after ATRA treatment of NB4 cells but not R4 cells. In NB4 cells engineered to overexpress BP1, proliferation was no longer inhibited and differentiation was reduced two- to three-fold. In patients, BP1 levels were increased in all pre-treatment APL patients tested, while BP1 expression was decreased in 91% of patients after combined ATRA and chemotherapy treatment. Two patients underwent disease relapse during follow up; one patient exhibited a 42-fold increase in BP1 expression, while the other showed no change. This suggests BP1 may be part of a pathway involved in resistance to therapy.

Prostate cancer Note Prostate cancer, another hormone dependent solid tumor, was examined for activation of BP1 (Schwartz et al., 2008). Significant BP1 immunoreactivity was identified in 70% of prostatic tumors, whether the analysis was performed on tissue sections (50 cases) or tissue microarray platforms (123 cases). We also observed low BP1 immunostaining in 42% of hyperplastic cells, similar to the 46% BP1 positivity in hyperplastic breast cells. Compared to normal and hyperplastic tissues, the malignant tissues consistently showed the highest number of BP1 positive cells and the highest intensity of BP1 immunostaining, similar to our observations in breast. In tissue sections, twelve cases with paired carcinoma and prostatic intraepithelial neoplasia (PIN) showed agreement, both components exhibiting strong immunoreactivity. Tumor proliferation, assayed with Ki67 immunostaining, was higher in cancer cells that were BP1 positive relative to those that were BP1 negative, in agreement with the data in breast cancer cells. These findings suggest that BP1 is an important upstream factor in the carcinogenic pathway of prostate cancer and that the expression of BP1 may reflect or directly contribute to tumor progression and/or invasion.

Non-small cell lung cancer (NSCLC) Note An interesting study by Yu et al. (2008a) demonstrated that high BP1 mRNA levels occur in NSCLC tumors, compared with adjacent normal cells or normal lung samples. High mRNA levels are associated with stage III tumors, lower disease free survival (DFS) and lower overall survival. In fact, high BP1 mRNA is an independent predictor of DFS.

References Nakamura S, Stock DW, Wydner KL, Bollekens JA, Takeshita K, Nagai BM, Chiba S, Kitamura T, Freeland TM, Zhao Z,



Minowada J, Lawrence JB, Weiss KM, Ruddle FH. Genomic analysis of a new mammalian distal-less gene: Dlx7. Genomics. 1996 Dec 15;38(3):314-24

Haga SB, Fu S, Karp JE, Ross DD, Williams DM, Hankins WD, Behm F, Ruscetti FW, Chang M, Smith BD, Becton D, Raimondi SC, Berg PE. BP1, a new homeobox gene, is frequently expressed in acute leukemias. Leukemia. 2000 Nov;14(11):1867-75

Fu S, Stevenson H, Strovel JW, Haga SB, Stamberg J, Do K, Berg PE. Distinct functions of two isoforms of a homeobox gene, BP1 and DLX7, in the regulation of the beta-globin gene. Gene. 2001 Oct 31;278(1-2):131-9

Chase MB, Fu S, Haga SB, Davenport G, Stevenson H, Do K, Morgan D, Mah AL, Berg PE. BP1, a homeodomain-containing isoform of DLX4, represses the beta-globin gene. Mol Cell Biol. 2002 Apr;22(8):2505-14

Fu SW, Schwartz A, Stevenson H, Pinzone JJ, Davenport GJ, Orenstein JM, Gutierrez P, Simmens SJ, Abraham J, Poola I, Stephan DA, Berg PE. Correlation of expression of BP1, a homeobox gene, with estrogen receptor status in breast cancer. Breast Cancer Res. 2003;5(4):R82-7

Man YG, Fu SW, Schwartz A, Pinzone JJ, Simmens SJ, Berg PE. Expression of BP1, a novel homeobox gene, correlates with breast cancer progression and invasion. Breast Cancer Res Treat. 2005 Apr;90(3):241-7

Stevenson HS, Fu SW, Pinzone JJ, Rheey J, Simmens SJ, Berg PE. BP1 transcriptionally activates bcl-2 and inhibits TNFalpha-induced cell death in MCF7 breast cancer cells. Breast Cancer Res. 2007;9(5):R60

Awwad RT, Do K, Stevenson H, Fu SW, Lo-Coco F, Costello M, Campbell CL, Berg PE. Overexpression of BP1, a

homeobox gene, is associated with resistance to all-trans retinoic acid in acute promyelocytic leukemia cells. Ann Hematol. 2008 Mar;87(3):195-203

Cavalli LR, Man YG, Schwartz AM, Rone JD, Zhang Y, Urban CA, Lima RS, Haddad BR, Berg PE. Amplification of the BP1 homeobox gene in breast cancer. Cancer Genet Cytogenet. 2008 Nov;187(1):19-24

Yu M, Wan Y, Zou Q. Prognostic significance of BP1 mRNA expression level in patients with non-small cell lung cancer. Clin Biochem. 2008a Jul;41(10-11):824-30

Yu M, Yang Y, Shi Y, Wang D, Wei X, Zhang N, Niu R. Expression level of beta protein 1 mRNA in Chinese breast cancer patients: a potential molecular marker for poor prognosis. Cancer Sci. 2008b Jan;99(1):173-8

Man YG, Schwartz A, Levine PH, Teal C, Berg PE. BP1, a putative signature marker for inflammatory breast cancer and tumor aggressiveness. Cancer Biomark. 2009;5(1):9-17

Schwartz AM, Man YG, Rezaei MK, Simmens SJ, Berg PE. BP1, a homeoprotein, is significantly expressed in prostate adenocarcinoma and is concordant with prostatic intraepithelial neoplasia. Mod Pathol. 2009 Jan;22(1):1-6

Kluk BJ, Fu Y, Formolo TA, Zhang L, Hindle AK, Man YG, Siegel RS, Berg PE, Deng C, McCaffrey TA, Fu SW. BP1, an isoform of DLX4 homeoprotein, negatively regulates BRCA1 in sporadic breast cancer. Int J Biol Sci. 2010 Sep 10;6(5):513-24


Berg PE, Kirolikar S. DLX4 (distal-less homeobox 4). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):658-661.

Gene Section Review




IL17A (interleukin 17A) Norimitsu Inoue, Takashi Akazawa

Department of Molecular Genetics, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka 537-8511, Japan (NI, TA)


Online updated version : http://AtlasGeneticsOncology.org/Genes/IL17AID40945ch6p12.html DOI: 10.4267/2042/46016


Identity Other names: CTLA8; IL-17; IL-17A; IL17

HGNC (Hugo): IL17A

Location: 6p12.2

Local order: Centromere-PKHD1 (polycystic kidney and hepatic diseases 1)-MIR206 (microRNA 206)-MIR133B (microRNA 133b)-IL17A-IL17F (interleukin 17F)-SLC25A20P1 (solute carrier family 25, member 20 pseudogene 1)-MCM3 (minichromosome maintenance complex component 3)-Telomere.

DNA/RNA Description 3 exons.

Transcription The transcript is 1859 bp and has a 45 bp 5' UTR, a 468 bp coding sequence, and a 1346 bp 3' UTR.

Pseudogene No pseudogene.

Protein Note The IL17A protein is a disulfide-linked homodimeric glycoprotein. Members of the IL17 protein family (IL17A to F) have four highly conserved cysteine residues on each of the monomeric peptides (Moseley et al., 2003; Kolls et al., 2004; Korn et al., 2009). Structural analysis of the IL17F protein indicates that these four cysteines participate in the characteristic cysteine-knot formation found in certain other growth factors such as nerve growth factor (NGF), bone morphogenetic proteins (BMPs), and transforming growth factor-beta1 (TGFbeta1) (Hymowitz et al., 2001). Two additional cysteine residues participate in homodimer formation via inter-chain disulfide-bonds. The IL17F peptide can also form a functional heterodimer with IL17A.

IL17A gene. The IL17A gene spans a region of 4252 bp composed of three exons (untranslated region (UTR), light blue; coding region, blue) and two introns (brown). Exons 1, 2, and 3 are 72 bp (45 bp 5' UTR plus 27 bp coding region), 203 bp (all coding region), and 1584 bp (238 bp coding region plus 1346 bp 3' UTR) in length, respectively. The two introns are 1144 bp and 1249 bp in length, respectively.

IL17A (interleukin 17A) Inoue N, Akazawa T


IL-17A protein. IL17A protein (155 amino acids) is composed of a signal peptide (light green, 23 amino acids) and a mature peptide (green, 132 amino acids). The four conserved cysteines (Cys) form the intra-chain disulfide bonds indicated by black lines (Cys94/Cys144 and Cys99/Cys146) (Hymowitz et al., 2001). The two cysteines indicated by asterisks (Cys33 and Cys129) participate in homodimer formation via inter-chain disulfide bonds. Asparagine 68 (Asn68, black circle) is predicted to be glycosylated.

Description Each IL17A monomer is a 19.9-kD peptide that consists of 155 amino acids. The IL17A peptide comprises a 23-amino acid signal peptide and a 132-amino acid mature peptide (IL17A homodimer, 35 kD).

Expression IL17A is secreted by CD4-positive T cells (Th17 cells), which also produce IL17F, IL21, and IL22 (Korn et al., 2009; Eyerich et al., 2010). CD8-positive T cells, gamma delta T cells, natural killer (NK) cells, NKT cells, and lymphoid tissue inducer (LTi) cells also secret IL17A. These leukocytes all express the retinoic acid receptor-related orphan nuclear receptor C (RORC, the human analogue of mouse RORgammat that is a splice variant of the Rorc gene). RORC is essential for IL17A production. Th17 cells are the third subset of helper T cells, with effector functions distinct from Th1 and Th2 cells. Th17 cells are differentiated from naïve T cells in the presence of IL6 plus TGFbeta1 (Bettelli et al., 2007; McGeachy et al., 2008; Awasthi et al., 2009). In the presence of TGFbeta1 alone, naïve T cells express the transcriptional factor forkhead box P3 (FOXP3) and differentiate into induced regulatory T cells (iTreg cells). In the presence of IL6 alone, the cells express the transcriptional factor BCL6 and differentiate into T follicular helper cells (Tfh cells) (Nurieva et al., 2009). Interleukin 21 is secreted from Th17 cells and amplifies Th17 cell generation by an autocrine mechanism. Interleukin 21 also induces the expression of the IL23 receptor in the Th17 cells (Bettelli et al., 2007; McGeachy and Cua, 2008; Awasthi and Kuchroo, 2009). Interleukin 23 is secreted from dendritic cells and macrophages following stimulation by Toll-like receptor ligands. IL23 in turn mediates the stabilization and maintenance of the Th17 cell phenotype, inducing IL17A production by Th17 cells (Stritesky et al., 2008; McGeachy et al., 2009). Interleukin 1beta is also involved in the induction of IL17A secretion and the promotion of Th17 differentiation (Chung et al., 2009). In addition to RORC and the aforementioned cytokines, signal transducer and activator of transcription 3 (STAT3), interferon regulatory factor 4 (IRF4), runt-related transcriptional factor 1 (RUNX1), and aryl

hydrocarbon receptor (AHR, a nuclear receptor for a number of low-molecular weight chemicals such as the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ)) all positively regulate Th17 cell differentiation (Korn et al., 2009; Hirahara et al., 2010). Moreover, prostaglandin E2, ATP, and C-type lectin ligands act on antigen-presenting cells to facilitate Th17 cell differentiation. In contrast, IL4, Interferon-gamma (IFNgamma), IL27, suppressor of cytokine signaling 3 (SOCS3), and STAT5 suppress Th17 cell differentiation. Finally, high levels of lactic acid secreted from tumors via the Warburg effect act on macrophages to mediate increased IL17A production but not Th17 cell differentiation (Shime et al., 2008; Yabu et al., 2011). Th17 cells in both the mouse and the human have recently been shown to differentiate from naïve CD4 T cells independently of TGFbeta1 signaling. These TGFbeta1-independent Th17 cells instead differentiate in the presence of IL6, IL23 and IL1beta (Hirahara et al., 2010; Ghoreschi et al., 2010). TGFbeta1-independent Th17 cells co-express RORgammat and T-bet (TBX21, T-box protein 21) and exhibit more pathogenic potential than TGFbeta1-dependent Th17 cells in the development of experimental allergic encephalomyelitis (EAE).

Function Interleukin 17A is a pro-inflammatory cytokine and act on a variety of cells (e.g., fibroblasts, epithelial cells, and monocytes) to induce the production of cytokines (IL6, tumor necrosis factor-alpha TNFalpha, granulocyte-macrophage colony-stimulating-factor (GMCSF), granulocyte colony-stimulating-factor (GCSF)), chemokines (chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, CXCL5, CXCL8) and matrix metalloproteinases (MMP2, MMP13) to mediate the recruitment, activation and migration of neutrophils and myeloid cells (Kolls and Linden, 2004; Eyerich et al., 2010). IL17A, IL17F, and the IL17A-IL17F heterodimer bind to a heteromeric receptor complex composed of IL17 receptor A (IL17RA) and IL17 receptor C (IL17RC). IL17RA is expressed at high levels in hematopoietic cells and at low levels in epithelial cells, fibroblasts and



endothelial cells (Gaffen, 2009). On the other hand, IL17RC is expressed at low levels in hematopoietic cells and at high levels in the adrenal gland, prostate, liver, and thyroid. Although cytokines secreted by most activated helper T cells generally stimulate the Janus kinase (JAK)/STAT pathway, the IL17 family cytokines stimulate signal pathways that are common in the innate immune system, such as the Toll-like receptor signaling pathway. IL17 receptors have a conserved domain termed the "similar expression to fibroblast growth factor/IL-17R (SEFIR)" domain in the cytoplasmic region. This domain is similar to the Toll-/IL-1R (TIR) domain (Gaffen, 2009). When the IL17 receptor is activated, the adaptor molecule actin related gene 1 (Act1, a U-box E3 ubiquitin ligase) is recruited to the SEFIR domain and mediates the lysine63-linked ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). Ubiquitinated TRAF6 then activates the transcriptional factor nuclear factor-kappaB (NFkappaB), various mitogen-activated protein (MAP) kinases including Erk and p38, and CCAAT/enhancer-binding proteins (C/EBP beta and C/EBP gamma).

Homology IL17A is a prototypical member of the IL17 family. This family includes six proteins, termed IL17A, IL17B, IL17C, IL17D, IL17E (also called IL25), and IL17F. Interleukin 17A to F are not homologous to any other known proteins. IL17A shows the highest homology with IL17F (55%). It is less similar to the other IL17 family members (IL17B, 29%; IL17C, 23%; IL17D, 25%; and IL17E, 17%) (Kolls and Linden, 2004).

Implicated in Various cancers Note Infiltration of IL17A-producing T cells in tumors. IL17A-producing T cells and/or IL17A expression are detected in many human tumor tissues, including ovarian, pancreatic, renal cell, prostate, gastric, and hepatocellular cancers (Zou et al., 2010; Maniati et al., 2010). Although IL17A-producing cells are not the dominant T cell subset in the tumor microenvironment, they are increased to greater extent in the tumor site than in the peripheral blood of the patients (Kryczek et al., 2009a). Anti-tumor effects. In some human tumors, such as ovarian and prostate cancer, IL17A and IL17A-producing cells are associated with antitumorigenic actions. Increased IL17A levels in ascites are well-correlated with better patient survival and lower grading stages of ovarian cancer (Kryczek et al., 2009a). An increased population of Th17 cells is also associated with lower grading stages of prostate cancer (Sfanos et al., 2008). In

addition, Immunotherapy is more effective in patients with prostate cancer that have a higher number of Th17 cells. In the mouse system, the overexpression of IL17A in tumor cells suppresses tumor growth in a cytotoxic T lymphocyte-dependent manner (Benchetrit et al., 2002). The transfer of tumor antigen-specific T cells polarized to the IL17-producing phenotype also induces the eradication of tumor cells by inducing strong CD8-positive T cell activation (Martin-Orozco et al., 2009). Furthermore, the deficiency of IL17A in mice promotes the growth and metastasis of tumors (Martin-Orozco et al., 2009; Kryczek et al., 2009b). Interleukin 17A-producing T cells are predicted to induce the recruitment of other effector cells (e.g., cytotoxic CD8-positive T cells and NK cells) to the tumors by inducing the expression of CXCL9 and CXCL10 by tumors (Kryczek et al., 2009a). Moreover, Th17 cells induce the expression of chemokine (C-C motif) ligand 20 (CCL20, a ligand for chemokine (C-C motif) receptor 6 (CCR6)) in tumor tissues. Chemokine (C-C motif) ligand 20 recruits dendritic cells to mediate anti-tumor effects in a CCL20/CCR6-depedent manner (Martin-Orozco et al., 2009). Pro-tumor effects. The proportion of Th17 cells in the peripheral blood is increased in patients with advanced stage gastric cancer compared with patients with early stage diseases (Zhang et al., 2008). In patients with hepatocellular carcinoma, increased intratumoral accumulation of IL17A-producing cells is significantly associated with a poor prognosis (Zhang et al., 2009). In the mouse system, the overexpression of IL17A in tumors facilitates tumor growth via the induction of angiogenesis in the tumor microenvironment (Numasaki et al., 2003; Numasaki et al., 2005). Furthermore, IL17A-deficient or IL17RA-deficient mouse models were used to show that IL17A was involved in the promotion of tumor growth via induction of myeloid derived suppressor cells (MDSC) (He et al., 2010), activation of IL6-STAT3 pathway (Wang et al., 2009), and production of IL17A by tumor-infiltrating gamma delta T cells (Wakita et al., 2010). The discrepancies between anti-tumor and pro-tumor effects may be due to distinct roles of IL17A and IL17A-producing cells in different tumors.

Gastric cancer Note The single nucleotide polymorphism (SNP) in the IL17A gene promoter region, which is located at a position -197 from the start codon (rs2275913, G/A SNPs, a position at 52051033 bp from pter), has been examined in Japanese gastric cancer patients (Shibata et al., 2009). The frequency of the A-allele (odds ratio, 1.42) and the A/A homozygote (odds ratio, 3.02) is significantly increased in gastric cancer patients compared with healthy controls.



Autoimmune and inflammatory diseases Note Interleukin 17A and IL17-producing cells are associated with the pathogenesis of many autoimmune and inflammatory diseases such as EAE/multiple sclerosis, inflammatory skin diseases/psoriasis, inflammatory bowel diseases, and experimental arthritis/rheumatoid arthritis in humans as well as mice (Korn et al., 2009; Awasthi and Kuchroo, 2009).

Infections Note Both IL17A and IL17F are preferentially produced during infections with the Gram-negative bacteria Klebsiella pneumonia, Borrelia burgdorferi, and Salmonella enterica enteritidis; the Gram-positive bacterium Listeria monocytogenes; the acid-fast bacterium Mycobacterium tuberculosis; and the yeast-like fungi Pneumocystis jirovecii and Candida albicans (Korn et al., 2009; O'Connor et al., 2010). In an early response to the infection, IL17A is predominantly secreted by gamma delta T cells (Roark et al., 2008; Cua et al., 2010). This results in the rapid recruitment of neutrophils to sites of infection for efficient pathogen clearance. Later, antigen-specific alphabetaTh17 cells contribute to the response.

References Hymowitz SG, Filvaroff EH, Yin JP, Lee J, Cai L, Risser P, Maruoka M, Mao W, Foster J, Kelley RF, Pan G, Gurney AL, de Vos AM, Starovasnik MA. IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding. EMBO J. 2001 Oct 1;20(19):5332-41

Benchetrit F, Ciree A, Vives V, Warnier G, Gey A, Sautès-Fridman C, Fossiez F, Haicheur N, Fridman WH, Tartour E. Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism. Blood. 2002 Mar 15;99(6):2114-21

Moseley TA, Haudenschild DR, Rose L, Reddi AH. Interleukin-17 family and IL-17 receptors. Cytokine Growth Factor Rev. 2003 Apr;14(2):155-74

Numasaki M, Fukushi J, Ono M, Narula SK, Zavodny PJ, Kudo T, Robbins PD, Tahara H, Lotze MT. Interleukin-17 promotes angiogenesis and tumor growth. Blood. 2003 Apr 1;101(7):2620-7

Kolls JK, Lindén A. Interleukin-17 family members and inflammation. Immunity. 2004 Oct;21(4):467-76

Numasaki M, Watanabe M, Suzuki T, Takahashi H, Nakamura A, McAllister F, Hishinuma T, Goto J, Lotze MT, Kolls JK, Sasaki H. IL-17 enhances the net angiogenic activity and in vivo growth of human non-small cell lung cancer in SCID mice through promoting CXCR-2-dependent angiogenesis. J Immunol. 2005 Nov 1;175(9):6177-89

Bettelli E, Korn T, Kuchroo VK. Th17: the third member of the effector T cell trilogy. Curr Opin Immunol. 2007 Dec;19(6):652-7

McGeachy MJ, Cua DJ. Th17 cell differentiation: the long and winding road. Immunity. 2008 Apr;28(4):445-53

Roark CL, Simonian PL, Fontenot AP, Born WK, O'Brien RL. gammadelta T cells: an important source of IL-17. Curr Opin Immunol. 2008 Jun;20(3):353-7

Sfanos KS, Bruno TC, Maris CH, Xu L, Thoburn CJ, DeMarzo AM, Meeker AK, Isaacs WB, Drake CG. Phenotypic analysis of prostate-infiltrating lymphocytes reveals TH17 and Treg skewing. Clin Cancer Res. 2008 Jun 1;14(11):3254-61

Shime H, Yabu M, Akazawa T, Kodama K, Matsumoto M, Seya T, Inoue N. Tumor-secreted lactic acid promotes IL-23/IL-17 proinflammatory pathway. J Immunol. 2008 Jun 1;180(11):7175-83

Stritesky GL, Yeh N, Kaplan MH. IL-23 promotes maintenance but not commitment to the Th17 lineage. J Immunol. 2008 Nov 1;181(9):5948-55

Zhang B, Rong G, Wei H, Zhang M, Bi J, Ma L, Xue X, Wei G, Liu X, Fang G. The prevalence of Th17 cells in patients with gastric cancer. Biochem Biophys Res Commun. 2008 Sep 26;374(3):533-7

Awasthi A, Kuchroo VK. Th17 cells: from precursors to players in inflammation and infection. Int Immunol. 2009 May;21(5):489-98

Chung Y, Chang SH, Martinez GJ, Yang XO, Nurieva R, Kang HS, Ma L, Watowich SS, Jetten AM, Tian Q, Dong C. Critical regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity. 2009 Apr 17;30(4):576-87

Gaffen SL. Structure and signalling in the IL-17 receptor family. Nat Rev Immunol. 2009 Aug;9(8):556-67

Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009;27:485-517

Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, Huang E, Finlayson E, Simeone D, Welling TH, Chang A, Coukos G, Liu R, Zou W. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood. 2009a Aug 6;114(6):1141-9

Kryczek I, Wei S, Szeliga W, Vatan L, Zou W. Endogenous IL-17 contributes to reduced tumor growth and metastasis. Blood. 2009b Jul 9;114(2):357-9

Martin-Orozco N, Muranski P, Chung Y, Yang XO, Yamazaki T, Lu S, Hwu P, Restifo NP, Overwijk WW, Dong C. T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity. 2009 Nov 20;31(5):787-98

McGeachy MJ, Chen Y, Tato CM, Laurence A, Joyce-Shaikh B, Blumenschein WM, McClanahan TK, O'Shea JJ, Cua DJ. The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat Immunol. 2009 Mar;10(3):314-24

Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD, Wang YH, Dong C. Bcl6 mediates the development of T follicular helper cells. Science. 2009 Aug 21;325(5943):1001-5

Shibata T, Tahara T, Hirata I, Arisawa T. Genetic polymorphism of interleukin-17A and -17F genes in gastric carcinogenesis. Hum Immunol. 2009 Jul;70(7):547-51

Wang L, Yi T, Kortylewski M, Pardoll DM, Zeng D, Yu H. IL-17 can promote tumor growth through an IL-6-Stat3 signaling pathway. J Exp Med. 2009 Jul 6;206(7):1457-64

Zhang JP, Yan J, Xu J, Pang XH, Chen MS, Li L, Wu C, Li SP, Zheng L. Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients. J Hepatol. 2009 May;50(5):980-9



Cua DJ, Tato CM. Innate IL-17-producing cells: the sentinels of the immune system. Nat Rev Immunol. 2010 Jul;10(7):479-89

Eyerich S, Eyerich K, Cavani A, Schmidt-Weber C. IL-17 and IL-22: siblings, not twins. Trends Immunol. 2010 Sep;31(9):354-61

Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O'Shea JJ. Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling. Nature. 2010 Oct 21;467(7318):967-71

He D, Li H, Yusuf N, Elmets CA, Li J, Mountz JD, Xu H. IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells. J Immunol. 2010 Mar 1;184(5):2281-8

Hirahara K, Ghoreschi K, Laurence A, Yang XP, Kanno Y, O'Shea JJ. Signal transduction pathways and transcriptional regulation in Th17 cell differentiation. Cytokine Growth Factor Rev. 2010 Dec;21(6):425-34

Maniati E, Soper R, Hagemann T. Up for Mischief? IL-17/Th17 in the tumour microenvironment. Oncogene. 2010 Oct 21;29(42):5653-62

O'Connor W Jr, Zenewicz LA, Flavell RA. The dual nature of T(H)17 cells: shifting the focus to function. Nat Immunol. 2010 Jun;11(6):471-6

Wakita D, Sumida K, Iwakura Y, Nishikawa H, Ohkuri T, Chamoto K, Kitamura H, Nishimura T. Tumor-infiltrating IL-17-producing gammadelta T cells support the progression of tumor by promoting angiogenesis. Eur J Immunol. 2010 Jul;40(7):1927-37

Zou W, Restifo NP. T(H)17 cells in tumour immunity and immunotherapy. Nat Rev Immunol. 2010 Apr;10(4):248-56

Yabu M, Shime H, Hara H, Saito T, Matsumoto M, Seya T, Akazawa T, Inoue N. IL-23-dependent and -independent enhancement pathways of IL-17A production by lactic acid. Int Immunol. 2011 Jan;23(1):29-41


Inoue N, Akazawa T. IL17A (interleukin 17A). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):662-666.





MYBBP1A (MYB binding protein (P160) 1a) Claudia Perrera, Riccardo Colombo

Department of Cell Biology-Oncology, Nerviano Medical Sciences, Viale Pasteur 10, Nerviano 20014, Italy (CP, RC)


Online updated version : http://AtlasGeneticsOncology.org/Genes/MYBBP1AID41467ch17p13.html DOI: 10.4267/2042/46000


Identity Other names: FLJ37886; P160; PAP2

HGNC (Hugo): MYBBP1A

Location: 17p13.2

DNA/RNA Note Total gene length 16491 bp, mRNA length 4122 bp, telomeric to SPNS2 and centromeric to GGT6. Two variants described. MYBBP1A gene is composed by 26 exones and 28 introns.

Description The human MYBBP1A gene is located on chromosome 17p13.3 (Keough et al., 1999).

Transcription The complete MYBBP1A cDNA is 4518 bp, including 25 bp of 5' UTR and 506 bp of 3' UTR up to the polyA tail.

Protein Description Human MYBBP1A is a 1328 aa long protein. Murine MYBBP1A was originally identified as a protein interacting with the leucine zipper of c-Myb (Favier et al., 1994). Subsequently, in 1998, the human gene homologue of MYBBP1A was cloned and its chromosomal location mapped to 17p13.3 (Keough et al., 1999).

Expression MYBBP1A is ubiquitously expressed (Tavner et al., 1998).

Localisation MYBBP1A is a nuclear protein, predominantly localized in the nucleolus (Keough et al., 2003). MYBBP1A has been confirmed as a resident protein of the nucleolus by three large-scale proteomic studies that have established a protein inventory of this sub-nuclear compartment

Schematic representation of Mybbp1A protein. aa 1-582 is the domain reported to interact in vitro with Myb. NLS: Nuclear and

nucleolar localisation signal. The indicated S, T and Y are phosphorylated residues identified in several phospho-proteomic studies.

MYBBP1A (MYB binding protein (P160) 1a) Perrera C, Colombo R


(Andersen et al., 2002; Scherl et al., 2002; Andersen et al., 2005). The nuclear/nucleolar localization signals are present in the C-terminal tail of MYBBP1A.

Function MYBBP1A functions have not yet been completely clarified. It was originally identified as a protein able to interact with the negative regulatory domain (NRD) of c-Myb; however, it was later shown to lack any significant effect in a Myb-dependent transcription reporter assay (Favier et al., 1994). MYBBP1A has been found to interact with and regulate several transcription factors: it binds and represses both Prep1-Pbx1, involved in development and organogenesis, and also PGC-1a, a key regulator of metabolic processes such as mitochondrial biogenesis and respiration and gluconeogenesis in liver (Fan et al., 2004; Diaz et al., 2007). MYBBP1A acts as a co-repressor for RelA/p65, a member of the NFkB family, by competing with the co-activator p300 histone acetyltrasferase for interaction with the transcription activation domain (TAD) of RelA/p65. It is also a co-repressor on the Period2 promoter, repressing the expression of Per2, an essential gene in the regulation of the circadian clock (Owen et al., 2007; Hara et al., 2009). Conversely, MYBBP1A is a positive regulator of the aromatic hydrocarbon receptor (AhR) which mediates transcriptional responses to certain hydrophobic ligands, such as dioxin, by enhancing the ability of AhR to activate transcription (Jones et al., 2002). MYBBP1A has also been reported to be a component of macromolecular complexes such as the B-WICH complex, a 3 MDa assembly made of proteins and RNAs, formed during active transcription (Cavellan et al., 2006) or part of large interactomes such as the SMN interactome (Fuller et al., 2010). MYBBP1A can be post-translationally processed in some type of cells to generate an amino-terminal fragments of 67 kDa (p67). Ribosomal stress induced by Actinomycin D (an inhibitor of ribosome biogenesis) treatment causes MYBBP1A processing and translocation from the nucleolus to the nucloplasm (Diaz et al., 2007; Yamauchi et al., 2008), indicating a possible MYBBP1A role in ribosome biogenesis. Several post-translational modifications have been described for MYBBP1A, even if their biological significance is not yet clarified. MYBBP1A is reported to be a heavily phosphorylated protein in cells, according to several large-scale mass spectrometry-based phosphoproteomic studies (Beausoleil et al., 2004; Beausoleil et al., 2006; Nousiainen et al., 2006; Olsen et al., 2006; Cantin et al., 2008; Daub et al., 2008; Dephoure et al., 2008; Imami et al., 2008). The majority of the phosphosites mapped in MYBBP1A in these studies (18 out of a total of 21) reside within the ~200 amino-acid long C-terminal portion of the protein, which has been shown to be relevant for its nuclear and nucleolar localization (Keough et al., 2003). Notably, MYBBP1A was also found to be also a

component of the proteome as well as the phospho-proteome of the human mitotic spindle. MYBBP1A contains several consensus motifs for several kinases, but until now, only Ser1303 has been proven in vitro and in HeLa cells to be indeed phosphorylated by Aurora B kinase (Perrera et al., 2010). In this work, it has been shown that MYBBP1A depletion by RNAi causes a delay in progression through mitosis and defects in mitotic spindle assembly and stability, indicating that, like other nucleolar proteins, MYBBP1A may have a role in insuring correct mitotic progression (Perrera et al., 2010). MYBBP1A has been reported to be also sumoylated upon MG132 treatment (Matafora et al., 2009).

Homology Orthologous genes for MYBBP1A sharing a high degree of similarity are present in rat and mice. Protein homologues have also been recognized in dog, bovine, and chicken and a MYBBP1A-like protein spanning 1269 residues and showing a 60% similarity to the human protein has been identified in zebrafish, suggesting that MYBBP1A is significantly conserved across vertebrate species (Amsterdam et al., 2004). MYBBP1A shares some homology to a yeast protein called POL5, reported to be an essential DNA polymerase in Saccharomyces cerevisiae (Yang et al., 2003).

Implicated in Various cancers Disease MYBBP1A maps at 17p13.3, a region frequently lost in many solid and haematological tumors, such as breast and ovarian cancer, medulloblastoma, astrocytoma, leukemias, etc. This indicates that this chromosomal band contains one or more tumor suppressor genes. However, MYBBP1A is unlikely a candidate for being a tumor suppressor gene, as it lies centromeric to the regions of LOH described (Keough et al., 1999).

References Favier D, Gonda TJ. Detection of proteins that bind to the leucine zipper motif of c-Myb. Oncogene. 1994 Jan;9(1):305-11

Tavner FJ, Simpson R, Tashiro S, Favier D, Jenkins NA, Gilbert DJ, Copeland NG, Macmillan EM, Lutwyche J, Keough RA, Ishii S, Gonda TJ. Molecular cloning reveals that the p160 Myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb. Mol Cell Biol. 1998 Feb;18(2):989-1002

Keough R, Woollatt E, Crawford J, Sutherland GR, Plummer S, Casey G, Gonda TJ. Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3. Genomics. 1999 Dec 15;62(3):483-9

Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M, Lamond AI. Directed proteomic analysis of the human nucleolus. Curr Biol. 2002 Jan 8;12(1):1-11

MYBBP1A (MYB binding protein (P160) 1a) Perrera C, Colombo R


Jones LC, Okino ST, Gonda TJ, Whitlock JP Jr. Myb-binding protein 1a augments AhR-dependent gene expression. J Biol Chem. 2002 Jun 21;277(25):22515-9

Scherl A, Couté Y, Déon C, Callé A, Kindbeiter K, Sanchez JC, Greco A, Hochstrasser D, Diaz JJ. Functional proteomic analysis of human nucleolus. Mol Biol Cell. 2002 Nov;13(11):4100-9

Keough RA, Macmillan EM, Lutwyche JK, Gardner JM, Tavner FJ, Jans DA, Henderson BR, Gonda TJ. Myb-binding protein 1a is a nucleocytoplasmic shuttling protein that utilizes CRM1-dependent and independent nuclear export pathways. Exp Cell Res. 2003 Sep 10;289(1):108-23

Yang W, Rogozin IB, Koonin EV. Yeast POL5 is an evolutionarily conserved regulator of rDNA transcription unrelated to any known DNA polymerases. Cell Cycle. 2003 Mar-Apr;2(2):120-2

Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N. Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12792-7

Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC, Gygi SP. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5

Fan M, Rhee J, St-Pierre J, Handschin C, Puigserver P, Lin J, Jäeger S, Erdjument-Bromage H, Tempst P, Spiegelman BM. Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1alpha: modulation by p38 MAPK. Genes Dev. 2004 Feb 1;18(3):278-89

Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M. Nucleolar proteome dynamics. Nature. 2005 Jan 6;433(7021):77-83

Beausoleil SA, Villén J, Gerber SA, Rush J, Gygi SP. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol. 2006 Oct;24(10):1285-92

Cavellán E, Asp P, Percipalle P, Farrants AK. The WSTF-SNF2h chromatin remodeling complex interacts with several nuclear proteins in transcription. J Biol Chem. 2006 Jun 16;281(24):16264-71

Nousiainen M, Silljé HH, Sauer G, Nigg EA, Körner R. Phosphoproteome analysis of the human mitotic spindle. Proc Natl Acad Sci U S A. 2006 Apr 4;103(14):5391-6

Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 2006 Nov 3;127(3):635-48

Díaz VM, Mori S, Longobardi E, Menendez G, Ferrai C, Keough RA, Bachi A, Blasi F. p160 Myb-binding protein interacts with Prep1 and inhibits its transcriptional activity. Mol Cell Biol. 2007 Nov;27(22):7981-90

Owen HR, Elser M, Cheung E, Gersbach M, Kraus WL, Hottiger MO. MYBBP1a is a novel repressor of NF-kappaB. J Mol Biol. 2007 Feb 23;366(3):725-36

Cantin GT, Yi W, Lu B, Park SK, Xu T, Lee JD, Yates JR 3rd. Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis. J Proteome Res. 2008 Mar;7(3):1346-51

Daub H, Olsen JV, Bairlein M, Gnad F, Oppermann FS, Körner R, Greff Z, Kéri G, Stemmann O, Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. Mol Cell. 2008 Aug 8;31(3):438-48

Dephoure N, Zhou C, Villén J, Beausoleil SA, Bakalarski CE, Elledge SJ, Gygi SP. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10762-7

Imami K, Sugiyama N, Kyono Y, Tomita M, Ishihama Y. Automated phosphoproteome analysis for cultured cancer cells by two-dimensional nanoLC-MS using a calcined titania/C18 biphasic column. Anal Sci. 2008 Jan;24(1):161-6

Yamauchi T, Keough RA, Gonda TJ, Ishii S. Ribosomal stress induces processing of Mybbp1a and its translocation from the nucleolus to the nucleoplasm. Genes Cells. 2008 Jan;13(1):27-39

Hara Y, Onishi Y, Oishi K, Miyazaki K, Fukamizu A, Ishida N. Molecular characterization of Mybbp1a as a co-repressor on the Period2 promoter. Nucleic Acids Res. 2009 Mar;37(4):1115-26

Matafora V, D'Amato A, Mori S, Blasi F, Bachi A. Proteomics analysis of nucleolar SUMO-1 target proteins upon proteasome inhibition. Mol Cell Proteomics. 2009 Oct;8(10):2243-55

Fuller HR, Man NT, Lam le T, Thanh le T, Keough RA, Asperger A, Gonda TJ, Morris GE. The SMN interactome includes Myb-binding protein 1a. J Proteome Res. 2010 Jan;9(1):556-63

Perrera C, Colombo R, Valsasina B, Carpinelli P, Troiani S, Modugno M, Gianellini L, Cappella P, Isacchi A, Moll J, Rusconi L. Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase. J Biol Chem. 2010 Apr 16;285(16):11775-85


Perrera C, Colombo R. MYBBP1A (MYB binding protein (P160) 1a). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):667-669.





PLCD1 (phospholipase C, delta 1) Xiaotong Hu

Biomedical Research Center, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China (XH)


Online updated version : http://AtlasGeneticsOncology.org/Genes/PLCD1ID43927ch3p22.html DOI: 10.4267/2042/46001


Identity HGNC (Hugo): PLCD1

Location: 3p22.2

Local order: The PLCD1 gene is located between the VILL gene and the DLEC1 gene.

DNA/RNA Description The PLCD1 gene is a functioning gene and contains 15 exons and spans 22.17 kb.

Transcription The variant 1 (NM_001130964) encodes the longer isoform 1 (NP_001124436). The variant 2 (NM_006225.3) contains an alternate 5' terminal exon compared to transcript variant 1, and initiates translation from an in-frame upstream AUG, resulting

in a shorter isoform 2 (NP_006216) with a different N-terminus compared to isoform 1.

Protein Description The deduced 777-amino acid isoform 1 (NM_001130964) and 756-amino acid isoform 2 (NP_006216) shares 95% sequence homology with the rat protein. They contain a N-terminal PH domain, 2 EF-hand1 domains, PI-PLC X-box, PI-PLC Y-box and C2 region.

Expression Expressed high or medium in CNS (brain), hematopoietic (blood), liver and pancreas, digestive (GI-tract), respiratory (lung), male and female tissues, placenta, urinary tract (kidney) skin and soft tissues but no expression in cardio vascular and endocrine tissues.

PLCD1 starts at 38048987 bp and ends at 38071253 bp from pter on Chr3p22-p21.3 and located between VILL and DLEC1 gene.

PLCD1 (phospholipase C, delta 1) Hu X


Closed and opened boxes represent coding and non-coding sequences of PLCD1 gene, respectively.

Protein domain organization of the mammalian PLCD1.

Localisation Intracellular: cytoplasm and nucleus.

Function Catalyzing the hydrolysis of phosphatidylinositol 4,5 biphosphate to generate diacylglycerol and inositol 1,4,5 triphosphate. Mediating a wide variety of cellular stimuli. Shuttling between the nucleus and the

cytoplasm, and nuclear import is mediated by its Ca2+-dependent interaction with importin beta 1. Playing an important suppressive role in the development and progression of cancers such as esophageal squamous cell carcinoma (ESCC) and gastric cancer (GC).

Homology The PLCD1 gene is conserved in dog, cow, mouse, rat, chicken, zebrafish, and A. thaliana.

Mutations

The mutation location is highlighted in red and this mutation occured in 17% (1/6) skin samples. AA Mutation: p.E226K (Substitution - Missense). CDS Mutation: c.676G>A (Substitution).



Implicated in Esophageal squamous cell carcinoma Prognosis Firstly, four commonly deleted regions (CDRs) at 3p26.3, 3p22, 3p21.3 and 3p14.2 were identified. Absent and down-regulated expression of several candidate TSGs, including CHL1, PCAF, RBMS3, PLCD1 and CACNA2D3, were detected in primary ESCC tumors and ESCC cell lines. These results provided evidence that minimal deleted regions at 3p26.3, 3p22, 3p21.3 and 3p14.2 containing potential TSGs may contribute to the pathogenesis of esophageal cancer. Secondly, absent expression of PLC delta 1 was detected in 26 of 50 (52%) primary ESCCs and 4 of 9 (44.4%) ESCC cell lines, which was significantly associated with DNA copy number loss and promoter hypermethylation (P < 0.05). Functional studies showed that PLC delta 1 was able to suppress both in vitro and in vivo tumorigenic ability of ESCC cells, including foci formation, colony formation in soft agar, and tumor formation in nude mice. The tumor-suppressive mechanism of PLC delta 1 was associated with its role in the cell cycle arrest at the G(1)-S checkpoint by up-regulation of p21 and down-regulation of phosphorylated Akt (Ser(473)). In addition, down-regulation of PLC delta 1 protein was significantly correlated with ESCC metastasis (P = 0.014), which was associated with its function in increasing cell adhesion and inhibiting cell mobility. These results suggest that PLC delta 1 plays an important suppressive role in the development and progression of ESCC.

Breast carcinoma Prognosis PLCD1 are more highly expressed in the transformed cell lines compared to MCF-10A. To test whether PLCd1 or PLCd3 played any role in tumor cell proliferation or cell migration. RNAi mediated knockdown of PLCD1 reduced proliferation of the MDA-MB-231 cells. Morphological changes including cell rounding, and surface blebbing and nuclear fragmentation were observed. These changes were accompanied by reductions in cell migration activities. On the other hand, PLCD1 knockdown failed to cause comparable morphological changes in the normal MCF-10A line, but did reduce cell proliferation and migration. Taken together, these data are consistent with the idea that PLCD1 support the growth and migration of normal and neoplastic mammary epithelial cells in vitro. However there is contrasted results published in another paper. Their results suggested that PLCD1 is a functional tumor suppressor inducing G(2)/M arrest and frequently methylated in breast cancer.

Gastric cancer Prognosis Located at the important tumor suppressor locus, 3p22, PLCD1 encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movements. We identified PLCD1 as a downregulated gene in aerodigestive carcinomas through expression profiling and epigenetic characterization. We found that PLCD1 was expressed in all normal adult tissues but low or silenced in 84% (16/19) gastric cancer cell lines, well correlated with its CpG island (CGI) methylation status. Methylation was further detected in 62% (61/98) gastric primary tumors, but none of normal gastric mucosa tissues. PLCD1 methylation was significantly correlated with tumor high stage. Detailed methylation analysis of 37 CpG sites at the PLCD1 CGI by bisulfite genomic sequencing confirmed its methylation. PLCD1 silencing could be reversed by pharmacological demethylation with 5-aza-2'-deoxycytidine, indicating a direct epigenetic silencing. Ectopic expression of PLCD1 in silenced gastric tumor cells dramatically inhibited their clonogenicity and migration, possibly through downregulating MMP7 expression and hampering the reorganization of cytoskeleton through cofilin inactivation by phosphorylation. Thus, epigenetic inactivation of PLCD1 is common and tumor-specific in gastric cancer, and PLCD1 acts as a functional tumor suppressor involved in gastric carcinogenesis.

Colon carcinomas Prognosis Decreased levels of the PLC delta 1 protein were seen in most colon carcinomas (12 of 13 paired samples) and PLC delta 1 protein was not detected in any of the carcinoma cell lines.

Rat colon neoplasms Prognosis The expression of PLC-delta expression in rat colon neoplasms induced by methylazoxymethanol (MAM) acetate was examined. Large-bowel neoplasms were observed in five of 10 rats given MAM acetate 40 weeks after treatment. PLC-delta expression in the neoplasms was not detected by northern blot analysis, and a low level of expression was detected by immunoblot analysis, although PLC-delta expression was apparent in the non-neoplastic colon mucosae of MAM acetate-treated rats as well as in the colon mucosae of control rats.

Insulinoma Note Insulinoma MIN6 cells.

Prognosis To study the effects of enhanced phosphoinositide



hydrolysis on insulin secretion, phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1) or PLCdelta1 was overexpressed in insulinoma MIN6 cells via adenoviral vectors. Inositol phosphate production stimulated by KCl or glucose in both PLCbeta1- and PLCdelta1-overexpressing cells were greater than that in control cells, reduced phosphatidylinositol-4,5-bisphosphate levels were observed in these cells stimulated by NaF or KCl. These data suggest that excessive phosphoinositide hydrolysis inhibits secretagogue-induced insulin release in MIN6 cells.

Pheochromocytoma Note Pheochromocytoma PC12 cells.

Prognosis PLCD1 is recruited from the cytoplasm to lipid rafts after CCH-induced Ca2+ mobilization following the activation of PLCbeta by GPCR and PLCD1 is activated only in lipid rafts by localized capacitative entry of extracellular Ca2+. PLCD1, p122RhoGAP and RhoA in combination could constitute a unique functional unit for the regulation of both phosphoinositide/Ca2+ signaling and the actin cytoskeleton in the periphery of specified membrane domains. This would provide further insights into the molecular mechanisms of cancer development.

References Yoshimi N, Wang A, Makita H, Suzui M, Mori H, Okano Y, Banno Y, Nozawa Y. Reduced expression of phospholipase C-delta, a signal-transducing enzyme, in rat colon neoplasms induced by methylazoxymethanol acetate. Mol Carcinog. 1994 Dec;11(4):192-6

Nomoto K, Tomita N, Miyake M, Xhu DB, LoGerfo PR, Weinstein IB. Expression of phospholipases gamma 1, beta 1,

and delta 1 in primary human colon carcinomas and colon carcinoma cell lines. Mol Carcinog. 1995 Mar;12(3):146-52

Ishikawa S, Takahashi T, Ogawa M, Nakamura Y. Genomic structure of the human PLCD1 (phospholipase C delta 1) locus on 3p22-->p21.3. Cytogenet Cell Genet. 1997;78(1):58-60

Ishihara H, Wada T, Kizuki N, Asano T, Yazaki Y, Kikuchi M, Oka Y. Enhanced phosphoinositide hydrolysis via overexpression of phospholipase C beta1 or delta1 inhibits stimulus-induced insulin release in insulinoma MIN6 cells. Biochem Biophys Res Commun. 1999 Jan 8;254(1):77-82

Fu L, Qin YR, Xie D, Hu L, Kwong DL, Srivastava G, Tsao SW, Guan XY. Characterization of a novel tumor-suppressor gene PLC delta 1 at 3p22 in esophageal squamous cell carcinoma. Cancer Res. 2007 Nov 15;67(22):10720-6

Qin YR, Fu L, Sham PC, Kwong DL, Zhu CL, Chu KK, Li Y, Guan XY. Single-nucleotide polymorphism-mass array reveals commonly deleted regions at 3p22 and 3p14.2 associate with poor clinical outcome in esophageal squamous cell carcinoma. Int J Cancer. 2008 Aug 15;123(4):826-30

Yamaga M, Kawai K, Kiyota M, Homma Y, Yagisawa H. Recruitment and activation of phospholipase C (PLC)-delta1 in lipid rafts by muscarinic stimulation of PC12 cells: contribution of p122RhoGAP/DLC1, a tumor-suppressing PLCdelta1 binding protein. Adv Enzyme Regul. 2008;48:41-54

Hu XT, Zhang FB, Fan YC, Shu XS, Wong AH, Zhou W, Shi QL, Tang HM, Fu L, Guan XY, Rha SY, Tao Q, He C. Phospholipase C delta 1 is a novel 3p22.3 tumor suppressor involved in cytoskeleton organization, with its epigenetic silencing correlated with high-stage gastric cancer. Oncogene. 2009 Jul 2;28(26):2466-75

Rebecchi MJ, Raghubir A, Scarlata S, Hartenstine MJ, Brown T, Stallings JD. Expression and function of phospholipase C in breast carcinoma. Adv Enzyme Regul. 2009;49(1):59-73

Xiang T, Li L, Fan Y, Jiang Y, Ying Y, Putti TC, Tao Q, Ren G. PLCD1 is a functional tumor suppressor inducing G(2)/M arrest and frequently methylated in breast cancer. Cancer Biol Ther. 2010 Sep;10(5):520-7


Hu X. PLCD1 (phospholipase C, delta 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):670-673.





PYY (peptide YY) Maria Braoudaki, Fotini Tzortzatou-Stathopoulou

University Research Institute for the Study and Treatment of Childhood Genetic and Malignant Diseases, University of Athens, "Aghia Sophia" Children's Hospital, Athens, Greece (MB), Hematology/Oncology Unit, First Department of Pediatrics, University of Athens, "Aghia Sophia" Children's Hospital, Athens, Greece (FTS)


Online updated version : http://AtlasGeneticsOncology.org/Genes/PYYID46182ch17q21.html DOI: 10.4267/2042/46002


Identity Other names: PYY1

HGNC (Hugo): PYY

Location: 17q21.31

DNA/RNA Description PYY gene is composed of 4 exons and 3 introns that span approximately 51732 bases (start 42030106 bp to end 42081837 bp from pter) oriented at the minus strand.

Transcription Two transcript variants (1048 bp and 1048 bp in length).

Protein Description Size: 97 amino acids; 11046 Da.

Expression PYY is expressed predominantly in endocrine L-cells that line the distal small bowel and colon.

Localisation Extracellular, subcellular location: secreted granules. Co-localized with proglucagon products, glicentin and glucagon-like peptide-1 (GLP-1) and GLP-2. PYY is a gastrointestinal track-derived hormone synthesized by endocrine cells of terminal ileum and colon, involved in the regulation of food intake.

Function Enteroendocrine L-cells release two circulating forms of PYY in the distal gut: PYY1-36 and PYY3-36. The latter form is considered the predominant form in both fasted and fed states and is produced by the cleavage of the N-terminal Tyr-Pro residues from PYY1-36 by dipeptidyl-peptidase IV (DPPIV).

Human peptide YY (PYY). Adapted from Shih et al., 2009.

PYY (peptide YY) Braoudaki M, Tzortzatou-Stathopoulou F


PYY exerts its inhibitory actions via various Y receptors, including Y1 receptor-mediated epithelial responses and Y2 receptor-mediated neuronal effects. It inhibits food intake via NPY-2 receptors expressed by neurons of the arcuate nucleus of the hypothalamus. Generally, it is considered to act in the hypothalamus as a signal of satiety. Other inhibitory actions include slowing gastric emptying; increasing nutrient absorption, inducing intestinal anion and electrolytic secretion as well as slowing small intestine motility. In addition, it has been shown to decrease exocrine pancreatic secretion and act as an appetite suppressant in the fasting state at physiological concentrations.

Homology PPY or PNP or PP and NPY.

Mutations Note Common polymorphisms: Arg72Thr, which has been associated with type-2 diabetes and in some cases with enhanced body mass. Other variants: Gln62Pro and Leu73Pro associated with body mass and obesity, respectively as well as A-23G,C888T and 3' UTR variant C+1134A. The latter has been related to enhanced body mass.

Implicated in Colon cancer Note Loss of PYY expression has been implicated in the development and progression to colon adenocarcinoma. PYY expression has been associated with elevated differentiation, whilst PYY treatment of colon cancers resulted in selected overexpression of enzymes frequently identified in the normal colonocytic phenotype. The colon cancer growth regulatory effects of PYY might be dose dependent.

Pancreatic cancer and pancreatitis Note PYY suppresses growth and levels of intracellular cyclic adenosine monophosphate (cAMP) in pancreatic adenocarcinoma. It is considered to have a therapeutic value for pancreatic cancer and pancreatitis, since it exerts its immune function by altering transcription factors vital for cell signaling pathways. In addition, administration of PYY has been shown to improve amylase and cytokine release in pancreatitis cases. It has also been suggested that PYY in combination with vitamin E exhibit a significantly increased inhibitory effect on pancreatic cancer in vitro.

Breast cancer Note PYY inhibits in vitro growth of breast cancer cells, however the exact mechanism of antitumor activity

remains unknown. Previous studies have proposed that PYY reduces intracellular levels of cAMP in breast carcinoma cells. Moreover, it has been reported that combination of PYY with vitamin E results in a significant additive inhibition of breast carcinoma cells.

Cancer cachexia Note Cancer cachexia is generally characterized by decreased protein synthesis and loss in the small bowel. PYY has been shown to increase small bowel weight and protein content. However, the exact role of PYY on cancer cachexia has not yet been clarified.

Body weight Note PYY-36 plays a role in long-term body weight regulation, due to the negative correlation between PYY concentrations and adiposity markers in humans, such that PYY levels increase with weight loss and when leptin levels are low.

Obesity and type II diabetes Note Low endogenous PYY levels in obese individuals, have previously suggested that PYY deficiency may contribute to hyperinsulinemia and insulin resistance and predispose obesity and type II diabetes.

References Conn MP, Melmed S.. Endocrinology: Basic and clinical principles. Humana Press. 1997.

Grise KR, Rongione AJ, Laird EC, McFadden DW.. Peptide YY inhibits growth of human breast cancer in vitro and in vivo. J Surg Res. 1999 Apr;82(2):151-5.

Heisler T, Towfigh S, Simon N, Liu C, McFadden DW.. Peptide YY augments gross inhibition by vitamin E succinate of human pancreatic cancer cell growth. J Surg Res. 2000a Jan;88(1):23-5.

Heisler T, Towfigh S, Simon N, McFadden DW.. Peptide YY and vitamin E inhibit hormone-sensitive and -insensitive breast cancer cells. J Surg Res. 2000b Jun 1;91(1):9-14.

Tseng WW, Liu CD.. Peptide YY and cancer: current findings and potential clinical applications. Peptides. 2002 Feb;23(2):389-95. (REVIEW)

Vona-Davis L, Yu A, Magabo K, Evans T, Jackson B, Riggs D, McFadden D.. Peptide YY attenuates transcription factor activity in tumor necrosis factor-alpha-induced pancreatitis. J Am Coll Surg. 2004 Jul;199(1):87-95.

Torekov SS, Larsen LH, Glumer C, Borch-Johnsen K, Jorgensen T, Holst JJ, Madsen OD, Hansen T, Pedersen O.. Evidence of an association between the Arg72 allele of the peptide YY and increased risk of type 2 diabetes. Diabetes. 2005 Jul;54(7):2261-5.

Ahituv N, Kavaslar N, Schackwitz W, Ustaszewska A, Collier JM, Hebert S, Doelle H, Dent R, Pennacchio LA, McPherson R.. A PYY Q62P variant linked to human obesity. Hum Mol Genet. 2006 Feb 1;15(3):387-91. Epub 2005 Dec 20.

Boey D, Heilbronn L, Sainsbury A, Laybutt R, Kriketos A, Herzog H, Campbell LV.. Low serum PYY is linked to insulin

PYY (peptide YY) Braoudaki M, Tzortzatou-Stathopoulou F


resistance in first-degree relatives of subjects with type 2 diabetes. Neuropeptides. 2006 Oct;40(5):317-24. Epub 2006 Oct 12.

Pfluger PT, Kampe J, Castaneda TR, Vahl T, D'Alessio DA, Kruthaupt T, Benoit SC, Cuntz U, Rochlitz HJ, Moehlig M, Pfeiffer AF, Koebnick C, Weickert MO, Otto B, Spranger J, Tschop MH.. Effect of human body weight changes on circulating levels of peptide YY and peptide YY3-36. J Clin Endocrinol Metab. 2007 Feb;92(2):583-8. Epub 2006 Nov 21.

Vona-Davis L, McFadden DW.. PYY and the pancreas: inhibition of tumor growth and inflammation. Peptides. 2007 Feb;28(2):334-8. Epub 2006 Dec 27. (REVIEW)

Cox HM.. Endogenous PYY and NPY mediate tonic Y1- and Y2-mediated absorption in human and mouse colon. Nutrition. 2008 Sep;24(9):900-6. Epub 2008 Jul 26.

Moschovi M, Trimis G, Vounatsou M, Katsibardi K, Margeli A, Dimitriadi F, Papassotiriou I, Chrousos G, Tzortzatou-Stathopoulou F.. Serial plasma concentrations of PYY and ghrelin during chemotherapy in children with acute lymphoblastic leukemia. J Pediatr Hematol Oncol. 2008 Oct;30(10):733-7.

Lomenick JP, Melguizo MS, Mitchell SL, Summar ML, Anderson JW.. Effects of meals high in carbohydrate, protein,

and fat on ghrelin and peptide YY secretion in prepubertal children. J Clin Endocrinol Metab. 2009 Nov;94(11):4463-71. Epub 2009 Oct 9.

Shih PA, Wang L, Chiron S, Wen G, Nievergelt C, Mahata M, Khandrika S, Rao F, Fung MM, Mahata SK, Hamilton BA, O'Connor DT.. Peptide YY (PYY) gene polymorphisms in the 3'-untranslated and proximal promoter regions regulate cellular gene expression and PYY secretion and metabolic syndrome traits in vivo. J Clin Endocrinol Metab. 2009 Nov;94(11):4557-66. Epub 2009 Oct 9.

Cox HM, Tough IR, Woolston AM, Zhang L, Nguyen AD, Sainsbury A, Herzog H.. Peptide YY is critical for acylethanolamine receptor Gpr119-induced activation of gastrointestinal mucosal responses. Cell Metab. 2010 Jun 9;11(6):532-42.

Kirchner H, Tong J, Tschop MH, Pfluger PT.. Ghrelin and PYY in the regulation of energy balance and metabolism: lessons from mouse mutants. Am J Physiol Endocrinol Metab. 2010 May;298(5):E909-19. Epub 2010 Feb 23. (REVIEW)


Braoudaki M, Tzortzatou-Stathopoulou F. PYY (peptide YY). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):674-676.

Gene Section Review




SIAH2 (seven in absentia homolog 2 (Drosophila)) Jianfei Qi, Ze'ev Ronai

Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, CA, 92037, USA (JQ, ZR)


Online updated version : http://AtlasGeneticsOncology.org/Genes/SIAH2ID46199ch3q25.html DOI: 10.4267/2042/46003


Identity Other names: hSiah2

HGNC (Hugo): SIAH2

Location: 3q25.1

DNA/RNA Description The human Siah2 gene is composed of 2 exons spanning a genomic region of about 22.4 kb.

Transcription The transcript length of human Siah2 is 2632 bp. The open reading frame of the coding region is 975 bp.

Pseudogene No pseudogene of Siah2 has been reported.

Protein Description Human Siah2 protein consists of 324 amino acids, with a molecular weight of 36 kDa. Siah protein consists of

an N-terminal ring domain, followed by two zinc finger motifs, and a C-terminal substrate binding domain (SBD). The ring domain is the catalytic domain that recruits E2 ubiquitin-conjugating enzymes, while the SBD mediates the binding of adaptor proteins or some Siah substrate proteins. The structure of murine Siah1a SBD has been solved. The structure reveals that Siah is a dimeric protein, and the SBD adopts an eight-stranded beta-sandwich fold (Polekhina et al., 2001). The substrate binding groove is formed by the beta-sandwich fold and the beta-strand that connects to the second zinc finger domain (House et al., 2005).

Expression Siah2 mRNA is widely expressed in the embryonic and adult mouse tissues. It is expressed at a higher level in the olfactory epithelium, retina, forebrain and proliferating cartilage of developing bone (Della et al., 1993). Siah2 mRNA is also expressed in most human tissues (Hu et al., 1997).

Localisation Siah protein can be localized in both cytoplasm and nucleus.

Genomic organization of human Siah2. The line indicates intron and boxes indicate coding regions (exon 1-2) of the gene. Exon and intron lengths, the ATG transcription start site and the TGA stop codon are indicated.

SIAH2 (seven in absentia homolog 2 (Drosophila)) Qi J, Ronai Z


Domains of human Siah2 protein.

Function Siah2 is the mammalian homolog of Drosophila SINA (seven in absentia), which interacts with transcriptional repressor Tramtrack via adaptor protein PHYL (Phyllopod) and induces the proteasomal degradation of Tramtrack, thereby determining R7 cell fate (Li et al., 1997; Tang et al., 1997). As a ring-finger E3 ubiquitin ligase, Siah targets the degradation of diverse substrates via ubiquitin-proteasome pathway, and affects multiple signaling pathways such as HIF (Nakayama et al., 2004), Ras (Nadeau et al., 2007; Schmidt et al., 2007), NF-kB (Polekhina et al., 2002; Habelhah et al., 2002), and beta-catenin (Liu et al., 2001; Matsuzawa and Reed, 2001). Siah2 transcription is upregulated by hypoxia (Nakayama et al., 2004); p38-mediated phosphorylation of mouse Siah2 on Thr24 and Ser29 alters its subcellular localization (Khurana et al., 2006); HIPK2-mediated phosphorylation of human Siah2 on Thr 26, Ser 28 and Ser 68 decreases the stability of Siah2 and impairs its interaction with HIPK2 (Calzado et al., 2009). Over 20 Siah substrates have been reported (Nakayama et al., 2009) and some of them can be degradated by Siah2, Siah1 or both of them. In contrast to Siah1a knockout mice which exibit growth retardation and spermatogenesis defect, Siah2 knockout mice display no apparent phenotype, whereas Siah2 and Siah1a double knockout mice are embryonic or neonatal lethal, suggesting that the two Siah homologs have both overlapping and distinct functions in vivo (Frew et al., 2003). Despite the diverse substrates of Siah identified in vitro, loss of Siah2 (or both Siah2 and Siah1a) in vivo largely has no effect on the levels of many Siah substrates and the physiological processes associated with these substrates (Frew et al., 2002; Frew et al., 2003). Siah2 is implicated in the regulation of hypoxia response through its effect on HIF prolyl hydroxylases or HIPK2 (Nakayama et al., 2004; Calzado et al., 2009). Siah2 knockout mice subject to hypoxia showed impaired respiratory response and defect to adjust levels of red blood cells (Nakayama et al., 2004). Siah2 has been shown to be required for development and progression of several types of cancers via its regulation of HIF or Ras pathways (House et al., 2009). Siah2-dependent degradation of Pard3A is found to control germinal zone exit of neuronal progenitors or immature neurons in mice (Famulski et al., 2010).

Homology Homologs: Human has two Siah genes (Siah1 and Siah2) (Hu et al., 1997), while mouse has three Siah genes (Siah2, Siah1a, Siah1b) (Della et al., 1993). Human Siah2 shares 77% identity with human Siah1 (Hu et al., 1997). Orthologs: Highly conserved Siah2 orthologs have been identified in all multicellular organisms examined (Nakayama et al., 2009).

Mutations Note No SIAH2 mutations have been reported.

Implicated in Lung cancer Note Ahmed et al. showed that Siah2 knockdown in human lung cancer cell lines (BZR, A549, H727, and UMC11) inhibited MAPK-ERK signaling, reduced cell proliferation and increased apoptosis; Siah2 knockdown also reduced anchorage-independent growth of A549 cells in soft agar, and blocked the growth of A549 xenograft tumors in nude mice (Ahmed et al., 2007).

Melanoma Note Qi et al. showed that inhibition of Siah2 activity using different inhibitory proteins blocked tumor formation or metastasis of SW1 melanoma cells in a syngeneic mouse model due to the inhibition of Ras and HIF pathways, respectively (Qi et al., 2008). Similary, Shah et al. showed that a putative chemical inhibitor of Siah2, menadione, decreased the levels of HIF-1alpha and phospho-ERK in human melanoma cell line UACC903 and abolished the growth of xenograft tumor in nude mice (Shah et al., 2009).

Breast cancer Note Möller et al. showed that inhibition of Siah in a mouse breast cancer cell line reduced the xenograft tumor growth and prolonged the survival of mice due to inhibition of HIF pathway (Möller et al., 2009). Behling et al. examined the SIAH staining in 65



patients of ductal carcinoma in situ (DCIS). Higher level of Siah staining was observed in tumors compared with the normal adjacent tissues, and in tumors with more aggressive features. There was also higher Siah staining in specimens from patients with recurrence as compared to patients without recurrence. This study stuggests that Siah may serve as a prognostic biomarker that predicts DCIS progression to invasive breast cancer (Behling et al., 2010).

Pancreatic cancer Note Schmidt et al. showed that inhibition of Siah activity attenuated MAPK-ERK signaling, blocked RAS-induced focus formation in fibroblasts, abolished anchorage-independent growth of human pancreatic cancer cells in soft agar and xenograft tumor growth in nude mice (Schmidt et al., 2008).

Prostate cancer Note Qi et al. showed that knockout of Siah2 in the TRAMP model abolished the formation of prostate neuroendocrine tumor, inhibition of Siah2 activity blocked hypoxia-induced neuroendocrine differentiation (NED) in prostate cancer cells or in the xenogaft tumors, and Siah2 protein levels were higher in high-grade PCa that expresss NE markers. This study suggests that Siah2 plays a key role in development of prostate NE tumor and NED of human PCa by controling a cooperation between HIF and NE-specific transcription factor FoxA2 (Qi et al., 2010).

Breakpoints Note There is no breakpoint reported.

References Hu G, Chung YL, Glover T, Valentine V, Look AT, Fearon ER. Characterization of human homologs of the Drosophila seven in absentia (sina) gene. Genomics. 1997 Nov 15;46(1):103-11

Li S, Li Y, Carthew RW, Lai ZC. Photoreceptor cell differentiation requires regulated proteolysis of the transcriptional repressor Tramtrack. Cell. 1997 Aug 8;90(3):469-78

Tang AH, Neufeld TP, Kwan E, Rubin GM. PHYL acts to down-regulate TTK88, a transcriptional repressor of neuronal cell fates, by a SINA-dependent mechanism. Cell. 1997 Aug 8;90(3):459-67

Liu J, Stevens J, Rote CA, Yost HJ, Hu Y, Neufeld KL, White RL, Matsunami N. Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein. Mol Cell. 2001 May;7(5):927-36

Matsuzawa SI, Reed JC. Siah-1, SIP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 responses. Mol Cell. 2001 May;7(5):915-26

Frew IJ, Dickins RA, Cuddihy AR, Del Rosario M, Reinhard C, O'Connell MJ, Bowtell DD. Normal p53 function in primary cells

deficient for Siah genes. Mol Cell Biol. 2002 Dec;22(23):8155-64

Habelhah H, Frew IJ, Laine A, Janes PW, Relaix F, Sassoon D, Bowtell DD, Ronai Z. Stress-induced decrease in TRAF2 stability is mediated by Siah2. EMBO J. 2002 Nov 1;21(21):5756-65

Polekhina G, House CM, Traficante N, Mackay JP, Relaix F, Sassoon DA, Parker MW, Bowtell DD. Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling. Nat Struct Biol. 2002 Jan;9(1):68-75

Frew IJ, Hammond VE, Dickins RA, Quinn JM, Walkley CR, Sims NA, Schnall R, Della NG, Holloway AJ, Digby MR, Janes PW, Tarlinton DM, Purton LE, Gillespie MT, Bowtell DD. Generation and analysis of Siah2 mutant mice. Mol Cell Biol. 2003 Dec;23(24):9150-61

Nakayama K, Frew IJ, Hagensen M, Skals M, Habelhah H, Bhoumik A, Kadoya T, Erdjument-Bromage H, Tempst P, Frappell PB, Bowtell DD, Ronai Z. Siah2 regulates stability of prolyl-hydroxylases, controls HIF1alpha abundance, and modulates physiological responses to hypoxia. Cell. 2004 Jun 25;117(7):941-52

House CM, Hancock NC, Möller A, Cromer BA, Fedorov V, Bowtell DD, Parker MW, Polekhina G. Elucidation of the substrate binding site of Siah ubiquitin ligase. Structure. 2006 Apr;14(4):695-701

Khurana A, Nakayama K, Williams S, Davis RJ, Mustelin T, Ronai Z. Regulation of the ring finger E3 ligase Siah2 by p38 MAPK. J Biol Chem. 2006 Nov 17;281(46):35316-26

Nadeau RJ, Toher JL, Yang X, Kovalenko D, Friesel R. Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2. J Cell Biochem. 2007 Jan 1;100(1):151-60

Schmidt RL, Park CH, Ahmed AU, Gundelach JH, Reed NR, Cheng S, Knudsen BE, Tang AH. Inhibition of RAS-mediated transformation and tumorigenesis by targeting the downstream E3 ubiquitin ligase seven in absentia homologue. Cancer Res. 2007 Dec 15;67(24):11798-810

Ahmed AU, Schmidt RL, Park CH, Reed NR, Hesse SE, Thomas CF, Molina JR, Deschamps C, Yang P, Aubry MC, Tang AH. Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth. J Natl Cancer Inst. 2008 Nov 19;100(22):1606-29

Qi J, Nakayama K, Gaitonde S, Goydos JS, Krajewski S, Eroshkin A, Bar-Sagi D, Bowtell D, Ronai Z. The ubiquitin ligase Siah2 regulates tumorigenesis and metastasis by HIF-dependent and -independent pathways. Proc Natl Acad Sci U S A. 2008 Oct 28;105(43):16713-8

House CM, Möller A, Bowtell DD. Siah proteins: novel drug targets in the Ras and hypoxia pathways. Cancer Res. 2009 Dec 1;69(23):8835-8

Calzado MA, de la Vega L, Möller A, Bowtell DD, Schmitz ML. An inducible autoregulatory loop between HIPK2 and Siah2 at the apex of the hypoxic response. Nat Cell Biol. 2009 Jan;11(1):85-91

Möller A, House CM, Wong CS, Scanlon DB, Liu MC, Ronai Z, Bowtell DD. Inhibition of Siah ubiquitin ligase function. Oncogene. 2009 Jan 15;28(2):289-96

Nakayama K, Qi J, Ronai Z. The ubiquitin ligase Siah2 and the hypoxia response. Mol Cancer Res. 2009 Apr;7(4):443-51

Shah M, Stebbins JL, Dewing A, Qi J, Pellecchia M, Ronai ZA. Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis. Pigment Cell Melanoma Res. 2009 Dec;22(6):799-808



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Gene Section Review




TP53BP2 (tumor protein p53 binding protein, 2) Kathryn Van Hook, Zhiping Wang, Charles Lopez

Department of Medicine, Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR, USA (KV, ZW, CL)


Online updated version : http://AtlasGeneticsOncology.org/Genes/TP53BP2ID42667ch1q42.html DOI: 10.4267/2042/46004


Identity Other names: 53BP2; ASPP2; BBP; P53BP2; PPP1R13A

HGNC (Hugo): TP53BP2

Location: 1q41

DNA/RNA Description The TP53BP2 gene spans about 66 kb on chromosome 1q42.1 on the minus strand (Yang et al., 1997). There are two transcripts as a result of alternative splicing (Takahashi et al., 2004). The transcript variant 1, which is shorter (4670 bp), does not contain exon 3 and gives rise to a longer form of the protein named TP53BPL (long) or ASPP2. The transcript variant 2, which is longer (4802 bp), contains exon 3 which harbors a stop codon. As a result, the transcription initiates at exon 6 giving rise to a shorter form of the protein named TP53BPS (short) or BBP.

Transcription ASPP2 is a serum inducible protein and subject to transcriptional regulation by E2F and its family members (Chen et al., 2005; Fogal et al., 2005).

Pseudogene Not known.

Protein Description ASPP2 is a pro-apoptotic protein with a predicted size of approximately 135 kDa. It is the founding member of a family of ASPP proteins that all share the common motifs of four Ankyrin-repeats, a Src-homology 3 (SH3) domain, and a Polyproline domain in their C-terminus (Iwabuchi et al., 1994). The N-terminus of ASPP2 is thought to be important for regulating its apoptotic function and contains a putative Ras-association domain as well as a ubiquitin-like fold (Tidow et al., 2007). ASPP2 has been most widely studied for its ability to interact with and stimulate the apoptotic function of the tumor suppressor p53 (and p63/p73) but several studies have also demonstrated p53-independent as well as apoptosis-independent functions for ASPP2 as well (Kampa et al., 2009a). ASPP2 was originally pulled out of a yeast two-hybrid screen using the p53-binding domain as bait as a partial C-terminal clone named 53BP2 (Iwabuchi et al., 1994).

TSS=transcription start site.

TP53BP2 (tumor protein p53 binding protein, 2) Van Hook K, et al.


ASPP2 protein domains. RA=Ras-association domain; PP=polyproline domain; AR=ankyrin repeats.

In 1996, Naumovski and Cleary determined that 53BP2 was a partial clone of a longer transcript they named Bcl-2 binding protein (Bbp or Bbp/53BP2) for its ability to bind the anti-apoptotic protein Bcl-2. It was later determined that Bbp is a splice isoform of the full length gene product from this locus, ASPP2 (Samuels-Lev et al., 2001).

Expression Northern blot analysis, using a C-terminal probe, shows elevated levels of ASPP2 mRNA in several human tissues including heart, testis, and peripheral blood leukocytes (Yang et al., 1999). ASPP2 protein levels are controlled by proteasomal degradation (Zhu et al., 2005).

Localisation ASPP2 contains a nuclear localization signal within its ankyrin repeat domain (amino acid residues 795-894) that when expressed alone or as a fusion with other proteins localizes in the nucleus of cells (Sachdev et al., 1998; Yang et al., 1999). Despite this signal however, full length ASPP2 is predominantly located in the cytoplasm and often seen near the cell periphery (Naumovski and Cleary, 1996; Iwabuchi et al., 1998; Yang et al., 1999).

Function Apoptosis. Before ASPP2 was known to be the full length gene product from the TP53BP2 locus, Yang and colleagues showed that overexpression of Bbp/53BP2 in cells induces apoptosis (Yang et al., 1999). In 2000, Lopez et al. demonstrated that Bbp/53BP2 was UV-damage inducible and that loss of this endogenous protein promotes cell survival in response to damage, thus implicating a function in the damage response pathway. In 2001, Samuels-Lev et al. provided evidence that not only does full length ASPP2 promote apoptosis but that it does so, at least in part, through a p53-mediated mechanism that may involve preferential binding of p53 to its apoptotic target genes. ASPP2 has also been shown to modulate the apoptotic activity of the p53 family members, p63/p73 (Bergamaschi et al., 2004), and is known to bind other proteins involved in apoptosis such as Bcl-2 and NF-kappaB (Naumovski and Cleary, 1996; Yang et al., 1999). However, the functional ramifications of these interactions remain unclear. Additionally, there is

evidence to indicate ASPP2 as a player in mitochondrial-mediated apoptosis (Kobayashi et al., 2005). Tumor suppressor. Several clinical studies demonstrate low ASPP2 expression in a variety of human tumors (breast, lung, lymphoma) and this low expression often correlates with poor clinical outcome, suggesting that ASPP2 may function as a tumor suppressor (Mori et al., 2000; Samuels-Lev et al., 2001; Lossos et al., 2002; Cobleigh et al., 2005). In support of this concept, Iwabuchi et al. demonstrated in 1998 that transfection of 53BP2 inhibits Ras/E1A-mediated transformation in rat embryonic fibroblasts. Since then two separate mouse models targeting the ASPP2 locus via homologous recombination have demonstrated that loss of only one copy of ASPP2 increases spontaneous and irradiation-induced tumor formation in vivo (Vives et al., 2006; Kampa et al., 2009b). Taken together these data strongly suggest that ASPP2 is a haplo-insufficient tumor suppressor. Cell cycle. Bbp, a splice isoform of ASPP2, can induce accumulation of cells in G2/M and thus impede cell cycle progression (Naumovski and Cleary, 1996). Additionally, ASPP2 appears to play a role in the G0/G1 cell cycle checkpoint in response to gamma-irradiation as murine thymocytes that lack one copy of the ASPP2 locus did not arrest at G0/G1 as efficiently as wild type thymocytes (Kampa et al., 2009b). Cell polarity. ASPP2 is often seen near the cell periphery and has been shown to co-localize with and bind to the tight junction protein PAR-3. Furthermore, loss of ASPP2 expression correlates with a loss of tight junction integrity and an impaired ability to maintain apical domains in polarized cells in culture (Cong et al., 2010). Interestingly these findings hold true in vivo as well. ASPP2 co-localizes with the PAR-3 complex and apical junctions in the brain and is necessary for tight junction integrity. Targeted deletion of ASPP2 in the mouse leads to defects associated with a loss of structural organization in the brain and retina (Sottocornola et al., 2010). Senescence. Senescence, a type of irreversible cell cycle arrest, is considered an intrinsic protective response against malignant transformation. Wang et al. recently identified ASPP2 as a mediator of Ras-induced senescence by demonstrating that mouse embryonic fibroblasts with a targeted deletion of



Potential functions and putative interacting partners of ASPP2. Modified from Kampa et al., 2009a.

exon 3 of the ASPP2 gene (TP53BP2) are less prone to senescence in the presence of activated Ras as compared to wild type fibroblasts (as measured by beta-galactosidase staining). Data also suggests that Ras-induced senescence may be mediated by ASPP2 through its ability to inhibit Ras from inducing accumulation of cyclin D1 in the nucleus (Wang et al., 2011).

Homology ASPP2 is a member of the ASPP family of proteins that share a significant amount of homology in their C-terminal domains. ASPP1, ASPP2, and the splice isoform of ASPP2, BBP, share homology in both their N-terminal and C-terminal domains while the family member iASPP only retains C-terminal homology (Samuels-Lev et al., 2001; Bergamaschi et al., 2003).

Mutations Note No mutations at the ASPP2 locus, TP53BP2, have been reported. However, single nucleotide polymorphisms in

TP53BP2 have been found associated with gastric cancer susceptibility (Ju et al., 2005) and epigenetic silencing of the promoter by methylation is frequently observed (Sarraf and Stancheva, 2004; Liu et al., 2005; Zhao et al., 2010).

Implicated in Breast cancer Note ASPP2 mRNA expression is frequently downregulated in human breast cancer samples as compared to adjacent normal tissue (Sgroi et al., 1999; Samuels-Lev et al., 2001; Cobleigh et al., 2005). Reduced levels of ASPP2 expression are seen in both invasive and metastatic breast tumor tissue (Sgroi et al., 1999) and ASPP2 downregulation may be favored in tumor cells expressing wild type but not mutant p53 (Samuels-Lev et al., 2001).

Prognosis Elevated levels of ASPP2 mRNA were correlated with a lower risk of distant recurrence of disease among a



panel of 78 patients with extensive lymph node involvement (Cobleigh et al., 2005).

Non-Hodgkin's lymphoma specifically diffuse large B-cell lymphoma, follicular center lymphoma, and Burkitt's lymphoma Note Overall, ASPP2 expression (as measured by Real-time RT-PCR) was found to be significantly higher in diffuse large B-cell lymphoma as compared to follicular center lymphoma. However, the variability of ASPP2 expression in diffuse large B-cell lymphoma was much greater than that seen in follicular center lymphoma. ASPP2 expression appeared inversely proportional to serum lactate dehydrogenase levels. Additionally, levels of ASPP2 expression are extremely low or undetectable in cell lines derived from Burkitt's lymphoma (Lossos et al., 2002).

Prognosis In general, patients with high ASPP2 expression tended to have a longer median survival than those with low ASPP2 expression (Lossos et al., 2002).

Gastric cancer Note Four single nucleotide polymorphisms within the ASPP2 gene locus, TP53BP2, show significant correlation with gastric cancer susceptibility (Ju et al., 2005).

Hepatitis B virus-positive hepatocellular carcinoma Note Downregulation of ASPP2 (and ASPP1) as a result of promoter hypermethylation (as measured by methylation-specific PCR) is frequently observed in human patient samples of HBV-positive hepatocellular carcinoma as compared to surrounding non-tumor tissue (Zhao et al., 2010).

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p53-binding proteins, 53BP1 and 53BP2. J Biol Chem. 1998 Oct 2;273(40):26061-8

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Mori T, Okamoto H, Takahashi N, Ueda R, Okamoto T. Aberrant overexpression of 53BP2 mRNA in lung cancer cell lines. FEBS Lett. 2000 Jan 14;465(2-3):124-8

Nakagawa H, Koyama K, Murata Y, Morito M, Akiyama T, Nakamura Y. APCL, a central nervous system-specific homologue of adenomatous polyposis coli tumor suppressor, binds to p53-binding protein 2 and translocates it to the perinucleus. Cancer Res. 2000 Jan 1;60(1):101-5

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Samuels-Lev Y, O'Connor DJ, Bergamaschi D, Trigiante G, Hsieh JK, Zhong S, Campargue I, Naumovski L, Crook T, Lu X. ASPP proteins specifically stimulate the apoptotic function of p53. Mol Cell. 2001 Oct;8(4):781-94

Lossos IS, Natkunam Y, Levy R, Lopez CD. Apoptosis stimulating protein of p53 (ASPP2) expression differs in diffuse large B-cell and follicular center lymphoma: correlation with clinical outcome. Leuk Lymphoma. 2002 Dec;43(12):2309-17

Bergamaschi D, Samuels Y, O'Neil NJ, Trigiante G, Crook T, Hsieh JK, O'Connor DJ, Zhong S, Campargue I, Tomlinson ML, Kuwabara PE, Lu X. iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nat Genet. 2003 Feb;33(2):162-7

Chen Y, Liu W, Naumovski L, Neve RL. ASPP2 inhibits APP-BP1-mediated NEDD8 conjugation to cullin-1 and decreases APP-BP1-induced cell proliferation and neuronal apoptosis. J Neurochem. 2003 May;85(3):801-9

Bergamaschi D, Samuels Y, Jin B, Duraisingham S, Crook T, Lu X. ASPP1 and ASPP2: common activators of p53 family members. Mol Cell Biol. 2004 Feb;24(3):1341-50

Cao Y, Hamada T, Matsui T, Date T, Iwabuchi K. Hepatitis C virus core protein interacts with p53-binding protein, 53BP2/Bbp/ASPP2, and inhibits p53-mediated apoptosis. Biochem Biophys Res Commun. 2004 Mar 19;315(4):788-95

Meek SE, Lane WS, Piwnica-Worms H. Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins. J Biol Chem. 2004 Jul 30;279(31):32046-54

Sarraf SA, Stancheva I. Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol Cell. 2004 Aug 27;15(4):595-605

Takahashi N, Kobayashi S, Jiang X, Kitagori K, Imai K, Hibi Y, Okamoto T. Expression of 53BP2 and ASPP2 proteins from



TP53BP2 gene by alternative splicing. Biochem Biophys Res Commun. 2004 Mar 5;315(2):434-8

Chen D, Padiernos E, Ding F, Lossos IS, Lopez CD. Apoptosis-stimulating protein of p53-2 (ASPP2/53BP2L) is an E2F target gene. Cell Death Differ. 2005 Apr;12(4):358-68

Cobleigh MA, Tabesh B, Bitterman P, Baker J, Cronin M, Liu ML, Borchik R, Mosquera JM, Walker MG, Shak S. Tumor gene expression and prognosis in breast cancer patients with 10 or more positive lymph nodes. Clin Cancer Res. 2005 Dec 15;11(24 Pt 1):8623-31

Fogal V, Kartasheva NN, Trigiante G, Llanos S, Yap D, Vousden KH, Lu X. ASPP1 and ASPP2 are new transcriptional targets of E2F. Cell Death Differ. 2005 Apr;12(4):369-76

Hershko T, Chaussepied M, Oren M, Ginsberg D. Novel link between E2F and p53: proapoptotic cofactors of p53 are transcriptionally upregulated by E2F. Cell Death Differ. 2005 Apr;12(4):377-83

Ju H, Lee KA, Yang M, Kim HJ, Kang CP, Sohn TS, Rhee JC, Kang C, Kim JW. TP53BP2 locus is associated with gastric cancer susceptibility. Int J Cancer. 2005 Dec 20;117(6):957-60

Kobayashi S, Kajino S, Takahashi N, Kanazawa S, Imai K, Hibi Y, Ohara H, Itoh M, Okamoto T. 53BP2 induces apoptosis through the mitochondrial death pathway. Genes Cells. 2005 Mar;10(3):253-60

Liu ZJ, Lu X, Zhang Y, Zhong S, Gu SZ, Zhang XB, Yang X, Xin HM. Downregulated mRNA expression of ASPP and the hypermethylation of the 5'-untranslated region in cancer cell lines retaining wild-type p53. FEBS Lett. 2005 Mar 14;579(7):1587-90

Takahashi N, Kobayashi S, Kajino S, Imai K, Tomoda K, Shimizu S, Okamoto T. Inhibition of the 53BP2S-mediated apoptosis by nuclear factor kappaB and Bcl-2 family proteins. Genes Cells. 2005 Aug;10(8):803-11

Zhu Z, Ramos J, Kampa K, Adimoolam S, Sirisawad M, Yu Z, Chen D, Naumovski L, Lopez CD. Control of ASPP2/(53BP2L) protein levels by proteasomal degradation modulates p53 apoptotic function. J Biol Chem. 2005 Oct 14;280(41):34473-80

Vives V, Su J, Zhong S, Ratnayaka I, Slee E, Goldin R, Lu X. ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth. Genes Dev. 2006 May 15;20(10):1262-7

Hakuno F, Kurihara S, Watson RT, Pessin JE, Takahashi S. 53BP2S, interacting with insulin receptor substrates, modulates insulin signaling. J Biol Chem. 2007 Dec 28;282(52):37747-58

Langton PF, Colombani J, Aerne BL, Tapon N. Drosophila ASPP regulates C-terminal Src kinase activity. Dev Cell. 2007 Dec;13(6):773-82

Tidow H, Andreeva A, Rutherford TJ, Fersht AR. Solution structure of ASPP2 N-terminal domain (N-ASPP2) reveals a ubiquitin-like fold. J Mol Biol. 2007 Aug 24;371(4):948-58

Sun WT, Hsieh PC, Chiang ML, Wang MC, Wang FF. p53 target DDA3 binds ASPP2 and inhibits its stimulation on p53-mediated BAX activation. Biochem Biophys Res Commun. 2008 Nov 14;376(2):395-8

Kampa KM, Bonin M, Lopez CD. New insights into the expanding complexity of the tumor suppressor ASPP2. Cell Cycle. 2009a Sep 15;8(18):2871-6

Kampa KM, Acoba JD, Chen D, Gay J, Lee H, Beemer K, Padiernos E, Boonmark N, Zhu Z, Fan AC, Bailey AS, Fleming WH, Corless C, Felsher DW, Naumovski L, Lopez CD. Apoptosis-stimulating protein of p53 (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage-response thresholds. Proc Natl Acad Sci U S A. 2009b Mar 17;106(11):4390-5

Uhlmann-Schiffler H, Kiermayer S, Stahl H. The DEAD box protein Ddx42p modulates the function of ASPP2, a stimulator of apoptosis. Oncogene. 2009 May 21;28(20):2065-73

Cong W, Hirose T, Harita Y, Yamashita A, Mizuno K, Hirano H, Ohno S. ASPP2 regulates epithelial cell polarity through the PAR complex. Curr Biol. 2010 Aug 10;20(15):1408-14

Sottocornola R, Royer C, Vives V, Tordella L, Zhong S, Wang Y, Ratnayaka I, Shipman M, Cheung A, Gaston-Massuet C, Ferretti P, Molnár Z, Lu X. ASPP2 binds Par-3 and controls the polarity and proliferation of neural progenitors during CNS development. Dev Cell. 2010 Jul 20;19(1):126-37

Zhao J, Wu G, Bu F, Lu B, Liang A, Cao L, Tong X, Lu X, Wu M, Guo Y. Epigenetic silence of ankyrin-repeat-containing, SH3-domain-containing, and proline-rich-region- containing protein 1 (ASPP1) and ASPP2 genes promotes tumor growth in hepatitis B virus-positive hepatocellular carcinoma. Hepatology. 2010 Jan;51(1):142-53

Liu CY, Lv X, Li T, Xu Y, Zhou X, Zhao S, Xiong Y, Lei QY, Guan KL. PP1 cooperates with ASPP2 to dephosphorylate and activate TAZ. J Biol Chem. 2011 Feb 18;286(7):5558-66

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Van Hook K, Wang Z, Lopez C. TP53BP2 (tumor protein p53 binding protein, 2). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):681-685.

Leukaemia Section Review




8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome) Paula Aranaz, José Luis Vizmanos

Department of Genetics, School of Sciences, University of Navarra, E-31008 Pamplona, Spain (PA, JLV)


Online updated version : http://AtlasGeneticsOncology.org/Anomalies/8p11inMPDID1091.html DOI: 10.4267/2042/46005


Identity Alias Stem cell leukemia-lymphoma syndrome (SCLL); 8p11 stem cell syndrome; 8p11 stem cell leukemia/lymphoma syndrome; Myeloid and lymphoid neoplasms FGFR1 abnormalities (WHO 2008 proposal)

Note Although "8p11 myeloproliferative syndrome" (EMS) (Macdonald et al., 1995) is the most frequent name for this disease in the literature, it must be designated as "myeloid and lymphoid neoplasm with FGFR1 abnormalities" under the current 2008 World Health Organization classification (Tefferi and Vardiman, 2008; Tefferi et al., 2009). This disease has been referred to as "stem cell leukemia/lymphoma" (SCLL) (Inhorn et al., 1995) which remark the coexistence of lymphoma, myeloid malignancy and lymphoblastic leukemia.

Clinics and pathology Note This disease is related to fusion genes between FGFR1, located in 8p11, and several partner genes. However there are some other aberrations affecting this chromosomal band and 8p12 in other neoplasms. Some acute myeloid leukemia (AML) cases have been described related to translocations affecting MYST3 (also known as MOZ) (Borrow et al., 1996; Carapeti et al., 1998; Chaffanet et al., 2000; Murati et al., 2007; Esteyries et al., 2008; Gervais et al., 2008) and WHSC1L1 (also known as NSD3) (Rosati et al., 2002; Romana et al., 2006; Taketani et al., 2009), both of them in 8p11. In addition, aberrations in 8p11-p12 are also frequent events in breast cancer, but the loci

responsible are not well known (Yang et al., 2004; Garcia et al., 2005; Gelsi-Boyer et al., 2005; Pole et al., 2006; Yang et al., 2006; Yang et al., 2010).

Disease Clinical entity defined by the disruption of the FGFR1 gene located at 8p11 with generation of a fusion gene between the 3' part of FGFR1 and the 5' part of the partner gene that also provides its promoter. The partner gene is always expressed in the haematopietic system and codes for a protein with oligomerization domains. As a result of oligomerization, chimeric proteins show constitutive and ligand-independent activation of FGFR1 kinase activity. The 8p11 myeloproliferative syndrome (EMS) is a myeloproliferative disease with multilineage involvement characterized by chronic myelomonocytic leukemia (CMML)-like myeloid hyperplasia, marked peripheral blood eosinophilia and associated with a high incidence of non-Hodgkin's lymphoma, usually of the T-cell lymphoblastic subtype. Occasional cases also show a B-cell lymphoproliferative disorder (Macdonald et al., 2002). EMS cases were already described in the 1970s and 1980s, but cytogenetic and molecular analyses were not available (Manthorpe et al., 1977; Kjeldsberg et al., 1979; Catovsky et al., 1980; Posner et al., 1982). In 1992, 3 cases of T-cell lymphoblastic lymphoma associated with eosinophilia that subsequently developed acute myeloid leukemia or myelodysplastic/myeloproliferative neoplasms were reported (Abruzzo et al., 1992). One of them showed a t(8;13) by conventional cytogenetics. Later it was shown that one of the breakpoints involved one 8p11 locus (Xiao et al., 1998). In the same year, Rao et al. reported a patient with t(8;13)(p11;q12) who presented with leukocytosis, monocytosis, myeloid hyperplasia of

8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome) Aranaz P, Vizmanos JL


bone marrow, and generalized lymphadenopathy due to T-cell lymphoblastic lymphoma (Rao et al., 1998). The term 8p11 myeloproliferative syndrome was suggested in 1995 by Macdonald et al. (Aguiar et al., 1995; Macdonald et al., 1995) and confirmed as clinical entity (SCLL) by Inhorn et al. (Inhorn et al., 1995).

Phenotype/cell stem origin The presence of the cytogenetic aberration in 8p11 both in myeloid and lymphoid cells, suggest a bilineage differentiation from a pluripotent and common stem cell (Macdonald et al., 2002).

Etiology The FGFR1 fusion proteins that result from chromosomal translocations affecting 8p11 have constitutive and ligand-independent FGFR1 enzymatic activity. FGFR1 is a receptor tyrosine kinase that dimerizes upon ligand binding, activating multiple signalling pathways like Ras/MAPK, PI3K, PLCgamma and STAT. These pathways could be abnormally activated as consequence of FGFR1 aberration (Macdonald et al., 2002) resulting in cell transformation. In fact, expression of ZMYM2-FGFR1 and BCR-FGFR1 fusions in immunodeficient mice are capable of initiating an EMS-like disease (Agerstam et al., 2010). Different fusion proteins could activate these pathways in a different way and could explain the phenotypic variability of the disease (Roumiantsev et al., 2004; Cross and Reiter, 2008; Jackson et al., 2010).

Epidemiology This is a very rare disease with less than 100 patients reported around the world and it can be found at any age. It has been reported at ages ranging from 3 to 84 years (median: 44 years). There is a slightly male-to-female predominance (Macdonald et al., 2002; Jackson et al., 2010).

Clinics Around 20-25% of patients show systemic and unspecific symptoms like fatigue, night sweats, weight loss and fever and around 20% are asymptomatic (and the disease is detected in routine analyses). Near two thirds of patients show lymphadenopathy, generalized or localized. Hepato- and/or splenomegaly are also frequent events in these patients. One of the distinctive features of this disease is the high frequency of lymphoblastic lymphoma, uncommon in other myeloproliferative neoplasms (Macdonald et al., 2002; Jackson et al., 2010).

Cytology It seems that neoplastic cells present in lymph nodes are predominantly small or medium lymphoblasts with a small cytoplasm (Jackson et al., 2010).

Pathology The blood counts reported are variable. More than 90% of patients have leukocytosis and less than 10% have

leukopenia but some cases have been reported with normal leukocyte counts. Eosinophilia is frequent, but monocytosis appears only in one third of patients. Basophils are increased only in cases with the t(8;22)(p11;q11). Blasts have been detected in half of the patients and some cases show blast counts typical of an acute leukemia. These blasts are mainly of a myeloid or myeloid and lymphoid (bilineal) lineage although some of them are also of an immature lymphoid lineage. Most of the patients show a hypercellular bone marrow that leads to a diagnosis of myeloid hyperplasia or a myeloproliferative neoplasm. But in some cases, the dysplastic features lead to a diagnosis of myelodysplastic syndrome or a myelodisplastic syndrome/myeloproliferative neoplasm. Most of the cases with lymph node biopsies reported had T-lymphoblastic lymphoma and the rest had myeloid sarcoma. In some cases evidence of bilineal T-cell/myeloid or B-and T- cell lymphoblastic lymphoma has been reported. For a review see Jackson et al., 2010.

Treatment This is a very aggressive disease with a high rate of progression to an AML resistant to conventional chemotherapy with a median survival time of less than 12 months (Macdonald et al., 2002; Cross and Reiter, 2008; Jackson, 2010). There are very few cases (Martinez-Climent et al., 1998; Zhou et al., 2010) responding to interferon alpha, this treatment could be useful at early stages. However, to date, only stem cell transplant remains effective to eradicate or suppress the malignant clone (Macdonald et al., 2002; Jackson et al., 2010). Median survival time for patients who received transplant after transformation to AML is 24 months (range 6-46 months) but median survival time is 12 months for the patients who did not received transplant (range 0-60 months) (Jackson et al., 2010). Currently there are no specific inhibitors for clinical use effective in this disease. Patients with FGFR1 fusions do not respond to drugs developed for other tyrosine kinases like imatinib, although several FGFR1 inhibitors have been tested, some of them with promising effects (Zhang et al., 2010; Zhou et al., 2010; Bhide et al., 2010; Risuleo et al., 2009; Ma et al., 2008; Cai et al., 2008; Chase et al., 2007; Kammasud et al., 2007; Klenke et al., 2007; Chen et al., 2004; Aviezier et al., 2000).

Evolution This disease has a chronic phase characterized by myeloid hyperplasia and overproduction of myeloid cells that can differentiate, but without treatment the disease progresses rapidly (1 to 2 years after diagnosis) to an acute myeloid leukemia (AML) or sometimes to a B-lineage ALL (Macdonald et al., 1995; Inhorn et al., 1995; Macdonald et al., 2002; Jackson et al., 2010).



Gain of an additional copy of chromosome 21 is a non-random cytogenetic event apparently associated with progression of this disease (Agerstam et al., 2007; Goradia et al., 2008) but its role remains unclear (Jackson et al., 2010). This abnormality is reported in only 5 of 47 (10.6%) karyotypes at the time of diagnosis but in 10 of 13 (76.9%) karyotypes reported in follow-up. These karyotypes were mostly derived during clinical deterioration. In addition, some findings at the time of transformation from EMS to acute leukemia include the addition of various marker chromosomes, as well as trisomy of chromosomes 8, 9, 12 or 19 and deletions of chromosome 7 or either the 7p or 7q arms, and derivative chromosome 9 (Jackson et al., 2010).

Prognosis As mentioned before, this is a devastating disease, which transforms to acute leukemia in a few months if left untreated, and in which the malignant clone cannot be eradicated by conventional chemotherapy. So at this moment, without specific FGFR1 inhibitors for clinical use, the stem cell transplant remains as the only possibility to a long-term survival (Macdonald et al., 1995; Inhorn et al., 1995; Macdonald et al., 2002; Jackson et al., 2010).

Genetics Note This disease is defined by the fusion of FGFR1 (8p11) with other partner genes, as consequence of a cytogenetic aberration, mainly chromosomal translocations. FGFR1 codes for a receptor tyrosine kinase. The gene fusion maintains the 3' terminal part of the FGFR1 gene (from exon 9) joined to the 5' terminal part of the partner gene. Partner genes are widely expressed and fusion genes have also this expression pattern. The chimeric gene codes for a protein which retains the TK domain from FGFR1 and oligomerization domains provided by the partner gene. This protein has a constitutive and ligand-independent activity and activates multiple signal transduction pathways.

Cytogenetics Variants t(8;13)(p11;q12) ZMYM2-FGFR1 This is the first translocation described (Xiao et al., 1998; Reiter et al., 1998; Smedley et al., 1998; Popovici et al., 1998) and the most common one. In fact, it has been described in 33 of the 65 cases described until now (Jackson et al., 2010). This translocation generates a fusion gene ZMYM2-FGFR1. Patients with this translocation develop lymphadenopathy and T-cell lymphoblastic lymphoma

(Macdonald et al., 2002; Cross and Reiter, 2008; Jackson et al., 2010). t(6;8)(q27;p11) FGFR1OP-FGFR1 This translocation was first described by Popovici et al. (1999) and fuses FGFR1OP (previously known as FOP -FGFR1 oncogene partner-) with FGFR1. As other FGFR1 fusion variants, the chimeric FGFR1OP-FGFR1 protein retains the N-terminus leucine-rich region of FGFR1OP (an oligomerization domain) fused to the catalytic domain of FGFR1 driving the abnormal oligomerization of the chimeric protein and a constitutive and ligand-independent activation. This translocation has been reported in 8 cases until date (Vizmanos et al., 2004). Eosinophilia is frequent in these patients. Four of these patients had features at presentation and/or a clinical course typical of EMS, but three showed polycythemia vera (PV) and another one B-ALL. t(8;9)(p12;q33) CEP110-FGFR1 This translocation was described in 1983 but molecularly characterized by Guasch et al. in 2000 (Guasch et al., 2000). This translocation has been reported in more than ten cases until date (Mozziconazzi et al., 2008; Jackson et al., 2010) and the MPD caused by this aberration transforms rapidly and always in myelomonocytic leukemia, with a possible B- or T-lymphoid involvement. In addition, tonsillar involvement and monocytosis also correlates with this variant (Mozziconazzi et al., 2008; Jackson et al., 2010). Recently a complete haematological and molecular remission has been reported after two years in a patient with this translocation treated early with interferon alpha (Zhou et al., 2010). t(8;22)(p11;q11) BCR-FGFR1 This translocation was reported simultaneously by two groups in 2001 (Fioretos et al., 2001; Demiroglu et al., 2001) and fuses BCR with FGFR1. As in the case of the other FGFR1 fusions, BCR is also widely expressed and BCR-FGFR1 retains oligomerization domains from BCR and the catalytic domain from FGFR1, leading to constitutive and ligand-independent activity of the chimeric protein. However it seems that BCR not only drives this oligomerization but could also play some role in triggering the downstream signalling pathways. Patients with BCR-FGFR1 fusions have a slightly different clinical phenotype from other FGFR1 fusion variants. In fact, these patients have a clinical and morphological picture similar to typical BCR-ABL positive chronic myeloid leukemia (Roumiantsev et al., 2004; Cross and Reiter, 2008; Baldazzi et al., 2010; Jackson et al., 2010). t(8;11)(p11;p15) NUP98-FGFR1 This translocation was described by Sohal et al. in 2001, in a patient with AML with additional cytogenetic aberrations (patient UPN6,



47,XY,t(8;11)(p11;p15),+8,-17,+i(17q)) (Sohal et al., 2001). Only interphase cells were available to perform FISH analysis, and the results obtained indicated a breakpoint within or in the vicinity of NUP98 but definite molecular characterization could not be done. Other patient with AML and the same translocation had been reported previously (Larson et al., 1983), indicating that this was a recurrent abnormality. However no new cases have been described since these reports so NUP98-FGFR1 fusion has not been confirmed definitely. NUP98 has a small coiled-coil region that could drive the constitutive activation of the chimeric NUP98-FGFR1 protein but it is possible that disruption of NUP98 was also involved in the oncogenic process. t(8;19)(p11;q13) HERVK-FGFR1 This aberration was firstly described in 2000, associated with loss of the Y chromosome in a man with an AML M0, probably secondary to a myeloproliferative disorder, who died 15 months after diagnosis (Mugneret et al., 2000). Later, the same group identified the chromosome 19 partner showing that a long terminal repeat of human endogenous retrovirus gene (HERV-K) was fused in frame with FGFR1 (Guasch et al., 2003). This fusion has been described only in this case. ins(12;8)(p11;p11p22) FGFR1OP2-FGFR1 This FGFR1 fusion is not caused by a chromosomal translocation but an inversion. Ins(12;8)(p11;p11p22) targeting FGFR1 was first described by Sohal et al. (2001) in a 75-years old patient diagnosed with a T-cell lymphoblastic lymphoma and marked lymph node infiltration with atypical eosinophils. Whole blood count was normal except for very mild eosinophilia and the bone marrow also showed some atypical eosinophils. After complete remission, this patient relapsed and transformed to an AML with the same chromosomal aberration and died. Later this aberration was molecularly characterized by the same group (Grand et al., 2004) as a fusion between FGFR1OP2 (from FGFR1 oncogene partner 2) located at 12p11.23 and FGFR1. Fusion structure was identical to other FGFR1 variants. This fusion has been reported only in one case (Sohal et al., 2001; Grand et al., 2004) but it has been also found in the cell line KG-1 (Gu et al., 2006; DSMZ ACC 14) that can be used to assay in vitro specific FGFR1 inhibitors (Gu et al., 2006; Chase et al., 2007). This cell line was derived from the bone marrow of a 59-year-old man with erythroleukemia transformed to AML at relapse in 1977 (Koeffler and Golde, 1978). t(7;8)(q34;p11) TRIM24-FGFR1 This translocation was described and molecularly characterized by Belloni et al. (2005) in a 49-year-old woman with a putative chronic MPD with eosinophilia which transformed to an AML-M4 and died in a few days. As other FGFR1 fusions, this is the only case reported to date.

The consequence of the t(7,8)(q34;p11) is the fusion gene TRIM24-FGFR1. t(8;17)(p11;q23) MYO18A-FGFR1 This translocation was described by Walz et al. (2005) in a 74-year-old female with a 2 years history of an unusual myelodysplastic/myeloproliferative disease (MDS/MPD) with thrombocytopenia, markedly reduced size and numbers of megakaryocytes and elevated numbers of monocytes, eosinophils and basophils. Her karyotype showed an additional trisomy 20 and she died after a treatment-resistant disease progression of two years. This translocation fuses MYO18A, located in 17q11.2 with FGFR1. However, the breakpoint in chromosome 17 was cytogenetically located to 17q23. FISH and molecular analysis showed that this fusion gene was consequence of a complex cytogenetic aberration with an additional inversion in 17q region between 17q11 and 17q23. t(8;12)(p11;q15) CPSF6-FGFR1 This translocation targeting FGFR1 was first described by Sohal et al. (2001). Later the same group identified the partner gene as CPSF6 (located at 12q15) (Hidalgo-Curtis et al., 2008) and again, only this case has been described. The patient was a 75-year-old female with lymphadenopathy, splenomegaly, neutrophilia and eosinophilia in peripheral blood and also an increase of eosinophils and eosinophil precursors in the bone marrow. After a rapid clinical deterioration the patient died in 10 weeks. t(2;8)(q37;p11) LRRFIP1-FGFR1 In 2009, Soler et al. identified and characterized the t(2;8)(q37;p11) in an 82-year-old man with 10% eosinophils, 2-4% myelocytes and metamyelocytes, and 8% circulating blasts and an hypocellular bone marrow with moderate dysgranulopoiesis and 15% blasts (Soler et al., 2009). Some years before, this patient had displayed pancitopenia and a bone marrow showing a refractory anemia with an excess of blasts (15%). The disease transformed to AML in one year and the patient died. FISH analysis on retrospective samples showed that the t(2;8)(q37;p11) was not present in early stages (pancitopenia) of the disease.

Genes involved and proteins ZMYM2 Location 13q12

Protein ZMYM2 (also known as ZNF198, RAMP -rearranged in atypical myeloproliferative disorder-, or FIM - fused in myeloproliferative disorder) codes for a zinc finger protein that may act as a transcription factor involved in ribosomal RNA transcription and also could be part of a BHC histone deacetylase complex. The chimeric



protein retains the proline-rich domain of ZMYM2 (an oligomerization domain) and the tyrosine kinase domain of FGFR1. The abnormal oligomerization of the chimeric protein leads to constitutive and ligand-independent activation. In addition, this abnormal protein is located in the cytoplasm and not in the membrane as native FGFR1.

FGFR1OP Location 6q27

Protein FGFR1OP, widely expressed, codes for a hydrophilic centrosomal protein that could be a member of a leucine-rich protein family, and it is involved in the anchoring of microtubules (MTS) to subcellular structures. FGFR1OP could play a role in lung cancer growth and progression and has been proposed as a prognostic biomarker for this disease (Mano et al., 2007).

CEP110 Location 9q33.2

Protein CEP110 encodes also a centrosomal protein with several leucine zipper motifs required for the centrosome to function as a microtubule organizing center. CEP110 is also widely expressed and CEP110-FGFR1 retains the leucine zipper motifs of CEP110 at its N-terminus which could mediate the consititutive activation of the FGFR1 catalytic domain at its C-terminus. In addition the CEP110-FGFR1 fusion protein has been found in the cytoplasm, whereas both CEP110 and FGFR1 wild-type proteins are centrosome and plasma membrane-bound proteins respectively (Guasch et al., 2000).

BCR Location 22q11.2

Protein BCR is, like ETV6, a common fusion partner of several tyrosine kinase genes rearranged in myeloid disorders (BCR-ABL, BCR-JAK2, BCR-PDGFRA and BCR-FGFR1 have been described to date). However function of the protein encoded by this gene is not clear and its name comes from breakpoint cluster region.

NUP98 Location 11p15

HERVK Location 7p22.1

Protein HERV-K is also ubiquitously expressed. The HERV-Ks are human specific endogenous retrovirus that have been proposed as etiological cofactors in some chronic diseases like cancer because they are mobile elements that could disrupt tumor suppressor and/or DNA repair genes. In this case, it seems that the part of the HERV-K sequence fused showed similarities with a retroviral envelope protein whose dimerization would induce the constitutive activation of the chimeric protein HERVK-FGFR1 (Guasch et al., 2003).

FGFR1OP2 Location 12p11.23

Protein As other FGFR1 partners, FGFR1OP2 is also widely expressed but its function is unknown. It could code for a cytoskeleton molecule (Lin et al., 2010). However, the putative protein coded by this gene has four potential coiled-coil domains and the first two are retained in the chimeric protein, so they could mediate its oligomerization and constitutive activation (Grand et al., 2004).

TRIM24 Location 7q34

Protein TRIM24 (previously known as TIF1) codes for a protein of the tripartite motif (TRIM) family that mediates transcriptional control by interaction with several nuclear receptors and localizes to nuclear bodies. The tripartite motif includes three zinc-binding domains - a RING, a B-box type 1 and a B-box type 2 - and a coiled-coil region that is retained in the chimeric protein so it could promote, as other FGFR1 fusion proteins, its constitutive and ligand-independent activation

MYO18A Location 17q11.2

Protein MYO18A is a widely expressed gene that codes for a protein of unknown function of the myosin superfamily. It has been recently described that this protein is a novel PAK2 (p21-activated kinase 2) binding partner (Hsu et al., 2010). PAK2 has many biological functions, including the regulation of actin reorganization and cell motility. MYO18A contains several functional motifs that are retained in MYO18A-FGFR1, including an N-terminal PDZ (PSD-95/Dlg/ZO-1) protein-protein interaction domain, a myosin head domain and a region that is predicted to



form multiple coiled-coils. Some of these coiled-coils could drive oligomerization of MYO18A-FGFR1, with consequent constitutive activation of the FGFR1 kinase activity. Recently, MYO18A has also been found fused to PDGFRB as consequence of a t(5;17)(q33-q34;q11) but with a different breakpoint in which all the predicted coiled-coil domains of normal MYO18A are retained in the chimeric protein (Walz et al., 2009).

CPSF6 Location 12q15

Protein The protein encoded by CPSF6 is the 68 kD subunit of a cleavage factor required for 3' RNA cleavage and polyadenylation processing. Unlike other partners of FGFR1, CPSF6 does not have any identifiable oligomerisation motifs. However RNA recognition motifs (RRM) such as the one retained in CPSF6-FGFR1, could mediate the dimerization needed for constitutive activation of the CPSF6-FGFR1 kinase activity.

LRRFIP1 Location 2q37.3

Protein LRRFIP1 (Leucine-rich repeat Flightless-Interacting Protein 1) is a ubiquitously expressed gene that encodes for a nuclear and cytoplasmatic protein with multiple functions. In the nucleus, it acts as a transcriptional repressor that decreases the expression of EGFR, PDGFRA and TNF. In the cytoplasm, it interacts with actin-binding proteins. It has an N-terminal coiled-coil domain that, as other FGFR1 partners, could drive the dimerization of LRRFIP1-FGFR1 leading to the constitutive activation of the kinase activity.

Result of the chromosomal anomaly Hybrid gene Detection Methods of detection 1. Conventional cytogenetics to identify translocations or other rearrangements involving 8p11. 2. Fluorescent in situ hibridization (FISH) with probes flanking or covering FGFR1 to demonstrate disruption of this gene. 3. 5' RACE PCR to identify FGFR1 partner gene. 4. RT-PCR with primers located in both genes fused.

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Cai ZW, Zhang Y, Borzilleri RM, Qian L, Barbosa S, Wei D, Zheng X, Wu L, Fan J, Shi Z, Wautlet BS, Mortillo S, Jeyaseelan R Sr, Kukral DW, Kamath A, Marathe P, D'Arienzo C, Derbin G, Barrish JC, Robl JA, Hunt JT, Lombardo LJ, Fargnoli J, Bhide RS. Discovery of brivanib alaninate ((S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate), a novel prodrug of dual vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 kinase inhibitor (BMS-540215). J Med Chem. 2008 Mar 27;51(6):1976-80

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Lin A, Hokugo A, Choi J, Nishimura I. Small cytoskeleton-associated molecule, fibroblast growth factor receptor 1



oncogene partner 2/wound inducible transcript-3.0 (FGFR1OP2/wit3.0), facilitates fibroblast-driven wound closure. Am J Pathol. 2010 Jan;176(1):108-21

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Aranaz P, Vizmanos JL. 8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):686-694.

Leukaemia Section Mini Review




i(5)(p10) in acute myeloid leukemia Nathalie Douet-Guilbert, Angèle Herry, Audrey Basinko, Marie-Josée Le Bris, Nadia Guéganic, Clément Bovo, Frédéric Morel, Marc De Braekeleer

Laboratory of Histology, Embryology, and Cytogenetics, Faculty of Medicine and Health Sciences, Université de Bretagne Occidentale, 22, avenue Camille Desmoulins, CS 93837, F-29238 Brest cedex 3, France (NDG, AH, AB, MJL, NG, CB, FM, MD)


Online updated version : http://AtlasGeneticsOncology.org/Anomalies/i5pID1376.html DOI: 10.4267/2042/46006

This article is an update of : Schoch C. i(5)(p10) in acute myeloid leukemia. Atlas Genet Cytogenet Oncol Haematol 2005;9(2):150-151 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

i(5)(p10) G-banding - Claudia Schoch (left), and R-banding - Nathalie Douet-Guilbert (right).

Note In literature, two types of i(5)(p10) are observed: Type 1: i(5)(p10) inducing a loss of the long arm of chromosome 5 (5q) and a trisomy of the short arm of the chromosome 5 (5p); Type 2: +i(5)(p10) (or supernumerary i(5)(p10) or gain of i(5)(p10)) inducing a tetrasomy of the short arm of chromosome 5 (5p). The i(5)(p10) occurred in addition to two normal chromosomes 5. The isochromosome of the short arm of chromosome 5 - i(5)(p10) - has only been described in a few cases of myeloid leukemia.

Clinics and pathology Phenotype/cell stem origin Type 1: classified as myelodysplastic syndrome (4 cases), acute myeloid leukemia (4 cases) predominantly AML M2;

Type 2: classified as acute myeloid leukemia (5 cases), predominantly AML M5a.

Etiology Unclear

Epidemiology Type 1: it is found in young adults in MDS (average age: 35 years; range: 19-67) and in older patients in AML (average age: 66 years; range: 50-85). Type 2: the +i(5)(p10) is found in patients with an average age of 48.5 years (range : 24-78).

Prognosis Prognosis of patients with i(5)(p10) seems to be poor compared to patients with del(5q), but it is unclear due to the very small number of cases and the usually associated complex chromosomal abnormalities.

i(5)(p10) in acute myeloid leukemia Douet-Guilbert N, et al.


A - FISH with partial chromosome painting 5p (pcp 5p) (Green signal) and 5q (pcp 5q) (Red signal). B - FISH with LSI 5p15.2 (Green signal) / 5q31 (Red signal). Nathalie Douet-Guilbert.

Cytogenetics Cytogenetics morphological The formation of i(5p) results from the loss of the long arm of chromosome 5 and duplication of its short arm inducing trisomy 5p and monosomy 5q in type 1 and tetrasomy 5p in type 2. A metacentric del(5q) could be an isochromosome of the short arm of chromosome 5. FISH technique with specific probes of chromosome 5p/5q used as a complement of conventional karyotype is necessary to identify i(5)(p10). The i(5p) is a variant of del(5q). The i(5p) is monocentric or dicentric.

Additional anomalies In one case, i(5)(p10) was the sole anomaly but rapidly evolved into a complex karyotype. Complex karyotypes were present in the other cases: -12/del12p (3 cases), -17/del17p (2 cases), del9q (2 cases). Supernumerary +i(5)(p10) was accompanied by several additional anomalies, especially trisomy 8

Genes involved and proteins Note Type 1: to explain the specific phenotype of i(5)(p10), loss of tumor suppressor genes in the deleted region (5q) associated with gene dosage effect of genes located on 5p is suggested. Type 2: gene dosage effect of genes located on the short arm of chromosome 5.

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Douet-Guilbert N, Herry A, Basinko A, Le Bris MJ, Guéganic N, Bovo C, Morel F, De Braekeleer M. i(5)(p10) in acute myeloid leukemia. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):695-696.

Leukaemia Section Mini Review




+20 or trisomy 20 (solely) 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/tri20ID1572.html DOI: 10.4267/2042/46007 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

Clinics and pathology Disease Myeloid and lymphoid malignancies (Shabtai et al., 1978; Kristoffersson et al., 1985; Michalová et al., 1987; Palka et al., 1987; Speaks et al., 1987; Attas et al., 1989; Cuneo et al., 1989; Nayak et al., 1990; Cuneo et al., 1992; Cuneo et al., 1995; Hashimoto et al., 1995; Rigolin et al., 1997; Jaing et al., 1999; Mauritzson et al., 2001; Tamura et al., 2001; Mikhail et al., 2002; Farag et al., 2006; Paulsson et al., 2008).

Note Trisomy 20 solely has also been reported in various benign and malignant solid tumours, in particular in desmoid fibromatosis, colonic adenomatous polyps, colorectal adenocarcinomas, fibroadenomas of the breast, breast adenocarcinoma, transitional cell carcinoma of the urinary tract, squamous cell carcinoma of the oro-pharynx and naso-pharynx; it has also been found more rarely in many other solid tumours (see records in the Mitelman Database).

Phenotype/cell stem origin Trisomy 20 solely has been described in 20 cases of hematological malignancies: This was a myeloid malignancy in 12 cases: six acute myeloid leukaemias (AML), four myelodysplastic syndromes (MDS), and two myeloproliferative disorders (MPD). They were: two M4-AML, two M5-AML, one M0-AML, one AML not otherwise specified (NOS), one refractory anaemia (RA), one RA with excess of blasts (RAEB), one chronic myelomonocytic leukaemia (CMML), one MDS-NOS, and two polycytemia vera (PV). One AML, a M5-AML, appeared to be treatment-related, in a 8-year-old girl with neuroblastoma, diagnosed 32 months before onset of the leukaemia. A cryptic rearrangement of MLL was found. Survival was short (Jaing et al., 1999).

The 8 lympoid cases were: three acute lymphoblastic leukaemias (ALL) (two of which involved the T-cell lineage), one chronic lymphocytic leukaemia (CLL), three non Hodgkin lymphomas (NHL) : one follicular (FL), one diffuse large B-cell (DLBL), and one T-cell lymphoma); and one Waldenstrom macroglobulinemia.

Epidemiology In the myeloid group, there were 7 male and 5 female patients, median age was 68-72 years (range: 8-79 years, 8 of the 10 documented cases were above 60 years). In the lymploid group, there was an unbalanced sex ratio: 6 male and 2 female patients; median age was 33-53 years (range: 7-76 years).

Prognosis Data is very scarce, and not conclusive, inasmuch as the genes involved in these cases are unknown, and as the trisomy 20 group is probably heterogeneous from that view point.

Genes involved and proteins Note Genes involved are unknown.

References Shabtai F, Weiss S, van der Lijn E, Lewinski U, Djaldetti M, Halbrecht I. A new cytogenetic aspect of polycythemia vera. Hum Genet. 1978 Apr 24;41(3):281-7

Kristoffersson U, Heim S, Heldrup J, Akerman M, Garwicz S, Mitelman F. Cytogenetic studies of childhood non-Hodgkin lymphomas. Hereditas. 1985;103(1):77-84

Michalová K, Kobylka P, Lukásová M, Neuwirt J. Cytogenetic study of circulating blasts in leukemias. Cancer Genet Cytogenet. 1987 Apr;25(2):329-39

Palka G, Spadano A, Geraci L, Fioritoni G, Dragani A, Calabrese G, Guanciali Franchi P, Stuppia L. Chromosome changes in 19 patients with Waldenström's macroglobulinemia. Cancer Genet Cytogenet. 1987 Dec;29(2):261-9

+20 or trisomy 20 (solely) Huret JL


Speaks SL, Sanger WG, Linder J, Johnson DR, Armitage JO, Weisenburger D, Purtilo D. Chromosomal abnormalities in indolent lymphoma. Cancer Genet Cytogenet. 1987 Aug;27(2):335-44

Attas L, Lichtman SM, Budman DR, Verma RS. Trisomy 20 in acute myelogenous leukemia. Cancer Genet Cytogenet. 1989 May;39(1):25-8

Cuneo A, Tomasi P, Ferrari L, Balboni M, Piva N, Fagioli F, Castoldi G. Cytogenetic analysis of different cellular populations in chronic myelomonocytic leukemia. Cancer Genet Cytogenet. 1989 Jan;37(1):29-37

Nayak BN, Sokal J, Ray M. Clonal chromosomal changes in chronic lymphocytic leukemia. Cancer Lett. 1990 Feb;49(2):99-105

Cuneo A, Fagioli F, Pazzi I, Tallarico A, Previati R, Piva N, Carli MG, Balboni M, Castoldi G. Morphologic, immunologic and cytogenetic studies in acute myeloid leukemia following occupational exposure to pesticides and organic solvents. Leuk Res. 1992 Aug;16(8):789-96

Cuneo A, Ferrant A, Michaux JL, Boogaerts M, Demuynck H, Van Orshoven A, Criel A, Stul M, Dal Cin P, Hernandez J. Cytogenetic profile of minimally differentiated (FAB M0) acute myeloid leukemia: correlation with clinicobiologic findings. Blood. 1995 Jun 15;85(12):3688-94

Hashimoto K, Miura I, Chyubachi A, Saito M, Miura AB. Correlations of chromosome abnormalities with histologic and immunologic characteristics in 49 patients from Akita, Japan with non-Hodgkin lymphoma. Cancer Genet Cytogenet. 1995 May;81(1):56-65

Rigolin GM, Cuneo A, Roberti MG, Bardi A, Castoldi G. Myelodysplastic syndromes with monocytic component: hematologic and cytogenetic characterization. Haematologica. 1997 Jan-Feb;82(1):25-30

Jaing TH, Yang CP, Hung IJ. Acute monoblastic leukemia in a child following chemotherapy for neuroblastoma. J Formos Med Assoc. 1999 Oct;98(10):688-91

Mauritzson N, Johansson B, Rylander L, Albin M, Strömberg U, Billström R, Ahlgren T, Mikoczy Z, Mitelman F, Hagmar L, Nilsson PG. The prognostic impact of karyotypic subgroups in myelodysplastic syndromes is strongly modified by sex. Br J Haematol. 2001 May;113(2):347-56

Tamura A, Miura I, Iida S, Yokota S, Horiike S, Nishida K, Fujii H, Nakamura S, Seto M, Ueda R, Taniwaki M. Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma. Cancer Genet Cytogenet. 2001 Aug;129(1):1-9

Mikhail FM, Serry KA, Hatem N, Mourad ZI, Farawela HM, El Kaffash DM, Coignet L, Nucifora G. AML1 gene over-expression in childhood acute lymphoblastic leukemia. Leukemia. 2002 Apr;16(4):658-68

Farag SS, Archer KJ, Mrózek K, Ruppert AS, Carroll AJ, Vardiman JW, Pettenati MJ, Baer MR, Qumsiyeh MB, Koduru PR, Ning Y, Mayer RJ, Stone RM, Larson RA, Bloomfield CD. Pretreatment cytogenetics add to other prognostic factors predicting complete remission and long-term outcome in patients 60 years of age or older with acute myeloid leukemia: results from Cancer and Leukemia Group B 8461. Blood. 2006 Jul 1;108(1):63-73

Paulsson K, Cazier JB, Macdougall F, Stevens J, Stasevich I, Vrcelj N, Chaplin T, Lillington DM, Lister TA, Young BD. Microdeletions are a general feature of adult and adolescent acute lymphoblastic leukemia: Unexpected similarities with pediatric disease. Proc Natl Acad Sci U S A. 2008 May 6;105(18):6708-13

Mitelman F, Johansson B and Mertens F (Eds.).. Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2011). http://cgap.nci.nih.gov/Chromosomes/Mitelman


Huret JL. +20 or trisomy 20 (solely). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):697-698.

Deep Insight Section Review




TMPRSS2:ETS gene fusions in prostate cancer Julia L Williams, Maisa Yoshimoto, Alexander H Boag, Jeremy A Squire, Paul C Park

Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada (JLW, MY, AHB, JAS), Kingston General Hospital, 76 Stuart Street, Douglas 4, Room 8-431, Kingston, Ontario, K7L 2V7 Canada (PCP)


Online updated version : http://AtlasGeneticsOncology.org/Deep/TMPRSS2ERGinCancerID20091.html DOI: 10.4267/2042/46008


Table of Contents I. Background I.i. Prostate cancer oncogenomics

II. Discovery of TMPRSS2:ETS fusion genes in prostate cancer III. Frequency of TMPRSS2:ETS gene fusions in prostate cancer IV. TMPRSS2:ETS gene fusions: genes and protein structure IV.i. TMPRSS2 and the androgen receptor

IV.ii. ETS transcription factors

V. Fusion variants VI. Detection and classification VI.i. FISH

VI.ii. Other methods of detection

VII. Fusion gene formation and chromosomal instability VIII. Heterogeneity of multifocal disease IX. Prognostic significance X. Clinical utility XI. Role of ETS in prostate tumourigenesis: Driver? XII. Concluding remarks

I. Background Prostate cancer (CaP) is the most commonly diagnosed male malignancy and a leading cause of cancer deaths in developed countries. With one in six men diagnosed, CaP remains a serious global public health issue (Jemal et al., 2008). CaP is a clinically heterogeneous disease, with manifestations ranging from a rapid and often fatal progression, to the typical, indolent disease which remains relatively insignificant to a patient's health.

Clinicopathological criteria, including Gleason grading, are not sufficient to differentiate men whose tumours require immediate and aggressive therapy from those that would suffice with vigilant clinical observation, thereby causing the latter group enormous amounts of unnecessary treatment (Yao and Lu-Yao, 2002). In this regard, the emerging data on the genetics of CaP hold great promise not only in stratifying this heterogeneous group of patients, but also in providing the groundwork for future development of targeted therapy.

TMPRSS2:ETS gene fusions in prostate cancer Williams JL, et al.


I. i. Prostate cancer oncogenomics Advances in cytogenetics and genomics facilitated the characterization of common genomic alterations in CaP, which are predominantly characterized by deletions (10q, PTEN; 13q, RB1; 8p, NKX3.1 (a prostate-specific tumour suppressor); 5q; 2q; 17p; and less commonly 6q; 7p; 16q; 18q) with only a small number of recurrent gains (8q, MYC; and chromosome 7). More complex patterns as well as an accumulation in the number of genomic gains and amplifications (Xq11.2-q12, androgen receptor (AR)) emerge as the disease advances. Genomic rearrangements leading to the formation of TMPRSS2:ETS gene fusions and deletion of the PTEN tumour suppressor are the two most frequent alterations observed in CaP (Tomlins et al., 2005; Yoshimoto et al., 2007; Yoshimoto et al., 2008). The TMPRSS2:ERG gene fusion is the principle genomic alteration and a characteristic signature in approximately half of prostatic malignancies.

II. Discovery of TMPRSS2:ETS fusion genes in prostate cancer The discovery of the TMPRSS2:ERG gene fusion exemplifies the current shift in the strategy in cancer genomics from experimental to bioinformatics approaches. By surveying 132 gene-expression CaP datasets from the Oncomine database (Compendia Bioscience) using the data transformation algorithm cancer outlier profile analysis (COPA), Chinnaiyan and colleagues identified genes with aberrant expression profiles in a subset of samples (Tomlins et al., 2005). COPA allowed the systematic investigation of cancer-related genes known to participate in chromosomal rearrangements in haematological malignancies and sarcomas. Two mutually exclusive Erythroblastosis virus E26 transformation-specific (ETS) transcription factors, ETS variant 1 (ETV1, 7p21.3) and ETS-related gene (ERG, 21q22.2), were identified as high-ranking outliers in several independent gene-expression profiling datasets. Exon-walking quantitative PCR (qPCR) of patient samples found the 3' regions of ERG and ETV1 to be overexpressed but the corresponding 5' regions were absent. 5' RNA ligase-mediated rapid amplification of cDNA ends identified the 5' end of these transcripts as the promoter sequences belonging to the prostate-specific, androgen regulated transmembrane protease, serine II (TMPRSS2, 21q22.3).

III. Frequency of TMPRSS2:ETS gene fusions in prostate cancer This breakthrough study reported that 79% of radical prostatectomy (RP) samples harboured a fusion of the 5' untranslated region (UTR) of TMPRSS2 with the coding sequences of either ERG or ETV1 (Tomlins et al., 2005). Interestingly, just months prior, Petrovics et al. (2005) reported that ERG was the most commonly overexpressed oncogene in CaP by microarray and

qPCR analysis. Subsequent studies showed that the frequency of the TMPRSS2:ERG fusion gene (or ERG rearrangement) is exceptionally variable and inconsistent in the literature, ranging from 27% to 79% in radical prostatectomy (RP) and biopsy samples, generally from prostate-specific antigen (PSA) screened cohorts (Tomlins et al., 2005; Yoshimoto et al., 2008; Mehra et al., 2007b; Watson et al., 2009; Barwick et al., 2010; Magi-Galluzzi et al., 2010). Some of the discrepancies in frequency relate to differences in the patient cohort (race as well as global geographical location or PSA-screened versus population based) or the type of specimen examined, as well as the technique used to detect the fusion gene. This concept is clearly illustrated when comparing population based cohorts, which have a lower reported frequency of 15-35%, as compared to RP cohorts (Attard et al., 2008a; Demichelis et al., 2007; Fitzgerald et al., 2008). The TMPRSS2:ERG gene fusion is found in approximately half of Caucasian patients, with a lower reported frequency in African-American men and is less common in Asian cohorts (Mosquera et al., 2009; Magi-Galluzzi et al., 2010; Lee K et al., 2010; Miyagi et al., 2010). An excellent example of discrepancies based on patient populations was demonstrated in a study that compared patients from the United Kingdom (UK) and China, wherein 41.3% of the UK patients but only 7.5% of Chinese cohort were found to harbour ERG rearrangements detected by FISH (Mao et al., 2010). The recent, contradicting report that 90% of the Chinese RP specimens harboured ETS rearrangements further emphasize the multifactorial nature of the variability in the frequency of this gene fusion (Sun et al., 2010). TMPRSS2:ERG gene fusions are reported in 10-21% of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions, but are identified almost exclusively adjacent to fusion-positive cancer (Cerveira et al., 2006; Perner et al., 2007; Carver et al., 2009b; Han et al., 2009; Zhang et al., 2010; Mosquera et al., 2008). Benign prostatic hyperplasia (BPH) and normal epithelium are negative for ERG rearrangements and fusion transcripts, with the exception of the report by Clark and colleagues (Rajput et al., 2007; Wang et al., 2006; Cerveira et al., 2006; Clark et al., 2007; Perner et al., 2007; Dai et al., 2008; Han et al., 2009; Mosquera et al., 2009; Zhang et al., 2010; Sun et al., 2010; Lu et al., 2009). This study found that eight of 17 (47%) normal epithelial samples adjacent to fusion-positive CaP were also positive for TMPRSS2:ERG rearrangements and found a 6% fusion-positive rate in BPH samples (Clark et al., 2007). Hormone refractory and/or metastatic CaP exhibits less variability in the occurrence of TMPRSS2:ERG rearrangements with reported frequencies ranging from 29-59% (Perner et al., 2007; Mehra et al., 2008; Gopalan et al., 2009; Han et al., 2009; Boormans et al., 2010; Saramaki et al., 2008; Attard et al., 2009; Stott et al., 2010). Additionally, minute prostatic adenocarcinoma, a form



of CaP that is considered less clinically significant, harbours ETS fusions in approximately half of reported cases, underscoring the necessity of examining all prostatic malignancies for aggressive features such as the fusion gene (Albadine et al., 2009). Investigators have also evaluated this rearrangement in the peripheral and transitional zones of the prostate. Nearly half of the peripheral tumours (43.3%) derived from RP samples were ERG rearrangement positive, whereas all 30 corresponding transitional tumours displayed a normal ERG locus by FISH (Guo et al., 2009). In contrast, other studies found TMPRSS2:ERG (13.3%) and ERG rearrangements (11.9%) are present in transitional zone tumours, despite being identified at a lower rate compared to peripheral zone lesions (Bismar and Trpkov, 2010; Falzarano et al., 2010). These data illustrate the enormous variability in the frequency of TMPRSS2:ERG fusion positivity with respect to the cohort, disease stage, origin of sample as well as the method of detection. Interestingly, a comprehensive study of patient samples from 54 tumour types, including sarcomas and haematological malignancies, for ETS gene fusions and ERG rearrangements by FISH found these alterations to be exclusive to CaP samples (Scheble et al., 2010). The same result was obtained when RT-PCR was performed to detect TMPRSS2:ERG and TMPRSS2:ETV1 fusion transcripts in gastric and colorectal carcinomas (Yoo et al., 2007). These findings provide strong evidence that TMPRSS2:ETS gene fusions are specific to CaP.

IV. TMPRSS2:ETS gene fusions: genes and protein structure IV. i. TMPRSS2 and the androgen receptor TMPRSS2, a 70 KDa serine protease family member is associated with physiological and pathological processes such as digestion, tissue remodelling, blood coagulation, fertility, inflammatory responses, tumour cell invasion and apoptosis. The normal function of this protein is not yet known but is composed of a type II transmembrane domain, receptor class A low density lipoprotein domain, scavenger receptor cysteine-rich domain, protease domain, and cytoplasmic domain (Paoloni-Giacobino et al., 1997). The 32 KDa serine protease domain undergoes autocleaveage, secretion into the prostate epithelia and interacts with cell surface proteins, the extracellular matrix and proteins of neighbouring cells (Vaarala et al., 2001; Wilson et al., 2005; Afar et al., 2001). The TMPRSS2 (21q22.3) gene is composed of 14 exons, with protein coding sequences only in the latter half. Importantly, TMPRSS2 harbours androgen responsive elements (ARE) in its 5' UTR. Prostate epithelial cells express TMPRSS2 at higher levels relative to other tissues, while TMPRSS2 gene expression is further elevated in CaP relative to BPH and normal prostatic epithelium (Afar et al., 2001). Androgens and the AR are essential

to the growth and development of the prostate gland but also play an important role in the initiation and progression of CaP (Balk et al., 2008). It is well established that despite chemical castration the AR functions to drive CaP, likely through a variety of mechanisms such as hypersensitivity to low levels of androgens, activation in absence of, or via unconventional ligands, or amplification of the AR gene locus, resulting in elevated levels of AR protein. All of these mechanisms could potentially allow AR activation in the presence of low androgen concentration (Ai et al., 2009; Kawata et al., 2010; Vis and Schroder, 2009). Consequently, TMPRSS2 in turn plays an important role in CaP progression in spite of hormonal ablation regimens as its promoter region drives the expression of the fused ETS gene.

IV. ii. ETS transcription factors Twenty-seven human ETS transcription factor family members have been identified, all of which share a conserved DNA binding domain that recognizes unique sequences containing GGA(A/T) (Nye et al., 1992). ERG (21q22.2) is the ETS transcription factor most commonly known to participate in CaP gene fusions. The ERG gene contains 11 exons, with the transcriptional start site in exon 3. The ERG protein can interact with ETS members as well as other transcription factors, such as Jun and Fos, through its protein-protein interacting domain, SAM-PNT (Carrere et al., 1998; Verger et al., 2001; Basuyaux et al., 1997). The conserved ETS DNA binding domain permits binding to purine rich DNA sequences (Reddy et al., 1991; Reddy et al., 1987), and in this manner, exert its effects in numerous cellular processes including membrane remodelling, angiogenesis, differentiation, proliferation, and tumourigenesis (Carver et al., 2009a; Kruse et al., 2009; Oikawa et al., 2003; Randi et al., 2009; Ellett et al., 2009; Birdsey et al., 2008; Mclaughlin et al., 2001; Sato et al., 2001). Emerging evidence suggests that formation of the fusion gene may promote prostatic tumourigenesis, progression, and invasive disease and is associated with CaP-related mortality (Demichelis et al., 2007; Klezovitch et al., 2008; Wang et al., 2008; Hawksworth et al., 2010). Functionally, ERG overexpression in CaP is highly implicated in promoting motility and invasiveness (Perner et al., 2007; Singh et al., 2002; Trojanowska et al., 2000; Tomlins et al., 2008a; Sreekumar et al., 2009; Schulz et al., 2010). In CaP, ERG expression has been associated with elevated levels of histone deactylase 1 (HDAC1) and subsequent down regulation of HDAC1 target genes, upregulation of WNT pathway proteins, and inhibition of apoptotic signalling (Iljin et al., 2006). HDAC1 upregulation is common in CaP, but was found to be uniformly increased in ERG rearranged tumours (Iljin et al., 2006; Bjorkman et al., 2008). Activation of the WNT pathway leads to transcription of numerous genes involved in tumourigenesis, including AR, MYC, JUN, cyclinD1, BMP4 and



MMP7 (Terry et al., 2006; Schweizer et al., 2008). Highlighting the importance of WNT pathway activation in fusion-positive CaP is the resultant increase in AR transcription and expression levels. Consequently transcription of the fusion gene is increased, further amplifying ERG expression. Moreover, beta-catenin and the AR interact in an androgen-dependent manner to regulate AR target genes, whereas in androgen-insensitive tumours, both beta-catenin and AR target genes are expressed (Schweizer et al., 2008). ERG overexpression has recently been shown to activate C-MYC and result in its overexpression. These experiments revealed that C-MYC is activated by ERG and together their co-overexpression results in the suppression of prostate-epithelial differentiation genes and shorter time to biochemical recurrence (Hawksworth et al., 2010; Sun et al., 2008). ERG can also induce ICAM2 expression, resulting in AKT activation via PDK1 and subsequent inhibition of BAD leading to suppression of apoptotic signals (Mclaughlin et al., 2001). In addition, ERG can bind BRCA1, a co-activator of the AR, and together were shown to regulate IGFR expression (Chai et al., 2001; Schayek et al., 2009). IGFR expression eventually leads to activation of AKT upon IGFR ligand-binding. ERG regulates MMPs thus influencing extracellular matrix (ECM) remodelling and the invasive potential of the cell (Ellett et al., 2009; Hawksworth et al., 2010; Singh et al., 2002; Schulz et al., 2010). Recently, Carver and colleagues have identified ETS binding sites in the promoter regions of CXCR4 and ADAMTS1, two genes involved in cellular motility and invasion (Carver et al., 2009a). Furthermore, it was shown that ERG directly upregulates the expression level of CXCR4 and ADAMTS1 (Carver et al., 2009b). Together, these studies provide a compelling evidence for a central, functional role of the fusion gene in the biology of prostatic carcinoma.

V. Fusion variants The TMPRSS2:ERG fusion gene constitutes the majority (>85%) of ETS rearrangements in CaP, likely owing to their close proximity (2.7 Mb) and identical orientation on chromosome 21. Although, the remainder of this review will predominately focus on the TMPRSS2:ERG rearrangement, numerous alternative ETS members also can fuse to TMPRSS2, albeit at a much lower frequency. Similarly, variability in 5' partners have also been identified (Table 1). An

intensive study examining ETV1 rearrangements found that only 9 of 23 samples had previously identified 5' partners, demonstrating the dramatic variability in fusion partners pairing with ETS transcription factors other than ERG (Attard et al., 2008b). The combined frequency of the remaining fusion variants accounts for approximately 10% of cases. Five prime partners are divided into classes based on their tissue specificity and sensitivity to androgens (Tomlins et al., 2007). Class I is reserved for TMPRSS2; Class II represents other prostate-specific androgen inducible 5' UTR or endogenous retroviral elements; Class III represents the prostate-specific but androgen repressed partners; Class IV represents the non-tissue-specific promoters that are ubiquitously expressed, (i.e. house-keeping genes-this class often forms a chimeric or fusion protein, unlike the previous divisions of gene fusions); and finally Class V consists of ETV1-specific rearrangements, including the localization of the entire ETV1 locus to prostate specific locus, 14q13.3-14q21.1 (Tomlins et al., 2007; Attard et al., 2008b). To date only a single study has identified fusion genes in CaP devoid of ETS transcription factor participation (Palanisamy et al., 2010).

VI. Detection and classification VI. i. FISH Two strategies for FISH experiments are frequently used to detect the TMPRSS2:ERG fusion gene in CaP. The three-colour break-apart strategy, developed by Yoshimoto et al. (2006), uses differentially labelled bacterial artificial chromosome (BAC) clones as probes for the 3' (RP11-476D17) and 5' (RP11-95I21) segments of ERG with an additional BAC probe specific for the 5' region of TMPRSS2 (RP11-535H11) or for the transcriptional regulatory sequences (telomeric) of TMPRSS2 (RP11-35C4, RP11-891L10; RP11-260O11; Figure 1) (Yoshimoto et al., 2006). Using this probe configuration enables not only the detection of ERG rearrangement, but also allows confirmation that ERG's coding sequences are juxtaposed to the transcriptional regulatory region of TMPRSS2. Moreover, the mechanism of rearrangement can also be deduced by this approach (Yoshimoto et al., 2006; Yoshimoto et al., 2007). Characterization of rearrangement method is essential as differential clinical impacts are observed with the various mechanisms resulting in ETS gene fusions.

1Table 1

5' partner Class 3' partner Initial reference

TMPRSS2 I ERG Tomlins et al.(2005)

TMPRSS2 I ETV1 Tomlins et al. (2005)

TMPRSS2 I ETV4 Tomlins et al. (2006)

HERV-K_22q11.23 II ETV1 Tomlins et al. (2007)



SLC45A3 II ETV1 Tomlins et al (2007)

C15orf21 III ETV1 Tomlins et al. (2007)

HNRPA2B1 IV ETV1 Tomlins et al. (2007)

KLK2 II ETV4 Hermans et al. (2008a)

CANT1 II ETV4 Hermans et al. (2008a)

ACSL3 II ETV1 Attard et al. (2008b)

SLC45A3 II ERG Han et al. (2008)

FLJ35294 II ETV1 Han et al. (2008)

DDX5 IV ETV1 Han et al. (2008)

TMPRSS2 I ETV5 Helgeson et al. (2008)

SLC45A3 II ETV5 Helgeson et al. (2008)

EST14 II ETV1 Hermans et al. (2008b)

HERVK17 II ETV1 Hermans et al. (2008b)

FOXP1 II ETV1 Hermans et al. (2008b)

SLC45A3 II ELK4 Rickman et al. (2009)

NDRG1 II ERG Pflueger et al. (2009)

SLC45A3 ETS neg BRAF* Palanisamy et al. (2010)

ESRP1 ETS neg RAF1* Palanisamy et al. (2010)

*Only 3' partners identified to date that are not a member of the ETS transcription factor family.

Figure 1: In house BAC probe configuration for three-colour break-apart TMPRSS2:ERG gene fusion FISH This schematic ideogram depicts the positions of differentially labelled bacterial artificial chromosome (BAC) clones specific for 3' and 5' regions of the ERG gene (RP11-476D17 in spectrum orange and RP11-95I21 in spectrum green, respectively), within the 21q22.2-3 region. Telomeric to this, the TMPRSS2 gene locus is represented by RP11-535H11 (spectrum red) which spans the gene, or by three BAC clones downstream (telomeric) of the TMPRSS2 gene (RP11-35C4, RP11-891L10 and RP11-260O11 in spectrum aqua). This probe design permits the accurate identification of TMPRSS2:ERG gene fusions as well as ERG rearrangements independent of fusion with TMPRSS2 fusion.

Work by Attard and colleagues classified the TMPRSS2:ERG rearrangement mechanisms according to the pattern of interphase FISH signals (Attard et al., 2008a). Class N describes the normal ERG locus, therefore co-localization of the two ERG probe signals in close proximity to the TMPRSS2 signal (less than one signal diameter) (Figure 2A). The majority of fusion-positive cases present with a heterozygous deletion at cytoband 21q22.2-3, termed Class Edel (Figure 2B). The deletion typically spans ERG, exons

1-3 and a partial deletion of exon 4, the known intervening genes (based on RefSeq Genes: NCRNA00114, ETS2, PSMG1, BRWD1, NCRNA00257, HMGN1, WRB, LCA5L, SH3BGR, C21orf88, B3GALT5, IGSF5, PCP4, DSCAM, C21orf130, MIR3197, BACE2, PLAC4, FAM3B, MX2, and MX1) and the coding exons of TMPRSS2. The three-colour FISH of an Edel rearrangement displays co-localization of the 3' ERG and TMPRSS2 signals, and absence of the 5' ERG signal. Less



frequently a genomic rearrangement leading to insertion of those sequences elsewhere in the genome to an unknown chromosome location can occur resulting in the separation of the 5' ERG signals from the co-localization of the 3' ERG and TMPRSS2 signals, thus described as ERG split or Class Esplit

(Figure 2C). In both types of TMPRSS2:ERG rearrangements the unaffected chromosome 21 generally display a Class N signal configuration. Finally, additional copies of TMPRSS2:ERG gene fusions is identified as Class 2+Edel (Attard et al., 2008a).



Figure 2: Classification of TMPRSS2:ERG gene fusion by interphase FISH.

Prostate cancer patient samples were hybridized with the three-colour probe set, described in Figure 1, and counterstained with DAPI. The nuclei of interest (in dashed boxes) are magnified in the insets. A) Class N, where no ERG rearrangement has occurred. Co-localization of 3' and 5' ERG probes (often visualized as a yellow signal), with the 5' TMPRSS2 BAC probe signals (red) indicates a normal Chr21q22.2-3 locus. Frequently, the signals for the TMPRSS2 probe, situated 2.7 Mb away from the ERG locus, may be separated from the ERG signals by up to one probe signal width. B) Class Edel is characterized by the co-localization of 3' ERG probe to the TMPRSS2 probe signals, and the absence of the 5' ERG signal. This represents rearrangement with the loss of the intervening sequence. The unaffected Chr21 displays Class N configuration. C) Class Esplit is characterized by the co-localization of the 3' ERG and TMPRSS2 signals, with the retention of the 5' ERG signal elsewhere in the nucleus. The unaffected Chr21 displays Class N configuration.

The second strategy employs a two-colour break-apart FISH design to identify rearrangements in specific ETS genes (Mehra et al., 2007; Zhang et al., 2010). By this method, confirmation of fusion between two genes and identification of the specific 5' partner is not possible because the probes are specific for a single gene, and therefore, this strategy is an indirect method for the detection of fusion genes in CaP.

VI.ii. Other methods of detection RT-PCR is another technique frequently employed to determine the fusion status of prostatic tissue samples. However, this approach is limited to the detection of the hybrid transcripts, and is unable to obtain important cytogenetic information such as the genomic mechanism that generated the gene fusion (Edel vs Esplit). To date, as many as 17 fusion transcripts and splice variations have been characterized, the most common fusion transcript is composed of exon 1 of TMPRSS2 fused to exon 4 of ERG (T1:E4 (Wang et al., 2006; Jhavar et al., 2008)). The majority of the remaining transcripts result in truncation of the ERG

protein product by 39-99 amino acids, while the few that initiate translation from the native start codon in exon 3 produce full length ERG (Tomlins et al., 2005; Soller et al., 2006; Wang et al., 2006; Clark et al., 2007; Clark et al., 2008a). One of the transcripts produces a genuine TMPRSS2:ERG fusion protein and eight contain premature stop codons and are unlikely to result in ERG overexpression (Tomlins et al., 2005; Soller et al., 2006; Wang et al., 2006; Clark et al., 2007; Clark et al., 2008a). These studies further revealed that elaborate heterogeneity exists in hybrid transcripts present, between foci and within individual tumour foci of the same patient. Variability in the translation start site consequentially affects the size of the mRNA transcript (373-885 bp) and therefore the protein product potentially modifying the functional capacity of the gene fusion product (Wang et al., 2006). Distinct transcript variants display differential prognostic influence based on the resultant biological activities (Hermans et al., 2009; Wang et al., 2008). A significant challenge remains in relating the clinical



prognosis to the fusion gene and will be addressed in the subsequent section. Indirect methods suggestive of gene fusions in CaP are being employed to broadly determine ETS rearrangement status with perhaps the anticipation of implementing a high-throughput method of detection. Analysis of array comparative genomic hybridization (aCGH) data, specifically the 21q22.2-3 region may permit identification of Class Edel TMPRSS2:ERG gene fusions (Watson et al., 2009; Ishkanian et al., 2009). Class Esplit retains the intervening sequences within the nuclei, in a copy neutral manner and consequently aCGH cannot identify this rearrangement. Overexpression of ETS transcription factors by gene expression microarrays may also imply an ETS rearrangement has occurred. Due to inconclusive results, FISH or RT-PCR assays are necessary to validate microarray findings pertaining to the ETS gene fusions. A novel multiplexing technology recently developed uses nanostructured microelectrodes integrated onto a chip and has the capability to detect disease-specific biomarkers, including differentiation of various fusion transcripts (Fang et al., 2009). More recently, immunohistochemistry (IHC) is also being explored as a means to detect the fusion by ERG protein overexpression, in the absence of confirming fusion at the genomic or transcript level (Furusato et al., 2010; Park et al., 2010). Of the numerous direct (three-colour FISH, RT-PCR) and indirect (two-colour FISH, microarrays, IHC) methods employed to determine fusion status to date, the three-colour FISH yields the most cytogenetic and genomic information. In addition, to identifying the fusion status and class, the inherent cell-by-cell analysis addresses the heterogeneous nature of the disease, and provides a biological context for such information.

VII. Fusion gene formation and chromosomal instability There is increasing research interest and effort focusing on the genomic events and attributes that lead to the formation of ETS gene fusions. A complex TMPRSS2:ERG rearrangement found in a single patient was meticulously examined and described by Yoshimoto et al. (2007). This patient had a microdeletion of the sequences from 5' ERG to and including the TMPRSS2 coding sequences with a concurrent translocation of the region immediately telomeric of 5' untranslated TMPRSS2 sequences. A detailed multicolour FISH assay mapped the region between ERG and TMPRSS2, revealing the complexity of chromosomal rearrangements that can lead to the formation of fusion genes in CaP. This case demonstrates the valuable information available through the use of complex multicolour FISH assays. The molecular mechanisms that underlie this recurrent translocation are just beginning to be understood. For example, fine mapping of the deletion breakpoints located within the ERG and TMPRSS2 loci, followed

by sequencing, revealed the presence of consensus sequences homologous to the human Alu-Sq and Alu-Sp subfamily (Liu et al., 2006). The presence of these consensus sequences within intronic regions correlated with the presence of the fusion gene and may be a factor contributing to the deletion at 21q22.2-3, resulting in the fusion gene. More recently, genotyping of familial CaP revealed that the fusion gene associates with polymorphisms in DNA repair genes, specifically POLI and ESCO1 (Luedeke et al., 2009). Using FISH, the AR was shown to induce chromosomal proximity of TMPRSS2 and ERG by binding to the promoter region of TMPRSS2 (Mani et al., 2009). Subsequently, LnCaP cells were irradiated to induce double-strand breaks inducing the formation of the TMPRSS2:ERG gene fusion upon dihydrotestosterone stimulation in the previously fusion-negative cell line (Mani et al., 2009). The DNA-bound AR is also implicated in chromatin architecture modifications that can cause double-strand breaks, commonly repaired by non homologous end joining machinery, and may result in the formation of gene fusions (Lin et al., 2009). Also recently, androgen signalling was shown to recruit topoisomerase II beta with the AR to the breakpoints resulting TMPRSS2:ERG fusion genes (Haffner et al., 2009). Undoubtedly, specific nucleotide sequences within the 21q22.2-3 region are of great importance and further elucidation as to their role in the formation of gene fusions is essential. The TMPRSS2:ERG gene fusion is an unique model to query sequence level polymorphisms that may lead to the formation of intra-chromosomal rearrangements largely due to the close proximity and orientation of the involved genes as well as the high rate of recurrence. Chromosomal instability, defined as the formation of novel chromosome alterations and rearrangements at an elevated rate, compared to normal cells may also be a factor contributing to the formation of ETS gene fusions in CaP. It is well established that deletion of the tumour suppressor PTEN (10q23.31), a common genomic aberration in CaP, results in an elevated level of chromosomal instability through activation of AKT. One of sequelae of this change is the phosphorylation and inhibition of the cell cycle check point kinase 1 (Chk1), an important kinase preventing cell cycle progression in response to DNA damage (Puc et al., 2005; Sanchez et al., 1997). Furthermore, nuclear PTEN interacts with kinetochore proteins and induces the expression of RAD51, a protein required to reduce the incidence of spontaneous double-strand breaks (Shen et al., 2007). PTEN deficiency ultimately alters multiple cell cycle checkpoints, which could potentially delay DNA damage repair and/or chromosome segregation (Gupta et al., 2009). Overall, PTEN has a variety of roles in maintaining chromosomal stability and integrity, and the recurrent PTEN loss in CaP may represent an important trigger in the events leading to the formation of TMPRSS2:ETS gene fusions.



VIII. Heterogeneity of multifocal disease CaP, one of the most heterogeneous epithelial carcinomas, is also notoriously multifocal in nature. As a multifocal disease, CaP permits the investigation and comparison of recurrent genomic events within distinct foci of individual patients. In 32 RP specimens with spatially distinct foci, ERG rearrangement status was examined using two-colour FISH (Barry et al., 2007). Nineteen samples displayed interfocal homogeneity, with 80% of the samples negative for ERG rearrangement. The remaining 13 samples exhibited interfocal heterogeneity but intrafocal homogeneity, with two samples harbouring three separate foci each with a different rearrangement status: Class N, Edel and Esplit. Similarly, whole mount examination of RP specimens for fusion transcripts yielded multiple fusion transcripts in individual CaP foci with an identical hybrid transcript profile in the adjacent HGPIN (Furusato et al., 2008). Two other studies examining fusion transcript variants found different transcript hybrids in discrete regions of a single prostate (Wang et al., 2006; Clark et al., 2007). Multiple TMPRSS2:ETS gene fusion-positive foci may arise independently and exhibit interfocal heterogeneity. Investigation of ERG rearrangement in multifocal CaP and corresponding metastases provides a glimpse of the potential biological impact of this aberration in the context of disease progression (Perner et al., 2009). This study reported that the metastatic lesion was always positive for ERG rearrangement through the same mechanism (Edel vs Esplit) as that present in at least one of the prostatic tumour foci (not necessarily the index focus). The authors suggest that this rearrangement may be a factor contributing to the development of metastases, regardless of clinicopathological criteria of the individual foci.

IX. Prognostic significance There exists appreciable controversy with respect to the prognostic significance of the TMPRSS2:ERG gene fusion in CaP, with studies suggesting that the fusion gene has a favourable (Winnes et al., 2007; Petrovics et al., 2005; Winnes et al., 2007; Saramaki et al., 2008), unfavourable (Yoshimoto et al., 2008; Mehra et al., 2007a; Perner et al., 2006; Wang et al., 2006; Nam et al., 2007a; Nam et al., 2007b; Attard et al., 2008a; Mehra et al., 2008; Reid et al., 2010; Demichelis et al., 2007; Rostad et al., 2009; Lapointe et al., 2007a) or no association (Mehra et al., 2008; Yoshimoto et al., 2006; Neill et al., 2007; Fletcher et al., 2008; Dai et al., 2008; Furusato et al., 2008; Darnel et al., 2009; Gopalan et al., 2009; Rubio-Briones et al., 2010; Fitzgerald et al., 2008; Lee et al., 2010; Sun et al., 2010; Rouzier et al., 2008) with clinical outcomes. When no association between clinicopathological criteria and the presence of the fusion gene was found, it was often attributable to small sample sizes. The most compelling evidence

suggests a trend towards unfavourable factors outcome in CaP progression. Notably, an interesting comparison of two studies looking at conservatively managed men had opposing results from an association with CaP-specific death (Demichelis et al., 2007) to no reduction in CaP-specific survival for patients harbouring this rearrangement (Fitzgerald et al., 2008). The role of deletion (Edel) or retention (Esplit) of intervening sequences in tumour biology has also been scrutinized with relatively uniform results suggesting that Edel is not only the more prevalent mechanism but is also associated with more aggressive disease (Mwamukonda et al., 2010; Attard et al., 2008a; Mehra et al., 2008). Edel was associated with worse prostate-specific survival and shorter time to biochemical recurrence than Class Esplit (Yoshimoto et al., 2006; Attard et al., 2008a). While, Class 2+Edel was associated with lethal disease and significantly worse clinical outcome, particularly when combined with prostate-specific clinicopathological criteria (Yoshimoto et al., 2006; Attard et al., 2008a). Another study also demonstrated that Edel rearrangements have a more aggressive tendency since all fusion-positive androgen-independent metastases had lost 5' ERG sequences (Mehra et al., 2008) and Edel was associated with clinically aggressive features of progression (Perner et al., 2006). These findings are consistent with elevated ERG expression and led investigators to speculate that the loss of 5' ERG is associated with aggressive CaP. It must be considered that the commonly deleted 2.7 Mb between ERG and TMPRSS2 could contain important tumour suppressor genes (Yoshimoto et al., 2006) and its loss in the Edel rearrangement leads to haploinsufficiency of a tumour suppressor gene that underlies the aggressive clinical course. One gene candidate for this effect within this region, HMGN1, a nucleosome binding protein has previously been associated with CaP progression (Birger et al., 2005). In agreement with this view, the Esplit rearrangement has not yet been shown to associate with any particular clinical outcome to date. However, given the low frequency of this event (~10% of TMPRSS2:ERG rearrangements), Esplit rearrangements cannot be excluded as a measure of prognosis until further studies are completed (Attard et al., 2008a). On the other hand, one investigation demonstrated no statistically significant association with clinicopathological criteria and Edel or Esplit, but evidence did suggest 2+Edel is a factor contributing to CaP-specific mortality (Fitzgerald et al., 2008). Conversely, TMPRSS2:ERG rearrangement alone were associated with lower histological grade, but no other clinical features or CaP-specific death (Gopalan et al., 2009). However, the same group also found that copy number increases (CNI) of the ERG locus with or without TMPRSS2:ERG gene fusions was associated with high grade and advanced stage, while cancers with CNI and 2+Edel rearrangements tended to be more



clinically aggressive (Fine et al., 2010). While the above results are conflicting to the previous studies that showed no association with extra copies of the unrearranged ERG locus (Attard et al., 2008a), the difference may be due to the deletion of the intervening genes potentially generating a more aggressive tumourigenic phenotype. SPINK1 (5q32) is a gene identified in a more recent COPA meta-analysis as being overexpressed in 10% of CaP, all of which are exclusively ETS fusion negative (Tomlins et al., 2008b). For this reason, SPINK1 expression and the TMPRSS2:ERG fusion gene were evaluated to determine their relative significance as prognostic biomarkers in biopsy samples from hormonally treated men (Leinonen et al., 2010). In this cohort, the fusion gene was associated with Ki-67 staining, age at diagnosis and tumour area, but not with any prostate-specific clinicopathological criteria (Leinonen et al., 2010). On the other hand, cases that overexpressed SPINK1 had a significantly shorter time to biochemical recurrence, but no association with any other criteria.

X. Clinical utility Promptly following the emergence of adverse clinical correlations of fusion-positive CaP Mosquera and colleagues evaluated a series of CaP cases to determine if there was a morphological phenotype associated with this genomic alteration (Mosquera et al., 2007). Blinded to ERG rearrangement status, the group identified five histologic criteria that were significantly related to ERG rearrangement positive samples: blue-tinged mucin, cribriform growth, macronucleoli, intraductal tumour spread, and signet-ring cell features. Only 24% of ERG rearranged samples did not identify with any of the above mentioned morphological features and 93% of samples with three features were positive for ERG rearrangement. The authors speculate that the morphological characteristics shared by ERG rearranged tumours could result from ETS dysregulated pathways and could be utilized in the routine assessment performed by pathologists. More recently, the same group published a similar set of morphological features with the addition of collagenous micronodules (Mosquera et al., 2009). A significant association between perineural invasion, blue-tinged mucin, and intraductal tumor spread with a positive gene fusion status has been documented (Nigwekar et al., 2008), while another study confirmed the association that ETS fusion-positive CaP samples were more likely mucin-positive than mucin-negative (Tu et al., 2007). These results should be taken, however, in the context of somewhat contradictory reports, such that the TMPRSS2:ERG gene fusion correlates with low Gleason grade and is inversely related to high-grade morphological features. These studies hold promise that morphological markers routinely examined by pathologists could be coupled with the current clinicopathological criteria for a more

comprehensive evaluation of biopsy or RP specimens providing well-informed judgments on diagnosis, prognosis and treatment decisions. In an attempt to optimize the clinical utility of this biomarker, investigators are turning to less invasive survey modes, such as urine specimens and circulating tumour cells (CTCs). Post-digital rectal exam (DRE) urine analyzed by qRT-PCR found 17.2% positive rate for ERG overexpression. Subsequent examination of the corresponding biopsy samples from the same cohort revealed a 40% positive rate (Rice et al., 2010). The clinical yield of this assay can be improved, as indicated by another study which found a 69% positive rate following prostatic message versus only 24% when no prostatic message was performed before collecting the urine samples (Rostad et al., 2009). Two additional studies report similar results (42% and 59%) of fusion-positive transcripts in post-DRE urine (Hessels et al., 2007; Laxman et al., 2006). Expressed prostatic secretion has also been a successful specimen for the non-invasive detection of fusion transcripts (Clark et al., 2008b). These studies demonstrate the potential for ETS gene fusion detection using non-invasive approaches following physical evaluation of the prostate by DRE, adding valuable information to disease stratification prior to radical treatments. A handful of studies have also used FISH to detect ERG rearrangement in CTCs in effort to monitor disease recurrence or treatment efficacy (Mao et al., 2008; Attard et al., 2009; Stott et al., 2010).

XI. Role of ETS in prostate tumourigenesis: Driver? Discrepancies regarding the fusion gene are not limited to its diagnostic potential and prognostic significance. Controversy also exists in the sequence of genomic and molecular alterations in CaP initiation and progression. Several groups speculate that the formation of the TMPRSS2:ERG fusion gene is required for CaP initiation in ETS positive CaP tumours, with other aberrations occuring later in the course of disease advancement and metastasis (Perner et al., 2007; Tomlins et al., 2007; Attard et al., 2009). Recently, Attard and colleagues demonstrated that CTCs from individual castration-resistant CaP patients were either clonally positive or negative for ERG rearrangement, however, loss of PTEN and increase in AR gene copy number was heterogeneous in the CTCs derived from a single patient (Attard et al., 2009). These results suggest that the formation of the fusion gene occurred before PTEN loss and gain of the AR locus. However, this study was performed on a very small cohort of castration-resistant CaP and may not necessarily reflect the sequence of accumulating genomic alterations in CaP. TMPRSS2:ETS fusion status in HGPIN occurs at a low frequency, and almost exclusively only when juxtaposed to fusion-positive CaP. These HGPIN lesions, however, do not exhibit the chromosomal copy



number changes seen in 42% of the paired CaP as assessed by CGH (Cerveira et al., 2006). These results propose that formation of the fusion gene may precede chromosomal-level alterations and is consistent with the literature indicating that few gross chromosome- or arm-level chromosomal copy alterations are present in localized CaP. Another study investigating a range of prostate tissues comprising benign, precursor, malignant and metastatic samples found that the majority of patient samples examined showed homogeneity with respect to fusion status and mechanism (Edel vs Esplit). The presence of fusion-positive HGPIN was interpreted as additional evidence that this entity is a true-precursor to CaP, a notion that has eluded indisputable proof (Perner et al., 2007). In contrast, a recent study that examined both PTEN genomic loss and fusion status by FISH observed PTEN loss in benign and HGPIN lesions, while ERG rearrangement was identified in HGPIN, albeit at a lower frequency than PTEN loss, and absent in BPH lesions (Bismar et al., 2010). Maintaining this model of progression and accumulation of genomic changes in CaP it is possible that heterozygous loss of PTEN may be a 'driver lesion', in a subset of CaP, and PTEN haploinsufficiency may facilitate the selective formation of the fusion gene (Figure 3). TMPRSS2:ERG gene fusion may occur as a secondary alteration and may function as an 'enhancer' permitting the cell to achieve a higher level of aggressiveness and invasiveness. When this sequence of events was emulated in transgenic mice, ERG overexpression resulted in marked acceleration of preneoplastic lesions to invasive CaP on a Pten deleted background, but ERG overexpression alone simply displayed slight atypical histology compared to control mice (Carver et al., 2009a; Carver et al., 2009b). In vitro experiments using cell lines demonstrated ERG overexpression provided enhanced motility without affecting proliferation, in agreement with Tomlins et al. (2007); Carver et al. (2009b). These findings corroborate the view that effectors of ERG overexpression affect cellular processes which are complimentary to unregulated

factors downstream of AKT as a result of PTEN deficiency, but on their own are not sufficient to provoke the transition from benign to neoplastic. The implication is not that PTEN loss is the driver in CaP, but that ETS gene fusions are enhancer alterations significantly affecting cellular processes further progressing prostatic tumourigenesis when the background is primed first by a driver event capable of initiating sufficient dysregulation leading to the development of a preneoplastic lesion. However, because PTEN loss and the presence of the fusion gene are significantly associated events in CaP (Yoshimoto et al., 2008; Carver et al., 2009a; Han et al., 2009; King et al., 2009; Bismar et al., 2010) it is likely that PTEN inactivation may be an important driver lesion for fusion-positive CaP. Together the driver event and ETS overexpression lead to a significantly aggressive and invasive lesion. Therefore, elucidation of the cellular pathways affected by ETS overexpression is fundamental to the comprehension of the aggressive nature observed in the majority of fusion-positive CaP, and to developing novel therapeutic strategies to specifically target this subset of CaP.

XII. Concluding remarks Difficulty in assigning prognostic significance, diagnostic and therapeutic utility to ETS gene fusions is a result of a myriad of factors including, the heterogeneity of the disease, the mechanism of rearrangement (Edel vs Esplit), the technique used to assess the presence of the fusion as well as the cohort examined. Nevertheless, continued controversy between positive and negative clinical associations of the gene fusion dictates further studies are required using larger cohorts to determine the absolute potential of this genomic aberration as a biomarker for CaP diagnostic utility, prognostic significance, and stratification of patients to aid in treatment decisions. Furthermore, comprehension of the pathways affected by ETS overexpression will aid in the potential of implementing ETS specific therapies to target this aggressive subtype of CaP and benefit many patients.



Figure 3: Model of prostate cancer progression showing ETS gene fusions as an enhancer lesion. Cooperation of unregulated pathways downstream of PTEN with effectors of ERG overexpression is likely a crucial event in the progression of an invasive and aggressive prostatic adenocarcinoma. Heterozygous genomic deletion of PTEN in benign prostatic precursors may represent an early event, and act as a driver lesion leading to proliferation, survival and genomic instability-all initial requisites of cancer. As a consequence of such heightened genomic instability, PTEN haploinsufficiency may facilitate the selective formation of the fusion gene with consequent acquisition of additional invasive properties. The presence of both rearrangements within a lesion is associated with accelerated disease progression and poor prognosis, indicating that synergistic molecular interactions exist between their complementary pathways. Continuing instability generates genotypic heterogeneity and diversity, such that subclones bearing PTEN homozygous deletions and amplified AR loci have further selective advantage for aggressive tumour progression, androgen escape and metastases.



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Pflueger D, Rickman DS, Sboner A, Perner S, LaFargue CJ, Svensson MA, Moss BJ, Kitabayashi N, Pan Y, de la Taille A, Kuefer R, Tewari AK, Demichelis F, Chee MS, Gerstein MB, Rubin MA.. N-myc downstream regulated gene 1 (NDRG1) is fused to ERG in prostate cancer. Neoplasia. 2009 Aug;11(8):804-11.

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Rickman DS, Pflueger D, Moss B, VanDoren VE, Chen CX, de la Taille A, Kuefer R, Tewari AK, Setlur SR, Demichelis F, Rubin MA.. SLC45A3-ELK4 is a novel and frequent erythroblast transformation-specific fusion transcript in prostate cancer. Cancer Res. 2009 Apr 1;69(7):2734-8. Epub 2009 Mar 17.

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Barwick BG, Abramovitz M, Kodani M, Moreno CS, Nam R, Tang W, Bouzyk M, Seth A, Leyland-Jones B.. Prostate cancer genes associated with TMPRSS2-ERG gene fusion and prognostic of biochemical recurrence in multiple cohorts. Br J Cancer. 2010 Feb 2;102(3):570-6. Epub 2010 Jan 12.

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Esgueva R, Perner S, J LaFargue C, Scheble V, Stephan C, Lein M, Fritzsche FR, Dietel M, Kristiansen G, Rubin MA.. Prevalence of TMPRSS2-ERG and SLC45A3-ERG gene fusions in a large prostatectomy cohort. Mod Pathol. 2010 Apr;23(4):539-46. Epub 2010 Jan 29.

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Fine SW, Gopalan A, Leversha MA, Al-Ahmadie HA, Tickoo SK, Zhou Q, Satagopan JM, Scardino PT, Gerald WL, Reuter VE.. TMPRSS2-ERG gene fusion is associated with low Gleason scores and not with high-grade morphological features. Mod Pathol. 2010 Oct;23(10):1325-33. Epub 2010 Jun 18.

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Hawksworth D, Ravindranath L, Chen Y, Furusato B, Sesterhenn IA, McLeod DG, Srivastava S, Petrovics G.. Overexpression of C-MYC oncogene in prostate cancer predicts biochemical recurrence. Prostate Cancer Prostatic Dis. 2010 Dec;13(4):311-5. Epub 2010 Sep 7.

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Lee K, Chae JY, Kwak C, Ku JH, Moon KC.. TMPRSS2-ERG gene fusion and clinicopathologic characteristics of Korean prostate cancer patients. Urology. 2010 Nov;76(5):1268.e7-13. Epub 2010 Aug 30.

Leinonen KA, Tolonen TT, Bracken H, Stenman UH, Tammela TL, Saramaki OR, Visakorpi T.. Association of SPINK1 expression and TMPRSS2:ERG fusion with prognosis in endocrine-treated prostate cancer. Clin Cancer Res. 2010 May 15;16(10):2845-51. Epub 2010 May 4.

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Williams JL, Yoshimoto M, Boag AH, Squire JA, Park PC. TMPRSS2:ETS gene fusions in prostate cancer. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):699-716.



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