“The importance of integrin-linked kinase in the regulation ofbladder cancer invasion”

National Chiayi University Dep. Of Microbiology, Immunology & Biopharmaceiticals.

International Journal of Cancer :130, 521–531 (2012)Oncology: 19/197

Impact factor: 6.198

Date: 21.03.2014

Advisor: Speaker: Yi-Wen Liu,PhD Mezbahul Haque

Professor Master Student

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INTRODUCTION

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INTEGRIN

Integrins are transmembrane receptors that mediate the attachment between a cell and its surroundings, such as other cells or the extracellular matrix (ECM).

Integrins consist of two non-covalently linked transmembrane glycoprotein subunits.

All are heterodimeric and consist of one alpha and one beta subunit. 18 alpha subunits identified 8 beta subunits identified

Both of the subunits contribute to the binding of ligand.

http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/CellAdhesion/integrinstructure.htm

From: http://www.scq.ubc.ca/

http://en.wikipedia.org/wiki/Integrin

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INTEGRIN ACTIVATION AND FUNCTION

Integrin functions: Attachment of the cell to the ECM Signal transduction from the ECM to the cell

http://en.wikipedia.org/wiki/Integrin

Also involve Immune patrolling, cell migration, and binding to cells by certain viruses, such as adenovirus, echovirus, hantavirus, and foot and mouth disease viruses.

Activation:Integrins can exist in different ligand affinity states - low, intermediate and high affinity.

a) Inside-out signalingb) Outside-in signaling

http://www.mechanobio.info/modules/go-0033622

From: www.mechanobio.inf

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INTEGRIN-LINKED KINASE

Integrin-linked kinase (ILK) is one kind of protein, originally identified while conducting a yeast-two hybrid screen with integrin β1 as the bait protein.

Activation: Cyclin D1 expression Activation of MMP9 Supression of E-cadherin AKT activation

Function: ILK has been shown to play crucial roles in actin rearrangement. Cell polarization, spreading, migration, proliferation and survival. It also reside in cell–cell adhesion sites, in centrosomes and in the nucleus. It demonstrating that ILK serves as a regulator protein rather than a kinase.

Journal of Cell Science 125, 1839–1843

Nature Reviews Cancer 5, 51-63 (January 2005)

Activator

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INTEGRIN-LINKED KINASE AND CANCER

ILK plays critical roles in development cell motility, adhesion- depending signaling, cytoskeleton recognization and tumor invasion. Cellular Microbiology (2006) 8 (2), 257–266

Dysregulation of ILK signaling is an important early event in the genesis of human colon cancer.

British Journal of Cancer (2003) 88(11), 1755 – 1762

Expression of integrin-linked kinase is closely correlated with invasion and metastasis of gastric carcinoma.

Virchows Arch (2003) 442:118–123

ILK expression increases dramatically with melanoma invasion and progression.

Clin Cancer Res 2003;9:4409-4414.

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EMT (Epithelial–mesenchymal transition)

From: Wiki

Epithelial cells lose their cell polarity and cell-cell adhesion.

Repression of E-cadherin expression and allow the cells increased mobility.

Gain migratory and invasive properties to become mesenchymal stem cells.

Transcriptional factors: Zeb1, Snail and Slug are repressors of E-cadherin, The expression of these transcription factors induces EMT.

Characteristics of Mesenchymal Cells: Lack regimented structure. Few tight intercellular adhesions. Weak adhesions which allow for ease of mobility. Forms irregular structures that are not uniform in composition or density. More extended and elongated in shape.

http://cnx.org/content/m36053/latest/?collection=col10523/latest

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EMT AND METASTASIS

Modified From: archive.osc.edu Modified From: www.discoverymedicine.com

Epithelial–mesenchymal transition (EMT) has been implicated as having a role in tumor invasion/migration and metastasis.Loss of E-cadherin expression is a hallmark of the EMT process.

Cell. 2008 May 16; 133(4): 704–715.

Ligand

ILK

APC

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E-cadherin

In epithelial cells, E-cadherin-containing cell-to-cell junctions are often adjacent to actin-containing filaments of the cytoskeleton.

E-cadherin downregulation decreases the strength of cellular adhesion within a tissue, resulting in an increase in cellular motility. This in turn may allow cancer cells to cross the basement membrane and invade surrounding tissues.

E-cadherin is also used by pathologists to diagnose different kinds of breast cancer.

http://en.wikipedia.org/wiki/CDH1_%28gene%29

Cadherins are a class of type-1 transmembrane proteins. E-cadherins are found in epithelial tissue. E-cadherin is a protein that in humans is encoded by the CDH1 gene. It is a tumor suppressor gene.

From: http://www.intechopen.com

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E-cadherin and Cancer

http://en.wikipedia.org/wiki/CDH1_%28gene%29

E- Cadherin in metastasis: E-cadherin acts as an invasion suppressor and a classical tumor

suppressor gene in pre-invasive lobular breast carcinoma. Mutations in this gene are correlated with gastric, breast,

colorectal, thyroid, and ovarian cancers. Loss of function is thought to contribute to progression in cancer

by increasing proliferation, invasion, and/or metastasis.

Cancer examples: Inactivation of CDH1 in 50% of diffuse gastric carcinomas. Complete loss of E-cadherin protein expression in 84% of lobular

breast carcinomas.

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BBN and bladder cancer

Butyl-(4-hydroxybutyl) nitrosamine (BBN) has been shown to be

a highly potent and specific bladder carcinogen.

Bladder carcinomas were found in all mice, other toxic effects were absent.

A 100% incidence of tumors can be induced by continuous and prolonged administration of BBN in drinking water. This compound can also be administered by oral gavages.

Eur J Cancer (1972)8:587–594.

IARC Sci Publ 1990; 99: 345–97

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BACKGROUND

Numerous cancers have now been described to undergo changes in levels of expression of ILK consequent upon acquisition of increasingly more malignant properties.

Nat RevCancer 2005;5:51–63

ILK also have been reported for colon, pancreas, prostate and gastric cancers, as well as melanoma.

Br J Cancer 2003; 88:1755–62 Clin Cancer Res 2003;9:4409–14

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OBJECTIVE

To evaluate the importance of integrin-linked kinase in bladder cancer progression.

To investigate the mechanism of the integrin-linked kinase signal in bladder cancer invasion.

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MATERIALS

Cell lines: 253J, TCCsup, KK47, J82, UMUC3, KU7, MGH-U3, 5637, T24, RT4, RT112. SV40-transformed urothelial cell line (SV-HUC1)

BBN Mouse model: Murine bladder cancers were induced by oral BBN.Six- to eight-weeks old C57BL/6 mice were given drinking water with 0.025% BBN. 8 weeks for bladder carcinoma in situ. 20 weeks for invasive bladder cancer.

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METHODS

1. Cell viability assay.2. Cell migration and invasion assay.3. Western blot analysis.4. Zymography5. Mouse bladder cancer specimen and tissue microarray immunohistochemistry.6. Statistical analyses.

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RESULTS

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FLOW CHART

Suppression of E-cadherin and promotion of cell invasion bladder cancer cells

Effect of Integrin-linked kinase suppression

Evaluation of the relationship between the pathological stage and ILK, E-cadherin and MMP-9 expression.

Integrin-linked kinase expression in cancer cell

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Expression of ILK and various epithelial and mesenchymal markers in bladder cancer cell lines and expression of ILK in a mouse model.

Bladder cancer cell lines BBN mouse model

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SUMMARY

Invasive bladder cancer cells tend to have high expression of integrin-linked kinase.

ILK expression may play some role in the EMT of bladder cancer through E-cadherin regulation.

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FLOW CHART

Integrin-linked kinase expression in cancer cell

Suppression of E-cadherin and promotion of cell invasion in bladder cancer cells

Effect of Integrin-linked kinase suppression

Evaluation of the relationship between the pathological stage and ILK, E-cadherin and MMP-9 expression.

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The change of EMT markers with ILK S343D over expression and cell viability.

ILK S343D : Active ILK expressing vector

253J cells : Transfection efficiency and epithelial characterization.

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Cell migration and Cell invasion assay

Cell migration Cell invasion

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Effect in inactivation of GSK3ß with LiCl

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SUMMARY

Overexpression of ILK suppresses E-cadherin expression and promotes cell invasion in 253J bladder cancer cells.

LiCl may reduce proliferation in general, obscuringphenotypic change from induced Zeb1.

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FLOW CHART

Suppression of E-cadherin and promotion of cell invasion bladder cancer cells

Effect of Integrin-linked kinase suppression

Evaluation of the relationship between the pathological stage and ILK, E-cadherin and MMP-9 expression.

Integrin-linked kinase expression in cancer cell

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The change in cell viability, migration and invasion of TCCsup and UMUC3 bladder cancer cells treated by ILK-siRNA.

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SUMMARY

ILK knockdown suppresses cell invasion in invasive bladder cancer cells through the regulation of E-

cadherin and MMP-9.

The ILK-GSK3b-Zeb1 pathway may be important inregulating the EMT of bladder cancer.

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FLOW CHART

Suppression of E-cadherin and promotion of cell invasion bladder cancer cells

Effect of Integrin-linked kinase suppression

Evaluation of the relationship between the pathological stage and ILK, E-cadherin and MMP-9 expression.

Integrin-linked kinase expression in cancer cell

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Immunohistochemical staining of ILK, E-cadherin and MMP-9 in a human bladder cancer TMA.

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SUMMARY

ILK expression correlates with the invasiveness of human bladder cancer.

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CONCLUTION

I L KI L K

I L KI L K

I L KI L K

I L KI L K

E-CadherinE-

CadherinE-CadherinE-

CadherinE-CadherinE-

CadherinE-CadherinE-

Cadherin

9MMP

9MMP

9MMP

9MMP

9MMP

9MMP

9MMP

9MMP

Integrin

ILK, E-Cadherin and 9MMP expression

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CONCLUTION

This study indicates that ILK is important in the EMT of bladder cancer, which regulates E-cadherin and MMP-9

expression.

This study also proposes that ILK may be a new target to suppress tumor progression.

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Thank you

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MMP9

Matrix metallopeptidase 9 (MMP-9), also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB), is an enzyme that in humans is encoded by the MMP9 gene.

FunctionProteases of the MMP family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, angiogenesis, bone development, wound healing, cell migration, learning and memory, as well as in pathological processes, such as arthritis, intracerebral hemorrhage, and metastasis. Most MMPs are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. The enzyme encoded by this gene degrades type IV and V collagens and other extracellular matrix proteins. Studies in rhesus monkeys suggest that the enzyme is involved in IL-8-induced mobilization of hematopoietic progenitor cells from bone marrow, and murine studies suggest a role in tumor-associated tissue remodeling.Thrombospondins, intervertebral disc proteins, regulate the effective levels of matrix metalloproteinases (MMPs) 2 and 9, which are key effectors of ECM remodeling.Clinical significanceMMP's play a role in inflammation associated with aortic aneurysms. Doxycycline suppresses the growth of aortic aneurysms through its inhibition of matrix metalloproteinase 9.MMPs such as MMP9 can be involved in the development of several human malignancies, as degradation of collagen IV in basement membrane and extracellular matrix facilitates tumor progression, including invasion, metastasis, growth and angiogenesis.

EMT IN TUMOR PROGRESSION

CHARACTERISTICS OF MESENCHYMAL CELLS

• Lack regimented structure• Few tight intracellular adhesions• Weak adhesions which allow for ease of mobility• Forms irregular structures that are not uniform in composition or density• More extended and elongated in shape

EMT MARKERS

• Proteins that increase in abundance

• N-cadherin• Vimentin• Fibronectin• Snail1 (Snail)• Snail2(Slug)• Twist• Goosecoid• FOXC2• Sox10• MMP-2• MMP-3• MMP9• Integrin vß6

• Proteins that decrease in abundance

• E-cadheren• Desmoplakin• Cytokeratin• Occludin• Proteins whose activity

increases• ILK• GSK-3ß• Rho• Proteins that accumulate

in the nucleus• ß-catenin• Smad-2/3• NF- ß• Snail1 (Snail)• Snail2 (Slug)• Twist

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ZYMOGRAPHY

Zymography is a electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. Three types of zymography are used; in gel zymography, in situ zymography and in vivo zymography

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TISSUE MICROARRAY

Tissue microarrays (also TMAs) consist of paraffin blocks in which up to 1000

separate tissue cores are assembled in array fashion to allow multiplex histological analysis.

ProcedureIn the tissue microarray technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin-embedded tissues such as clinical biopsies or tumor samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced, array pattern. Sections from this block are cut using a microtome, mounted on a microscope slide and then analyzed by any method of standard histological analysis. Each microarray block can be cut into 100 – 500 sections, which can be subjected to independent tests. Tests commonly employed in tissue microarray include immunohistochemistry, and fluorescent in situ hybridization. Tissue microarrays are particularly useful in analysis of cancer samples.

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WOUND HEALING