1. “The importance of integrin-linked kinase in the regulation of
bladder 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 signaling
b) 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. Zymography
5. 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, obscuring
phenotypic 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 in
regulating 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 K
I L K
I L K
I L K
I L K
I L K
I L K
I 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.
Function
Proteases 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 significance
MMP'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.

35. EMT IN TUMOR PROGRESSION

36. 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

37. 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

38. ZYMOGRAPHY
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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

39. TISSUE MICROARRAY
39
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.
Procedure
In the tissue microarray technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from region
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.

40. WOUND HEALING
40