“The importance of integrin-linked kinase in the regulation of
bladder cancer invasion”
National Chiayi University
Dep. Of...
2
INTRODUCTION
3
INTEGRIN
Integrins are transmembrane receptors
that mediate the attachment between a
cell and its surroundings, such as ...
4
INTEGRIN ACTIVATION AND FUNCTION
 Integrin functions:
 Attachment of the cell to the ECM
 Signal transduction from th...
5
INTEGRIN-LINKED KINASE
Integrin-linked kinase (ILK) is one kind
of protein, originally identified while
conducting a yea...
6
INTEGRIN-LINKED KINASE AND CANCER
ILK plays critical roles in development cell motility, adhesion-
depending signaling,...
7
EMT (Epithelial–mesenchymal transition)
From: Wiki
 Epithelial cells lose their cell polarity and
cell-cell adhesion.
...
8
EMT AND METASTASIS
Modified From: archive.osc.edu Modified From: www.discoverymedicine.com
Epithelial–mesenchymal trans...
9
E-cadherin
 In epithelial cells, E-cadherin-containing cell-to-cell junctions are
often adjacent to actin-containing fi...
10
E-cadherin and Cancer
http://en.wikipedia.org/wiki/CDH1_%28gene%29
E- Cadherin in metastasis:
 E-cadherin acts as an i...
11
BBN and bladder cancer
 Butyl-(4-hydroxybutyl) nitrosamine (BBN) has been shown to be
a highly potent and specific bla...
12
BACKGROUND
Numerous cancers have now been described to undergo
changes in levels of expression of ILK consequent upon
...
13
OBJECTIVE
To evaluate the importance of integrin-linked
kinase in bladder cancer progression.
To investigate the mechan...
14
MATERIALS
Cell lines:
253J, TCCsup, KK47, J82, UMUC3, KU7, MGH-U3, 5637,
T24, RT4, RT112.
SV40-transformed urothelial c...
15
METHODS
1. Cell viability assay.
2. Cell migration and invasion assay.
3. Western blot analysis.
4. Zymography
5. Mouse...
16
RESULTS
17
FLOW CHART
Suppression of E-cadherin and promotion of cell
invasion bladder cancer cells
Effect of Integrin-linked kina...
18
Expression of ILK and various epithelial and mesenchymal markers in
bladder cancer cell lines and expression of ILK in ...
19
SUMMARY
Invasive bladder cancer cells tend to have high
expression of integrin-linked kinase.
ILK expression may play s...
20
FLOW CHART
Integrin-linked kinase expression in cancer cell
Suppression of E-cadherin and promotion of cell
invasion in...
21
The change of EMT markers with ILK S343D over expression
and cell viability.
ILK S343D :
Active ILK expressing vector
2...
22
Cell migration and Cell invasion assay
Cell migration Cell invasion
23
Effect in inactivation of GSK3ß with LiCl
24
SUMMARY
Overexpression of ILK suppresses E-cadherin expression
and promotes cell invasion in 253J bladder cancer cells....
25
FLOW CHART
Suppression of E-cadherin and promotion of cell
invasion bladder cancer cells
Effect of Integrin-linked kina...
26
The change in cell viability, migration and invasion of TCCsup and
UMUC3 bladder cancer cells treated by ILK-siRNA.
27
SUMMARY
ILK knockdown suppresses cell invasion in invasive
bladder cancer cells through the regulation of E-
cadherin a...
28
FLOW CHART
Suppression of E-cadherin and promotion of cell
invasion bladder cancer cells
Effect of Integrin-linked kina...
29
Immunohistochemical staining of ILK, E-cadherin and MMP-9 in
a human bladder cancer TMA.
30
SUMMARY
ILK expression correlates with the invasiveness of human
bladder cancer.
31
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-
Ca...
32
CONCLUTION
This study indicates that ILK is important in the EMT of
bladder cancer, which regulates E-cadherin and MMP-...
33
Thank you
34
MMP9
Matrix metallopeptidase 9 (MMP-9), also known as 92 kDa type IV collagenase, 92 kDa gelatinase or
gelatinase B (GE...
EMT IN TUMOR PROGRESSION
CHARACTERISTICS OF
MESENCHYMAL CELLS
• Lack regimented structure
• Few tight intracellular adhesions
• Weak adhesions whic...
EMT MARKERS
• Proteins that increase in
abundance
• N-cadherin
• Vimentin
• Fibronectin
• Snail1 (Snail)
• Snail2(Slug)
• ...
ZYMOGRAPHY
38
Zymography is a electrophoretic
technique for the detection of
hydrolytic enzymes, based on
the substrate re...
TISSUE MICROARRAY
39
Tissue microarrays (also
TMAs) consist of paraffin
blocks in which up to 1000
separate tissue cores a...
WOUND HEALING
40
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  • Integrins are specialized integral membrane proteins that take part in communication between the cell and the outside world. Extracellular signaling molecules attach to the receptor, triggering changes in the function of the cell.
    There are many types of integrin, and many cells have multiple types on their surface. Integrins are of vital importance to all animals and have been found in all animals investigated, from sponges to mammals. Integrins have been extensively studied in humans.
    The alpha subunits all share some homology to each other, as do the beta subunits. The receptors always contain one alpha chain and one beta chain and are thus called heterodimeric.
  • This low affinity structure undergoes rapid, reversible conformational changes to increase ligand affinity, termed "activation“
  • Two-hybrid screening (also known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
    In biology, scaffold proteins are crucial regulators of many key signaling pathways. Although scaffolds are not strictly defined in function, they are known to interact and/or bind with multiple members of a signaling pathway, tethering them into complexes. In such pathways, they regulate signal transduction and help localize pathway components (organized in complexes) to specific areas of the cell such as the plasma membrane, the cytoplasm, the nucleus, the Golgi, endosomes, and the mitochondria.
  • 1. The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis for cancer progression.
  • MMP9
  • integrin-linked kinase

    1. 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
    2. 2. 2 INTRODUCTION
    3. 3. 3 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
    4. 4. 4 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
    5. 5. 5 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
    6. 6. 6 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.
    7. 7. 7 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
    8. 8. 8 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
    9. 9. 9 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
    10. 10. 10 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.
    11. 11. 11 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
    12. 12. 12 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
    13. 13. 13 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.
    14. 14. 14 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.
    15. 15. 15 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.
    16. 16. 16 RESULTS
    17. 17. 17 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
    18. 18. 18 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
    19. 19. 19 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.
    20. 20. 20 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.
    21. 21. 21 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.
    22. 22. 22 Cell migration and Cell invasion assay Cell migration Cell invasion
    23. 23. 23 Effect in inactivation of GSK3ß with LiCl
    24. 24. 24 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.
    25. 25. 25 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
    26. 26. 26 The change in cell viability, migration and invasion of TCCsup and UMUC3 bladder cancer cells treated by ILK-siRNA.
    27. 27. 27 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.
    28. 28. 28 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
    29. 29. 29 Immunohistochemical staining of ILK, E-cadherin and MMP-9 in a human bladder cancer TMA.
    30. 30. 30 SUMMARY ILK expression correlates with the invasiveness of human bladder cancer.
    31. 31. 31 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
    32. 32. 32 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.
    33. 33. 33 Thank you
    34. 34. 34 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. 35. EMT IN TUMOR PROGRESSION
    36. 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. 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. 38. ZYMOGRAPHY 38 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. 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. 40. WOUND HEALING 40

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