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Regulation of transcription factors by integrin-linked kinase during oncogenesis Tan, Clara Chia-Hua 2002

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R E G U L A T I O N O F T R A N S C R I P T I O N F A C T O R S B Y I N T E G R I N - L I N K E D K I N A S E D U R I N G O N C O G E N E S I S By Clara Chia-Hua Tan Bachelor of Science, McGil l University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In F A C U L T Y OF GRADUATE STUDIES (DEPARTMENT OF BIOCHEMISTRY AND M O L E C U L A R BIOLOGY) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA J A N U A R Y 2002 © Clara Chia-Hua Tan s 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada DE-6 (2/88) 1 Abstract Integrin-linked kinase is a serine-threonine kinase that was identified by a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of Pi-integrin. Overexpression of ILK in epithelial cells promotes cell proliferation, anchorage-independent growth, epithelial to mesenchymal transformation, inhibition of anoikis, increased invasiveness and tumourgencity in nude mice. This thesis aims to identify downstream effectors of ILK, and to elucidate the pathway by which ILK regulates the activity of transcription factors. In the first part of the study, I demonstrate that ILK inhibits the activity of glycogen synthase kinase-3 (GSK-3) and that I L K can promote the phosphorylation of GSK-3 either directly or indirectly through ILK-dependent activation of Protein kinase B (PKB/Akt). Furthermore, ILK activity is stimulated upon cell adhesion to fibronectin (FN), leading to inhibition of GSK-3 activity. This in turn prevents GSK-3 from phosphorylating the D N A binding site of c-Jun, resulting in the positive regulation of the AP-1 transcriptional activity. It was previously shown that over-expression of I L K results in increased stability of P-catenin and as well as translocation of P-catenin to the nucleus. Since GSK-3 can phosphorylate P-catenin and promote its degradation, the role of ILK-mediated inhibition of GSK-3 and P-catenin stability was investigated. In the second part of this thesis, I studied the effect of inhibiting I L K activity in the human colon carcinoma cell lines, SW-480 and DLD-1, which lack functional APC. I found that I L K is able to regulate p-catenin pools, and modulate P-catenin/TCF/LEF transcription activity in an APC independent manner. It was previously observed that ILK overexpression caused a concomitant decrease in the expression of E-cadherin. This was proposed to account for the morphological changes seen upon I L K expression. The expression of E-cadherin is regulated by E-boxes in the E-cadherin promoter, which serve as regions of repressor binding. Therefore, binding of transcriptional repressor factors to the E-boxes results in repression of transcription and expression of E-cadherin. The transcriptional repressors that bind to E-boxes are members of the Snail family. This study shows that ILK upregulates the expression of the Snail transcriptional repressor, which in turn suppresses the expression of E-cadherin. Transfection with dominant negative ILK or exposure to the small molecule ILK inhibitor was able to reverse the effects of ILK. Together, these data demonstrate that ILK is involved in regulating the activity of transcription factors p-catenin/TCF/LEF, and AP-1. I L K regulates the activity of AP-1 and P-catenin-TCF/LEF through the inactivation of GSK-3 and the regulation of P-catenin pools. 11 Table of Contents Abstract i i Table of contents i i i List of Figures v List of Abbreviations . .vii Preface viii Acknowledgement ix Introduction 1 1.1 Integrin-linked kinase 2 1.2 Regulation of P-catenin and E-cadherin by I L K in colon carcinoma 4 1.3 Indirect regulation of E-cadherin expression by ILK 8 Materials and Methods 11 2.1 Cell lines U 2.2 Cell Culture 12 2.3 Maintenance of stable transfected cell lines 13 2.4 Transient Transfection 13 2.5 Adhesion Assays 13 2.6 Integrin-linked kinase (ILK) kinase assay 14 2.7 Glycogen synthase kinase-3 (GSK-3) assay 14 2.8 Western blot analysis 15 2.9 Luciferase assay 18 2.10 Nuclear Extracts 18 2.11 Whole cell extracts 19 2.12 Cell growth in the presence of ILK inhibitor 20 2.13 Annexin V/FITC staining protocol 20 2.14 Purification of recombinant proteins 21 in Results 23 3.1.1 Effects of ILK on GSK-3 23 3.2.1 Upon adhesion to fibronectin, ILK stimulates the activity of AP-1 in a GSK-3 dependent manner _ 29 3.3.1 Role of ILK in the regulation of P-catenin/TCF activity and in E-cadherin expression in colon carcinoma 38 3.4.1 Inhibition of I L K promotes the expression of E-cadherin in a dose-dependent manner _ 54 Discussion 64 4.1 The regulation of GSK-3 by ILK kinase activity in a PKB/Akt dependent and independent manner .....65 4.2 Stimulation of ILK activity by adhesion onto fibronectin is capable of regulating AP-1 transcription activity in a GSK-3 dependent manner 66 4.3 Regulation of P-catenin/TCF/LEF activity and E-cadherin expression by Integrin-linked kinase in colon carcinomas 67 4.4 U K inhibitor 69 Conclusion 71 List of References 72 iv List of Figures Figure 1 ILK regulates GSK-3 activity in IEC-18 cells 25 Figure 2 Transient overexpression of ILK wild-type inhibits GSK-3 :HA activity while over-expression of ILK kinase dead enhances GSK-3 :HA activity, in HEK-293 cells 26 Figure 3 I L K phosphorylates GSK-3 in vitro 28 Figure 4 The effect of fibronectin on ILK and GSK-3 activities 30 Figure 5 Densitometric analysis of ILK and GSK-3 activities following adhesion to fibronectin 31 Figure 6 AP-1 activity is stimulated by adhesion to fibronectin 33 Figure 7 The effects of GSK-3 overexpression on fibronectin-induced AP-1 activity 35 Figure 8 Fibronectin-induced AP-1 activity is sensitive to LY294002 37 Figure 9 Dose-dependent inhibition of TCF transcriptional activity by ILK(KD):V5 40 Figure 10 Inhibition of cyclin D l promoter activity by expression of ILK(KD):V5 42 Figure 11 Beta-catenin/TCF activity regulates cyclin D l expression 43 Figure 12 KPSD-1 inhibits beta-catenin/TCF activation in a dose-dependent manner 45 Figure 13 Cyclin D l expression and inhibition of GSK-3 phosphorylation by KPSD-1 in a dose-dependent manner 47 Figure 14 KPSD-1 decreases the growth rate of SW-480 and DLD-1 cells in a dose-dependent manner 49 Figure 15 KPSD-1 decreases the number of colonies formed by SW-480 and DLD-1 in a dose-dependent manner 50 Figure 16 KPSD-1 induces cell apoptosis after 72 hours 51 Figure 17 Inhibition of ILK activity in SW-480 cells increases the expression of E-cadherin and decreases the nuclear translocation of beta-catenin in a dose-dependent manner 53 Figure 18 Inhibition of ILK activity increases the E-cadherin promoter activity in a dose-dependent manner 56 Figure 19 Lack of ILK activity decreases the human Snail promoter activity 58 Figure 20 Overexpression of ILK wild-type decreases E-cadherin promoter activity 60 Figure 21 Functional ILK is involved in enhancing Snail promoter activity 61 Figure 22 Dose-dependent activation of E-cadherin promoter activity by KPSD-1 63 vi List of Abbreviations APC Adenopolyposis coli DCS Donor Calf Serum FBS Fetal Bovine Serum GSK-3 Glycogen synthase kinase ILK Integrin-linked kinase LEF-1 Lymphocyte enhancing factor -1 M E M Minimum Essential Media PKB Protein Kinase B TCF T-Cell Factor Preface The thesis contains work from previously published material. I am the second author in the following two articles. Some of the figures are from Delcommenne, M . , Tan, C , Gray, V. , Ruel, L. , Woodgett, J. and S. Dedhar. Proceeding of the National Academy of Science of U . S. A. 95(19), 11211-11216, 1998. Protein Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B / A K T by the integrin-linked kinase. Some of the figures are from Troussard, A. A. , Tan, C., Yoganathan, T.N. and S. Dedhar. Molecular and Cellular Biology. 19(11), 7420-7427, 1999. Cell-extracellular matrix interactions stimulate the AP-1 transcription factor in an integrin-linked kinase- and glycogen synthase kinase 3-dependent manner. I am the primary author in the following article. Some of the figures are from Tan, C , Costello, P., Sanghera, J., Dominguez, D., Baulida, J., de Herreros, A .G . and S. Dedhar. Oncogene. 20(1), 133-140, 2001. Inhibition of integrin linked kinase (ILK) suppresses beta-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor, snail, in APC-/- human colon carcinoma cells. We attest that the above statements are accurate. Virginia Gray Armelle Troussard Tertiary Author Primary Author v i i i Acknowledgements I would like to thank the past and present members of Shoukat Dedhar's Lab (Armelle Troussard, Julia Mills, Marc Delcommenne, Nasrin Mawji, Sarah Attwell, Severine Cruet, Sujata Persad, Sujata Syam, Tim McPhee, and Virginia Gray), Dr. Shoukat Dedhar, family (Mommy, Papa and Maymay), friends (Eric Tarn, Aruna Somasiri, Laurent Ruel, David Dominguez, Jason Bush, Jason Wong, Marty Boulanger, Michael Page, Raj Hundal, Angela Chang and May Woo), committee members (Dr. Alice Mui and Dr. Ross MacGillvray), Dr. George Mackie, Dr. Hans Clever, Dr. Jim Woodgett, Dr. Antonio Garcia de Herreros, Dr. Penny Costello and Dr. Nathan Yoganathan. i x I. Introduction There are many types of cancers, which are categorized according to their tissue of origin. These include carcinomas, sarcomas and leukemias. Carcinomas, which originate from epithelial cells comprise over 90% of the cancers diagnosed in North America. The hallmarks of carcinomas are that they proliferate at a much higher rate than normal cells, do not under go apoptosis at the appropriate time, do not remain attached to the basement membrane, and often lose polarity, rendering them capable of metastasis or migration to a distant site where they continue to proliferate. These processes are modulated by extracellular stimuli, which initiate intracellular signals by activating receptors, such as integrins and growth factor receptors. Integrin-stimulated signaling pathways that control the cell cycle and cell survival, include those involving Mitogen activated protein (MAP) kinase, the Wnt/Wingless pathway, and Akt/Protein kinase B (Akt/PKB). Dysregulation of these pathways can result in the overexpression of oncogenes or the suppression of tumor suppressor genes. Since the overexpression of Integrin-linked kinase (ILK) results in the increased anchorage independent survival and epithelial to mesenchymal transformation as observed in cancer cells, we wanted to investigate the role of I L K in cancer. The goal of this thesis was to elucidate downstream signaling pathways by which Integrin-linked kinase (ILK) regulates gene expression. Overexpression of I L K in epithelial cells lead to the transformed phenotypes observed in cancer cells. In addition, I L K is overexpressed and dysregulated in many cancers. However, little is known about how I L K regulates downstream signaling events, what are the immediate substrate of ILK, how ILK regulates transcription factors and in turn, 1 modulates the expression of oncogenes and tumor suppressors. Since altered expression of oncogenes and/or tumor suppressors can result in cancer, delineating the molecular pathways by which I L K regulates these genes is important for understanding the role of I L K in both physiological and pathological processes. 1.1. Integrin-linked kinase Integrin linked kinase was discovered by a yeast 2-hybrid screen, which showed that it can interact with the cytoplasmic domain of the p i and P3 integrin subunits (Hannigan et al., 1996). Integrins are heterodimeric transmembrane proteins consisting of a and P subunits and are involved in bi-directional transduction of signals between the extracellular and intracellular environments. I L K is expressed at high levels in muscle and pancreas, and binds to the cytoplasmic tails of the P integrins. In doing so, I L K couples the engagement of integrins to downstream signal transduction events (Hannigan et al., 1996). I L K is a 59 kDa serine/threonine kinase which consists of four ankyrin repeats, a phosphoinositide binding motif domain, a kinase domain and a p integrin binding domain (Hannigan et al., 1996). Homologous protein kinases have been found in C. elegans, drosophila, mice, and humans (Dedhar, 2000). I L K is a cytoplasmic protein whose localization can be regulated by PINCH, an adaptor protein composed of five L I M domains. PINCH binds to the ankyrin repeats of I L K (Li et al., 1999). The fourth L I M domain of PINCH interacts with NCK2, which is a protein that consists of SH2 and SH3 (Src Homology) domains. The NCK2 protein associates with the insulin receptor substrate (IRS-1). Together, PINCH and NCK-2 form a physical bridge that brings I L K and IRS-1 into close proximity. When IRS-1 is 2 bound to the cytoplasmic tail of a stimulated growth factor receptor, it recruits PI-3 kinase. Phosphatidylinositol-3 (PI-3) kinase produces phosphatidylinositol-3,4,5-trisphosphate (PIP3) which regulates ILK activity (Dedhar, 2000; L i et al., 1999). These proteins form a physical bridge to bring other signaling proteins into close proximity and coordinate their activities. The overexpression of ILK in epithelial cells also promotes anchorage-independent cell growth (Radva et al., 1997), inhibits anoikis (Attwell et al., 2000), inhibits apoptosis (Radeva et al., 1997), and promotes epithelial to mesenchymal transformation (Novak et al., 1998). Overexpression of ILK also changes the protein expression and morphology of the cell and induces tumorgenesis. In addition, I L K is overexpressed in colon polyps and carcinomas, melanomas and Ewing's sarcoma (Marotta et al., in press; Janji et al., 2000; Chung et al., 1998). The mechanism by which I L K regulates gene transcription remains unclear. The first goal of this thesis is to identify downstream effectors of ILK. The components involved in the Akt/PKB and WNT/ Wingless signaling pathways were candidates for I L K substrates, because the activation of Akt/PKB or WNT signaling pathways increased cell survival and promotes epithelial-mesenchymal transformation, just as I L K does. Thus it is possible that I L K may be a link between integrins and these pathways. I L K is capable of activating Akt/PKB through phosphorylation of serine 473 on Akt/PKB PI-3 kinase dependent manner. Thus ILK may be PDK-2 (Delcommenne et al., 1998; Persad et al., 2000; Lynch et al., 2000; Morimoto et al., 1999). The activation of Akt/PKB in turn inhibits GSK-3 activity. In addition, it has been demonstrated that I L K can act upon GSK-3 independently of Akt/PKB. There is some evidence suggesting that 3 I L K can directly phosphorylate GSK-3. GSK-3 is a serine/threonine kinase, which is regulated by phosphorylation, and exists as two isoforms (a=52 kDa and b=47 kDa) which are encoded by separate genes. GSK-3 was first described as a kinase that phosphorylates glycogen synthase and regulates metabolism and cell survival. Recently, GSK-3 has been shown to be important in development and to be regulated by phosphorylation (Stambolic and Woodgett, 1997). GSK-3 is activated upon tyrosine phosphorylation and inhibited upon phosphorylation on serine 9 (GSK-3 (3) or serine 21 (GSK-3oc). In the Wnt/wingless signalling pathway, GSK-3 activity is inhibited via the Dishevelled protein in a Akt/PKB-independent manner. Upon Wnt stimulation, GSK-3 becomes inactivated and is no longer able to phosphorylate P-catenin. This increases the stability of P-catenin and allows it to translocate to the nucleus where it acts as a transcriptional co-activator. Ultimately, these events result in an increase in the transcription of mesenchymal genes as well as cyclin D l (Tetsu and McCormick, 1999). Several other substrates of GSK-3 have been identified. These include cyclin D l . GSK-3 phosphorylates cyclin D l on threonine 286 and thereby regulates the stability of the cyclin D l protein. Another substrate of GSK-3 is c-jun. GSK-3 phosphorylates the c-jun D N A binding domain and in this way negatively regulates AP-1 transcriptional activity (de Groot et al., 1993). Thus GSK-3 may be an important target of both Akt/PKB and I L K signaling. 4 1.2. Regulation of p-catenin and E-cadherin by ILK in colon carcinoma The transformation from epithelial to mesenchymal cell fate is important during gastrulation in embryonic development, and in the maturation of ovaries (Morin 1999). In cancer, this transformation from epithelial cell fate to mesenchymal cell fate in adult tissues contribute to the oncogenic and invasive properties of tumor cells derived from epithelial origins. Many of these cancers may be due to inappropriate activation of the WNT/p-catenin signaling pathway. In human colon carcinomas and melanomas, P-catenin is translocated to the nucleus and constitutive activation of TCF/p-catenin is oncogenic (Korinek et al., 1997; Morin et al., 1997; Rubinfeld et al., 1997). In the case of colorectal cancer, alterations in the Wnt/Wingless signaling pathways, including A P C mutations, promote the adenoma to carcinoma transition. A similar epithelial to mesenchymal transformation phenotype is also observed in cells overexpressing ILK. Overexpression of ILK promotes P-catenin translocation to the nucleus, E-cadherin expression loss and epithelial to mesenchymal cell transformation. Because activation of the Wnt pathway promotes epithelial to mesenchymal transformation and overexpression of ILK promotes the same phenotype, this led to the hypothesis that ILK regulates the p-catenin signaling pathway. In addition, I L K has been observed to be highly active, and highly expressed in polyps and colon carcinoma (Marotta et al., in press). In order to elucidate how I L K might regulate the stability of P-catenin, and promote the development of colon carcinoma cell lines, such as SW-480 and DLD-1 were used as models to study the role of I L K in the regulation of P-catenin/TCF/LEF, because they have dysregulated expression of Adenopolyposis coli 5 tumor suppressor (APC) and therefore were a physiologically relevant model for colon carcinoma. P-catenin is part of the catenin family of proteins, along with a and y (plakoglobin) catenin. p and y catenin are homologous proteins originally discovered as cytoplasmic components of cell-cell adherens junctions. P- and y- catenin, however, have different functions (Ozawa et al., 1989; Ben-Ze'ev and Geiger, 1998). In adherens junctions, P-and y -catenin independently bind to the cytoplasmic domain of cell-cell adhesion receptors of the cadherin family, and by physical association with a-catenin, are linked to the actin cytoskeleton (Adams and Nelson, 1998). p-catenin consists of an N -terminal site which contains serine residues, which when phosphorylated, target the P-catenin for degradation, 13 armadillo repeats, which are necessary for protein-protein interactions to E-cadherin, a-catenin, and nuclear localization sequences, one at the N -terminus and one at the C-terminus. In addition to their structural role in adherens junctions, p-catenin (Armadillo; Drosophila homologue) is part of the Wnt/Wingless signaling pathway (Woodarz and Nusse, 1998; Peifer et al., 1993). There are many lines of evidence in genetic models that demonstrate the importance of p-catenin in development (Haegel et al., 1995; Hulsken et al., 1994). Signaling by P-catenin is believed to involve the nonjunctional pool of this protein, as recruitment of P-catenin to adherens junctions (Fagotto et al., 1996; Sanson et al., 1996; Simcha et al., 1998), or degradation of this protein blocks its signaling function (Orford et al., 1997; Aberle et al., 1997). It has been demonstrated that P-catenin is associated with the F-box/WD-repeat ubiquitin ligase p-TrCP. Upon phosphorylation at the N-terminus, P-catenin is ubiquitinated and is recogonized and degraded by the proteasome system (Winston et al., 6 1999; Kitagawa et al., 1999; Hart et al., 1999). When p-catenin is not phosphorylated at the N-terminus it is not targeted for degradation, and P-catenin levels become elevated (Papkoff et al., 1996). Thereafter, P-catenin translocates to the nucleus and subsequently promotes the degradation of Groucho. P-catenin forms complexes with the LEF (Huber et al., 1996) and/or TCF (Molenaar et al., 1996) transcription factors, forming an active factor comples in which p-catenin contributes the transactivation domain. CBP binds this complex (Takemaru and Moon, 2000) and together promotes the transcription of target genes (Behrens et al., 1996). One means of regulating this epithelial and mesodermal cell fate is with proteins involved in Wnt signalling. These ligands activate a signaling pathway that results in the stabilization of the cytosolic pool of P-catenin (Aberle et al., 1997). In epithelial cells, P-catenin forms a complex with APC (Munemitsu et al., 1995; Rubinfeld et al., 1996), Axin/Conductin (Figure 16) (Ikeda et al., 1998), protein phosphatase 2A (Seeling et al., 1999; Hsu et al., 1999) and GSK-3/dishevelled (Li et al., 1999; Smalley et al., 1999). APC and Axin/Conductin may act as scaffolds that co-localize GSK-3 and p-catenin, allowing GSK-3 to phosphorylate B-catenin, and target it for degradation by the ubiquitone-proteasome pathway. This prevents the formation of an active p-catenin/TCF/LEF transcription factor complex. TCF/LEF is part of a family of transcription factors that are cell-type specific. They play an important role in cell fate signal transcription pathway and their levels have been shown to increase in I L K overexpressing cells. LEF-1 does not contain a transactivation domain, however, it can transactivate gene expression by forming transcription factor complexes in which LEF-1 provides the DNA-binding moiety, and P-catenin or another protein contributes a 7 transactivation domain (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996; Takemaru and Moon, 2000). In the presence of Wnt, specifically Wnt-1, GSK-3 activity is inhibited, and thus P-catenin does not get phosphorylated and is not targeted for degradation. The P-catenin/TCF/LEF bipartite complex then transcribes mesenchymal transformation genes, such as homeobox proteins (Carnac et al., 1996; Moon et al., 1997; Brannon et al., 1997; Fagotto et al., 1996). We chose to investigate the role of GSK-3 in regulating P-catenin pools in relation to ILK's ability to phosphorylate and inhibit GSK-3 activity because overexpression of I L K inhibits GSK-3, promotes the translocation of P-catenin into the nucleus (Delcommenne et al., 1998; Novak et al., 1998) and promotes the expression of mesenchymal genes. 1.3. Indirect regulation of E-cadherin expression by ILK The cytoplasmic domain of E-cadherin interacts with molecules of the catenin family, which consists of a-, p- and y-catenins (Ozawa et al., 1989). The cadherin family of transmembrane glycoproteins includes E-cadherin, N-cadherin, P-cadherin and L -C A M . These receptors mediate homophilic, Ca2+-dependent cell-cell interactions, and are involved in cell sorting, morphogenesis and compact cell associations (Nagafuchi et al., 1987). The cadherins range in size from 120 to 130kDa, and contain an extracellular domain, a transmembrane domain and a highly conserved cytoplasmic domain. The extracellular domain consists of three to four unique repeated sequences that are thought to be responsible for calcium-binding and binding specificity (Ozawa et al., 1989). In development, the expression of each member of the cadherin family is spatio-temporally 8 regulated, correlating with morphogenetic events in which adhesion or dispersion of cells is involved. For example, high expression of E-cadherin maintains the normal epithelial phenotype by initiating the formation of adherens junctions (Nelson et al., 1990) (Figure 6). Several studies have demonstrated a strong correlation between loss of E-cadherin/catenin expression, loss of epithelial phenotype and increased invasive phenotype (Rasbride et al., 1993; Shiozaki et al., 1991). More interestingly, overexpression of E-cadherin in several tumor cell lines and epithelial cells having fibroblastic morphology re-establishes the epithelial phenotype and decreases the invasive phenotype (Frixen et al., 1991; Chen et al., 1997). The transcription of E-cadherin is regulated by two E-boxes in the E-cadherin promoter region, which are almost identical in mouse and human (Cano et al., 2000). A transcription factor known to bind to E-boxes is Snail. Snails comprise a family of transcription factors that repress transcription upon binding to E-boxes. Recently, it has been demonstrated that epithelial cells that express a transfected Snail gene adopt a fibroblast phenotype and acquire tumorigenic and invasive properties (Batlle et a l , 2000). The endogenous Snail protein is present in invasive mouse and human carcinoma cell lines as well as tumors in which E-cadherin expression has been lost (Cano et al., 2000). Since the down regulation of E-cadherin has been observed in both I L K overexpressing cells and Snail overexpression cells, we wanted to investigate if I L K indirectly inhibits E-cadherin expression by regulating Snail expression. Besides regulating P-catenin, GSK-3 is able to regulate AP-1 activity as well. It has recently been shown that overexpression of ILK upregulates the activity of AP-1 transcription heterodimer (Troussard et al., 1999). The AP-1 transcription factors 9 consists of two members, c-jun and c-fos, which combine to form homodimeric or heterodimeric complexes. AP-1 complexes can only consist of c-jun and c-jun homodimers, or c-jun and c-fos heterodimers. These dimers recognize and bind to a specific D N A sequence, the palindromic tetradecanoyl phorbol actate (TPA)-response element T G A C / G T C A (Angel et al., 1987), which is present in the promoters of many genes. C-jun, the crucial component of these dimers, is a nuclear proto-oncogen that is expressed in many cell types at low levels (Karin et al., 1997). It consists of a D N A -binding/leucine zipper domain (C-terminus) and a transactivation domain (N-terminus) (Karin, 1995; Karin et al., 1997). AP-1 activity is regulated at both the transcriptional and post-translational levels. At the post-translational level, the phosphorylation of serine residues 63 and 73 by c-Jun N terminal kinase (JNK) in the N-terminus promotes AP-1 transactivation (Boyle et al., 1991; Smeal et al.,1991). In addition, the phophorylation of serine and threonine in the C-terminus by GSK-3 inhibits the ability of AP-1 to bind to D N A (Boyle et al., 1991; Goode et al., 1992; Nikolakaki et alk, 1993; Papavassiliou et al., 1992). Because ILK can inhibit GSK-3 activity (Troussard et al., 1999) and promote AP-1 activity we tested the hypothesis that I L K regulates AP-1 via the inhibition of GSK-3. 10 II. Materials and Methods 2.1. Cell lines The IEC-18 rat intestinal epithelial cell line was obtained from American Type Culture Collections (ATCC). Clones of IEC-18 cells stably transfected with cDNA wild-type I L K (ILK-13 Ala3 and A4a), I L K antisense D N A (ILK 14) or catalytically-inactive I L K (ILK-KD) were made by Chung Yee Leung-Hagjestein (University of Toronto). Human embryonic kidney cells (HEK-293) were obtained from Dr. M . Moran (University of Toronto) and were transformed with the E2F of adenovirus5. SW480 is one of eleven colorectal adenocarcinoma cell lines, which were isolated by A . Leibovitz and associates from 1971-1975. It is from a 51 years old, male, Caucasian, and the tissue is from a colorectal adenocarcinoma. More specifically, it is from a tumor of Grade III-IV adenocarcinoma of the colon. These cells are epithelial and are tumorogenic in mice. These cells also express wild-type b-catenin and mutant, truncated A P C . They do not express E-cadherin protein at high rates nor do they express COX-2 nor COX-1. DLD-1 is one of the two colorectal adenocarcinoma cell lines that were isolated by D.L. Dexter and associates during a period from 1977-1979. The original tumor was an adenocarcinoma of the colon. DLD-1 is a characterized cell line of human colonic tumor origin which has been employed in studies on the effects of polar solvent on cell characteristics. These cells have wild-type p-catenin and mutant APC. They also express E-cadherin. 11 2.2. Cell Culture IEC-18 cells were routinely cultured in a-mimimal essential medium (a-MEM) (Gibco BRL) supplemented with 5 % fetal bovine serum (FBS) (Gibco BRL) , glucose (3.6 mg/mL) and insulin (10 pg/mL). HEK-293, and SW480 cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco BRL) supplemented with 10 % donor calf serum (DCS) (Gibco BRL) . Scp2 cells were cultured in DMEM/F12 medium (1:1, Gibco BRL) supplemented with FBS (5%) and insulin (5 ug/mL). DLD-1 cells were cultured in RPMI-1640 (Gibco BRL) supplemented with FBS (10%). A l l cells were grown at 37°C, in a 99% humidified atmosphere of 5% C O 2 in air. Cells were frozen in D M E M containing 20% dimethylsulfoxide (DMSO) (Sigma) and 25% FBS, and were stored in liquid nitrogen (-198°C). Cells were seeded at approximately 106cells/100 mm tissue culture dish. When the cells were approximately 80% confluent, cells were exposed to trypsin (1% trypsin (Gibco/BRL)/5 m M EDTA (Sigma)) for 2 minutes, detached from the dish and removed from the tissue culture dish. Cell stocks were maintained in D M E media containing 10% FBS and 15% DMSO (Sigma). 2.3. Maintenance of stable transfected cell lines These cells were grown under the same conditions as those the parental cell line, except for the addition of geneticin (G418) (40 to 80 pg/mL) (Gibco BRL) to maintain selection. 12 2.4. Transient transfection Cells were transiently transfected by either calcium phosphate precipitation or lipofection. Cells at 50 % confluency were transfected with plasmids (see below) using the following lipofection kits: Fugene (Boehringer Manheim), Lipofectamine or Lipofectin (Gibco BRL) . Transfections were carried out for six hours to overnight in serum-free medium. The transfection medium was then replaced by serum-containing medium for at least 3 hours before initiating an experiment. 2.5. Adhesion assays Cells were serum starved for 18 hours. Subsequently, cells were detached by incubation with phosphate buffered saline (PBS) containing 5 m M E D T A and then scraped with a cell scraper. The cells were centrifuged at 800 x g and the cell pellet was resuspended in serum free media. Single cells were obtained by passing the cell suspension through a cell separator (VWR). Tissue culture plates (150 mm) were coated with fibronectin (FN; 10 ug/mL in PBS) (Gibco BRL) or bovine serum albumin (BSA; 2.5 mg/mL in medium) (Sigma) overnight at 4 °C. The following day, all plates were blocked with B S A (2.5 mg/mL in medium) for 2 hours at 37 °C. Cells were then seeded on prepared plates and maintained in medium for the indicated times. Adhesion assays involving transiently transfected cells were performed 48 hours post-transfection. 13 2.6. Integrin-linked kinase (ILK) kinase assay Kinase assays were carried out as previously described (Hannigan et al., 1998). The cells were lysed in NP-40 lysis buffer (50 m M HEPES (pH 7.5), 150 m M NaCl, 1 % Nonidet P-40, 0.5 % sodium deoxycholate, 10 pg/mL leupeptin, 2.5 pg/ml of aprotinin, 1 m M phenylmethylsulfonly flouride (PMSF), 5 m M sodium flouride, and 5 m M sodium orthovanadate). Equivalent amounts of protein for each cell lysate were pre-cleared with nonspecific immunoglobulin G (IgG) which was bound to protein A-Sepharose. The supernatants were immunoprecipitated with the anti I L K antibody ((ILK rabbit polyclonal antibody 550-592) (Upstate Biotechnology Institute) or made by the Dedhar lab (Delcommenne et al., 1998)). The precipitates were washed and the reactions were carried out as described (Hannigan et al., 1996). Myelin basic protein (MBP) was used as a substrate. Phosphorylated M B P was resolved by 15 % sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by autoradiography. The autoradiographic signals were quantified by densitometery using a densitometer (Bio-Rad Laboratories). 2.7. Glycogen synthase kinase - 3 (GSK-3) kinase assay This assay was performed as the I L K Kinase assay with the following modifications. The antibody was an anti-GSK-3 rabbit polyclonal (gift from the Woodgett Lab). Glycogen synthase (GS-1) was used as a substrate for GSK-3. Phosphorylated GS-1 was resolved by Tricine gel electrophoresis (Schagger et al., 1987). 14 2.8. Western Blot analysis Cells were cultured on tissue culture dishes. To harvest, the cells were rinsed three times with PBS, and lysed with NP-40 lysis buffer (as described in I L K kinase assay) for 10 minutes on ice. Cells were scraped off the plate and the cell suspensions were vortexed for 10 seconds before centrifugation at 14000 rpm at 4°C. Supernatants were collected and stored at -70°C. Protein concentrations were measured using a BioRad Bradford protein assay kit. Lysates were normalized for equal amount of protein by NP-40 buffer and then an equal volume of 2X sample buffer (20 % glycerol, 0.1 M Tris pH 6.8, 4 % SDS, 0.004 % bromophenol blue, optional 10 % 2-mercaptoethanol) was added. Equivalent amounts of protein were resolved by 7 or 7.5 or 8 or 10 or 12 % SDS-PAGE. Proteins were then transferred to PVDF (Immobilion-P Millipore) membranes. Membranes were incubated for 1 hour with blocking buffer (5% skim milk powder in Tris buffered saline (TBS)-Tween-20 (0.01%)) to block the nonspecific sites. The protein was detected by incubating the membrane with a primary antibody diluted in blocking buffer and rocked overnight at 4°C. Unbound primary antibodies were removed by washing five times for 15 minutes each with TBS-T (50 m M Tris bufferd saline pH 8.0 with Tween-20), followed by incubation with horse radish peroxidase-conjugated species specific IgG secondary antibody (diluted in blocking buffer) for 1 hour and 30 minutes at room temperature for 1 hour in the blocking buffer. Unbound secondary antibodies were removed by washing 5 X 1 5 minutes each with TBS-T. Bands were visualized with enhanced chemiluminescent (ECL) (Amersham) reagents. The following are the antibodies use in the experiments. 15 Antigen Species Origin I L K Rabbit Polyclonal Dedhar Lab I L K Rabbit Polyclonal Upstate Biotechnologies (UBI) GSK-3 Rabbit Polyclonal Woodgett Lab Hemalglutinin (HA) Mouse Monoclonal Babco A K T phosphoserine 473 Mouse Monoclonal New England Biolabs (NEB) A K T Rabbit Polyclonal NEB GSK-3 phosphoserine 9/21 Mouse Monoclonal NEB GSK-3 Rabbit Polyclonal NEB P-catenin Mouse Monoclonal NEB Cyclin D l /2 Mouse Monoclonal Transduction Labs Histone Rabbit Polyclonal UBI E-Cadherin Mouse Monoclonal NEB V5/HIS Mouse Monoclonal Transduction Labs LEF-1 Rabbit Polyclonal Grosschedl Lab TCF-4 Rabbit Polyclonal UBI Inducible Nitric Oxide Synthase Mouse Monoclonal NEB I K B Rabbit Polyclonal NEB I K B phosphoserine 32 Rabbit Polyclonal NEB 16 5. Constructs and Plasmids The constructs and plasmids used in the experiments are the following. Construct Vector PKB/Akt wild type:HA pCMV6 PKB/Akt A A A mutant:HA pCMV6 PKB/Akt kinase dead mutant:HA pCMV6 GSK-3 wild type:HA pcDNA3 GSK-3 kinase dead:HA pcDNA3, GSK-3 kinase dead:GST pGEX I L K wild type:GST pGEX I L K wild type:V5/HIS pcDNA3.1 I L K kinase dead:V5/HIS pcDNA3.1 I L K anti-sense:V5/HIS pcDNA3.1 I L K wild type p C M V I L K kinase dead p C M V I L K antisense p C M V Empty:V5/HIS pcDNA3.1 Cyclin D l promoter full length pGL3 Cyclin D l mutant TCF site full length promoter pGL3 Snail promoter full length -996/+116 fragment of human pGL3 17 E-Cadherin promoter full length -178/+92 fragment of human pGL3 TCF/LEF-(3-catenin response element pTOPFlash Mutant pTOPFLASH control pFOPFlash AP-1 Response Element pGL3 Control pGL3 N F - K B Response Element pGL3 2.9. Luciferase Assay Luciferase assays were performed on transiently transfected cells according to the manufacturer's instructions (Promega Corp). A l l assays were normalized for transfection efficiency by measuring P-galactosidase enzymatic activity or a modified luciferase activity (pRenilla; dual luciferase; Promega). Protein concentrations were determined by Bradford assay. Results are expressed in relative light units (RLU) per microgram of protein. 2.10. Nuclear extracts Nuclear extracts were prepared by the miniextraction method as previously described (Andrews et al., 1991). Cells were washed with ice-cold PBS and scraped into 1.5 mL of PBS. The cells were collected by centrifugation and resuspended in 400 uL of 10 m M HEPES-potassium hydroxide (pH 7.9) - 1.5 mM magnesium chloride - 10 mM potassium chloride - 0.5 m M dithiothreitol - 0.2 m M PMSF. After 10 minute of incubation on ice, samples were spun for 10 seconds and the nuclei were collected by 18 centrifugation. The nucleus were then resuspended in 50 pL of 20 m M HEPES-potassium hydroxide (pH 7.9) - 25% glycerol - 420 m M sodium chloride - 1.5 m M magnesium chloride - 0.2 m M EDTA-0.5 m M dithiothreitol - 0.2 m M PMSF. After 20 minutes on ice, and the tubes were centrifuged to remove insoluble material. The supernatant was collected and the extracts were stored at -70°C. 2.11. Whole cell extracts Whole cell extracts were made by three methods. 2.11.1. Method 1. Cells were freeze thawed two times with NP-40 lysis buffer, scraped, and incubated on ice for fifteen minutes. The cell suspension was resuspended and centrifuged at 14 000 rpm for 15 minutes at 4°C. Supernatants were collected and stored at -70°C. 2.11.2. Method 2. Cells were lysed on ice with P K B / A K T lysis buffer. This buffer contained 20 m M Tris HC1 (pH 7.5), 150 m M NaCl, 1 mM EDTA, 1 m M EGTA, 1 % Triton X-100, 2.5 m M sodium pyrophosphate, 1 m M P-glycerolphosphate, microcystin, 1 m M N a 3 V 0 4 , 1 pg/mL leupeptin, 1 pg/mL aprotinin, and 1 m M PMSF. The cell suspension was scraped into tubes, resuspended, and incubated on ice for 10 minutes. The cell suspension was collected by centrifugation and stored as above. 19 2.11.3. Method 3. Cells were lysed with Triple-detergent lysis buffer (50 mM Tris Cl (pH 8.0), 150 m M NaCl, 0.1 % SDS, 100 ug/mL PMSF, 1 pg/mL aprotinin and leupeptin, 1 % Nonidet P-40 (NP-40) and 0.5 % sodium deoxycholate). A l l lysis buffers were filtered before use and store at 4°C until the time of use. 2.12. Cell Growth In the Presence of ILK Inhibitor Cells were seeded in 12-well plate (105/well) for 24 hours before the addition of the indicated inhibitors. A l l cells (floating and attached) were collected and counted following incubations of 24, 48, 72, 120 hours in the presence of inhibitor. For colony formation assays, wells (24-well plate) were pre-coated with 100 uL of neutralized collagen (50 % type I collagen, 40 % culture medium and 0.75 % NaHCOs) and polymerized at 37 °C for 1 hour. Cells (104) were then resuspended in 160 uL of neutralized collagen and added to the pre-coated wells and polymerized. Each well was then covered with 1 mL of culture medium. Nine days after seeding, cells were visualized with 0.05 % crystal violet (Sigma) containing 10% buffered formalin (Sigma) and counted. 2.13. Annexin V/FITC Staining Protocol Cells were grown in tissue culture plates until 70 % confluency, and the described treatments carried out. A l l cells, floating and attached, were collected. Cells were washed once with cold PBS and once with lx binding buffer (lOmM Hepes-NaOH, pH 7.4, 140 m M NaCl, 2.5 m M CaCl 2). Annexin V-FITC reagent (15 pi) (Pharmingen) was 20 then added to aliquots of 10 cells. The cells were incubated for 15 minutes at room temperature (20-25°C) in the dark. Subsequently, lx binding buffer (400 pi) was added and the cells were analyzed by flow cytometry within 60 minutes. Unstained cells served as a control. 2.14. Purification of Recombinant Proteins 2.14.1. ILK fusion proteins P G E X plasmids were transformed into Escherichia.coli (E. coli) cells (BL21) cells (Pharmacia), cultured and plated on agar plates containing amipicillin (10 pg/ml). Bacteria were grown at 37 °C overnight. Single colonies were selected and transferred to Luria Broth (LB) (50 ug/mL ampicillin) (Sigma) cultures. Liquid cultures were expanded to 100 ml. IPTG (0.5mM) was added to the bacteria at log phase growth and the culture was incubated for an additional 2 hours at 30°C. Cultures were centrifuged 700 x g and the cell pellet was resuspended in Lysis Buffer (PBS, 1 mg/ml lysozyme, 1 % Triton-X-100, 25 % sucrose, 1 m M EDT A, 5 m M DTT, 1 % aproptinin, and 1 m M PMSF) and incubated for 30 minutes on ice. Cell lysates were then sonicated and centrifuged at 1000 x g for 10 minutes. The supernatant was removed and incubated with 1 ml of pre-washed Glutathione-Sepharose (50 % slurry) (Pharmacia) for 18 hrs at 4 °C with constant agitation. The resin was collected by centrifugation and washed 3 x with 20 bed volumes of Wash Buffer (PBS, 1 % Triton X-100, 1% aprotinin, ImM PMSF). The protein was eluted with (3-mercaptoethanol or GST. A l l of the fusion protein was then resolved by SDS-PAGE. The fusion protein band was then excised and transferred 21 into a dialysis bag by electroelution. The protein is concentrated and degraded fragments are removed with a Centricon 60 protein concentrater conical vial (Amicon). 2.14.2. GSK-3 fusion protein Fusion protein was isolated using the methods above, with a few modifications. The induction was for 4 to 7 hours at 30°C with a final concentration of 1 m M IPTG.. The bacterial pellet from 100 ml culture was resuspend by vortexing in 10 ml of PBST (150 m M NaCl, 16 m M Na 2 HP0 4 , 4 m M NaH 2 P0 4 , 0.1% Triton X-100, 100 p M N a V 0 4 , 5 pg/ml leupeptin, 50 m M NaF, 1 m M Benzamide). The cells were lysed by snap freeze and thaw at 37°C, vortexing in between 3 times. To further ensure lysis and shearing of the D N A , the sample was sonciated at 50 % amplitude for 2 minute in 30 second intervals. The bacterial cell wall and other non-soluble fragments were removed by centrifugating at 13000rpm for 10 minutes at 4°C. The protein was collected using Glutathione agarose beads (Sigma G-4510). The beads were washed and pre-swollen beads, 2 to 3 times, with PBS before adding the bacterial supernatant. The supernatant (10 ml) was added to 0.5 ml of beads. This mix was rotated for 2 hours to overnight at 4°C. Spin at 3 K speed for 30 seconds. The supernatant was then removed but not discarded. The beads were washed 4 times with 1 ml of PBS by inverting. The protein was eluted on a column with glutathione solution or P-mercaptoethanol and then dialyzed overnight at 4°C. 22 III. Results i The results that will be presented in this section have been published in journals already, with the exception of figure 16. Figures 1 to 3 have been published in volume 95(19) of the 1998 edition of the journal published by the Proceedings of the National Academy of Science USA. The title of the article is 'Phosphoinositide-3-OH kinase-dependent regulation of glycogen-synthase kinase 3 and protein kinase B / A K T by the integrin-linked kinase'. Figures 4 to 8 have been published in the 19(11) volume of the 1999 edition of the journal of Molecular and Cellular Biology. The title of the article is 'Cell-extracellular matrix interactions stimulate the AP-1 transcription factor in an integrin-linked kinase- and glycogen synthase kinase 3-dependent manner'. I was the second author in the two previously mentioned articles. Figures 9 to 22 can be found in pages 133 to 140 of volume 20(1) of the 2001 edition of the journal Oncogene. The title of the article is 'Inhibition of integrin linked kinase (ILK) suppresses B-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor, snail, in APC-/ -human colon carcinoma cells'. 3.1.1. Effects of ILK on GSK-3 To determine the role of I L K in intracellular signaling, the identification of the downstream effectors is necessary. Because I L K overexpression in epithelial cells results in the translocation of P-catenin to the nucleus (Novak el al.1998), and this is also observed in cells stimulated by Wnt-1 during development, I wanted to determine whether I L K regulates the phosphorylation and localization of P-catenin by regulating GSK-3, a kinase that destabilizes P-catenin by phosphorylating it. 23 3.1.2. Inhibition of ILK indirectly increases GSK-3 activity via the inactivation of AKT/PKB in intestinal epithelial cells. As illustrated in Figure 1, GSK-3 kinase activity was found to be inhibited in I L K overexpressing IEC-18 ileum epithelial cells compared to cells transfected with I L K antisense D N A (Figure 1). The IEC-18 clones used in the thesis are the following: I L K overexpressing (ILK-13, Ala3 , A4a), I L K anti-sense (ILK-14), I L K kinase dead (ILK-K D , GHR31RH). To determine whether this inhibition of GSK-3 activity was due to ILK, I performed transient transfection assays in human embryonal kidney epithelial cells (HEK-293), because their transfection efficiency is high. As shown in Figure 2, co-transfection of HA-tagged GSK-3 together with wild-type ILK, resulted in inhibition of GSK-3 activity, demonstrating that catalytically active ILK can inhibit GSK-3 activity. Co-transfection with kinase-deficient I L K resulted in increased GSK-3 activity over basal levels (Figure 2), suggesting that this form of ILK may behave as a dominant-negative. Concurrently, the increase in I L K activity corresponded to an increase in phosphorylation of Akt/PKB on serine 473 (Figure 2). These data suggest that I L K can either directly or indirectly phosphorylate and activate Akt/PKB, in turn inhibiting GSK-3 activity. 24 ILK Activity GSK-3 Activity Stable Transfectants Figure. I. I L K regulates GSK-3 activity in IEC-18 cells. ILK and GSK-3 kinase activities were measured in IEC-18 cells using myelin basic protein (MBP) or glycogen synthase fragment (GS-1) respectively, as substrates. ILK and GSK-3 were immunoprecipitated from IEC-18 stably overexpressing wild-type ILK (ILK-13), kinase deficient ILK (ILK-KD, GH31RH) or ILK anti-sense (ILK-14). Overexpression of wild type ILK results in high activity and inhibition of GSK-3 activity. The level of expression of GSK-3 protein is similar in cell clones, as determined by Western blot analysis (Delcommenne et al., 1998). This is a representative of six independent experiments. 25 GSK-HA ILK-WT ILK-KD + + + + + G S K - 3 Activity GS-1 AKT + + + « AKT-SER473® BLOT: ANTI-SER473® AKT Figure. 2. Transient overexpression of I L K wild-type inhibits GSK-3 :HA activity while over-expression of I L K kinase dead enhances GSK-3 :HA activity, in HEK-293 cells. Co-expression of wild-type results in inhibition of GSK-3 :HA activity whereas co-expression of kinase dead ILK (ILK-KD)results in enhanced GSK-3 activity relative to cells not transfected with ILK. Transfection efficiency was determined by monitoring GSK-3 :HA expression by Western blot analysis by using an anti-HA antibody (Delcommenne et al., 1998). Phosphorylation level of Akt/PKB antibody (This blot was done by Marc Delcommenne). This is representative of six independent trials. 26 3.1.3. I L K is capable of directly phosphorylating GSK-3 in vitro. GSK-3 activity is inhibited when it is phosphorylated on serine 9 (in (3) or 21 (in a) (Stambolic and Woodgett, 1994). In order to determine i f I L K can modulate the activity of GSK-3 in an Akt/PKB independent manner, recombinant ILK was incubated with recombinant GSK-3 kinase deficient mutant which autophosphorylates minimally. GSK-3 can be regulated by I L K through direct phosphorylation or through ILK's ability to regulate Akt/PKB activity (Delcommenne et al., 1998; Persad et al., 2000; Lynch et a l , 2000), which in turn phosphorylates GSK-3. As shown in Figure 3, I L K is capable of phosphorylating GSK-3 directly in vitro. 27 GST-ILK (pi) 0 1.25 2.5 5 10 20 GST-GSK-3 (KD) (pi) + + + + + + Figure. 3. I L K phosphorylate GSK -3 in vitro. Recombinant kinase-deficient GST-GSK-3 coupled to glutathione-Sepharose beads was incubated with increasing volumes of GST-ILK (7.5 ng/pl) in the presence of 3 2 P-y-ATP. After a 2 hour incubation at 30°C, phosphorylated GSK-3 was detected by SDS/PAGE and autoradiography. ILK did not phosphorylate GST. This is representative of four independent experiments. 28 3.2.1. Upon adhesion to fibronectin, ILK stimulates the activity of AP-1 in a GSK-3 dependent manner. It has recently been demonstrated that the attachment of IEC-18 cells to F N stimulates I L K activity (Delcommenne et al., 1998). Since ILK can directly phosphorylate GSK-3 in vitro and inhibits GSK-3 activity when overexpressed in cells (Delcommenne et al., 1998), we examined whether adhesion to F N results in an inhibition of GSK-3 activity. 3.2.2. ILK activity increases and GSK-3 activity decreases upon adhesion to fibronectin. Attachment of IEC-18 cells to fibronectin (FN) led to increased I L K kinase activity, concomitant with decreased GSK-3 kinase activity, compared to attachment to bovine serume albumin (BSA) as a control as shown in figure 4. Maximal stimulation of I L K activity occurred at 30 minutes after plating, and this corresponds to the maximal inhibition of GSK-3 activity. As shown previously (Delcommenne et al., 1998), I L K activity declines after 30 minutes and is substantially lower at 45 minutes after plating. The expression levels of I L K and GSK-3 did not change during the course of cell adhesion to F N or BSA. This shows that the kinase activity changes are not due to the modulation of protein expression (Figure 4). At the 45 minute time point, the GSK-3 activity rebounds. The autoradiographic signals were quantified by densitometric analysis, and the data are shown in figure 5. 29 ILK Activity GSK-3 Activity GS-1 30 min 45 min Blots: Anti-ILK Anti-GSK-3 Figure. 4. The effect of fibronectin on I L K and GSK-3 activities. ILK and GSK-3 kinase activities were measured following an adhesion assay. IEC-18 cells were starved of serum for 18 hours, harvested, and seeded on fibornectin (FN) or bovine serum albumin (BSA) for 30 or 45 minutes. Cells were harvested and lysed. Lystates were immunoprecipiated for I L K or GSK-3 and kinase assays were performed. ILK and GSK-3 activities were determined using M B P and GS-1 as the substrates, respectively. Immunoblots of I L K and GSK-3 show that equivalent amounts of these proteins were present in each extract. This is representative of four independent experiments. 30 CN E E "3 Q O CO z 111 Q UJ > UJ a: 5 4.5 -4 -3.5 -3 2.5 -j 2 1.5 1 0.5 -j 0 • ILK-1 : • GSK-3! B S A 30MIN F N 30MIN B S A 45MIN F N 45MIN Figure. 5. Densitometric analysis of ILK and GSK-3 activities following adhesion to FN. Data is expressed optical density units (ODu) and are the mean +/- standard deviation of septuplicates. Data was collected an analysed with a densitometer (BioRad). 31 3.2.3. AP-1 Activity increases upon adhesion to fibronectin. Glycogen synthase kinase-3 (GSK-3) has been shown to phosphorylate c-Jun and thereby regulate AP-1 (de Groot et a l , 1993). The increase in GSK-3 activity decreases the AP-1 activity. ILK also regulates GSK-3 (Delcommenne et al., 1998, Troussard et al., 1999 or refer to section 1.1. and 1.2.). In addition, it has been previously shown that overexpression of ILK stimulates AP-1 activity (Troussard et al., 1999). Experiments were performed to determine whether cell attachment to fibronectin (FN) resulted in the stimulation of AP-1 activity, and whether I L K and GSK-3 were upstream of this activation. Since IEC-18 have poor transfection efficiency, the experiments were done with HEK-293 (human embryonic kidney) cells, because these cells have a higher efficiency of transient transfection. Moreover, the transient transfection of HEK-293 cells with wild-type I L K results in an increase in AP-1 activity (Troussard et al., 1999). AP-1 activity was measured with a luciferase reporter assay after transient transfected HEK-293 cells with a plasmid containing the AP-1 responsive element were adhered for various time points on F N or BSA. Figure 6 shows AP-1 activity was maximally stimulated 30 minutes after attachment to FN. 32 c • 1—* <D -t—> o o 5fJ 20000 17500 15000 12500 10000 7500 5000 2500 0 i 30 40 60 Time of Adhesion (Minutes) Figure. 6. AP-1 activity is stimulated by adhesion to FN. HEK-293 cells were transfected by calcium phosphate with 50pg of pGL3-AP-l containing the AP-1 responsive promoter and luciferase reporter gene. Cells were then seeded on BSA or F N for the indicated times and lysed. A luciferase assay was performed on the cell extracts. Results represent the difference in AP-1 activity when either F N or B S A was used as a substrate. The data are expressed in relative light units (RLU) and are mean +/- standard deviation of four independent experiments. 33 3.2.4. AP-1 activity is regulated in a GSK-3 dependent manner. Co-transfection of the AP-1 reporter gene with increasing amounts of wild-type HA-tagged GSK-3 cDNA in HEK-293 cells resulted in an inhibition of AP-1 activity induced by 30 minute adhesion to F N (Figure 7). This suggests that inhibition of GSK-3 activity is required for the attachment stimulated AP-1 activity. Collectively, these results correlate that attachment of cells to F N increases I L K kinase activity, resulting in the inhibition of GSK-3 activity, and thus increase in AP-1 transcriptional activity. 34 o -4—» O Ix a. <*-o =L D - l 120000 100000 80000 60000 40000 20000 0 jug of GSK-3 plasmid Lig of empty plasmid • B S A • F ibronect in 0 6 15 24 24 18 9 0 anti-HA Figure. 7. The effects of GSK-3 overexpression on FN-inducd AP-1 activity. HEK-293 cells were co-transfected with 50pg of pGL3-AP-l and varying amounts of pcDNA-HA-tagged GSK-3 or empty vector. Cells were plated on FN or B S A for 30 minutes, lysed and assayed for AP-1 activity by luciferase assay. GSK-3 expression in HEK-293 cells is shown by Western blot analysis using an anti-HA antibody. Data are mean +/-standard deviation of four independent experiments. 35 3.2.5. AP-1 activity is regulated in a PI-3 kinase dependent manner. It has recently been shown that ILK inhibits GSK-3 activity in a PI 3-kinase-dependent manner (Delcommenne et al. 1998). Several studies have implicated PI 3-kinase in AP-1 transactivation. TPA (Huang et al., 1997), epidermal growth factor and insulin (Huang et al.,1996) have all been shown to activate AP-1 in a PI 3-kinase-dependent manner. Therefore, the effect of PI 3-kinase-specific inhibitors LY294002 (Vlahos et al., 1994) on F N adhesion-dependent and ILK-induced AP-1 activity was investigated. F N adhesion-stimulated AP-1 activity was inhibited by LY294002 (Figure 8). These data suggest that PI-3 kinase, is involved in adhesion mediated, I L K activation of AP-1 (Troussard et al., 1999). Collectively the data describe a pathway for fibronectin adhesion stimulated AP-1 activity involving PI-3 kinase, I L K and GSK-3. 36 0 LIM 10 uM 25 JLIM Concentration of LY294002 Figure. 8. FN-induced AP-1 activity is sensitive to LY294002. FTEK-293 cells transfected by calcium phosphate with 50 ug of pGL3-AP-l were plated on B S A or FN for 30 minutes in the presence of the indicated concentration of LY294002. AP-1 activity was evaluated by a luciferase assay. Data are meant +/- standard deviation of four independent trials. 37 3.3.1. Role of ILK in the regulation of P-catenin/TCF activity and in E-cadherin expression in colon carcinoma. In ILK-overexpressing epithelial cells, there is an accumulation and translocation of P-catenin into the nucleus as well as inhibition of E-cadherin expression (Novak et al., 1998). This results in high levels of LEF-1/TCF4-P-catenin transcription factor activity and the epithelial-mesenchymal transformation (Novak et al., 1998). The regulation of LEF-l/TCF4-P-catenin activity involves the regulation of P-catenin levels and localization, the levels of LEF-1 and TCF4, and perhaps other mechanisms independent of the Wnt signalling pathway. In most human colon cancers, loss of functional adenomatous polyposis coli (APC) tumour-suppressor genes occur and thus causes the accumulation and translocation of P-catenin (Morin et al., 1997), since the P-catenin cannot be targeted for degradation. The subsequent accumulation of P-catenin activates genes that are responsive to transcription factors of the TCF/LEF family, with which the p-catenin interacts. The hypothesis that P-catenin accumulation is the critical step in intestinal tumorigenesis is further bolstered by the finding that a majority of colon tumors and colon tumor cell lines with no APC mutations instead have mutations in the P-catenin gene which increase the protein's stability (Morin it al., 1997; Sparks et al., 1998). However, it is entirely possible that an APC independent pathway also plays a role in the stimulation of P-catenin/TCF activity in colon carcinomas. One of these pathways could involve I L K since overexpression of ILK stimulates P-catenin/TCF activity. 38 3.3.2. Inhibition of ILK suppresses B-catenin/TCF activities in A P C - / - colon carcinoma cell lines. Because colon carcinoma cell lines contain mutations in the Wnt signalling pathway, they are good models and controls to use for deciphering the role of I L K in the regulation of P-catenin pools in a physiologically relevant system. To determine i f I L K is involved in regulating B-catenin-TCF transcriptional activity, in the presence or absence of APC, pTOPFlash (P-catenin-TCF/LEF binding response element cloned proximal to the luciferase gene) and increasing amounts of dominant negative ILK(KD):V5 were cotransfected into SW480 and DLD-1 (APC -/- cells), and assayed for TCF/LEF-p-catenin activity. The experiments revealed that increasing amounts of ILK(KD):V5 expression in SW480 and DLD-1 cells resulted in a dose-dependent inhibition in TCF/LEF-P-catenin activity (Figure 9). 39 120 CO. i 80i .£ & c w ^ CD 55 —) CO —' CO — —J o m a: o ^ 0 ILK(KD):V5 (ug) Empty:V5 (ug) anti-V5 160 C CD | S 3 120 C Q . O $ LU -c .5 _J O CD 80 O co 40 DLD-1 ILK(KD):V5 (ug) 0 1 Empty:V5 (ug) 4 3 anti-V5 2 2 4 0 Figure. 9. Dose-dependent inhibition of TCF transcriptional activity by ILK(KD):V5. Expression of increasing amounts of ILK(KD):V5 resulted in a dose-dependent inhibition of TCF transcriptional activity as determined by activity of the minimal TCF response element promoter. SW-480 and DLD-1 cells were co-transfected with 2 ug of pTOPFlash (•) (Wild-type TCF response element conjugated to luciferase reporter gene) of pFOPFlash ( M ) (Mutant TCF promoter) and the indicated amount of ILK(KD):V5 and Empty:V5. Forty-eight hours post-transfection, the cells were assayed for luciferase activity. Data are meant +/- standard deviation of six independent trials. Cell transfection efficiency was normalized with pRenilla (a constitutively expressing mutant luciferase). ILK(KD):V5 expression in SW-480 and DLD-1 cells is shown by Western blot analysis using an anti-V5 antibody. 40 3.3.3. Inhibition of ILK decreases the activity of the cyclin Dl promoter. It has recently been demonstrated that the expression of cyclin D l in colon carcinoma cells is regulated by p-catenin (Tetsu and McCormick, 1999). The cyclin D l promoter contains a TCF/LEF-P-catenin binding site and this has been suggested to be an important site for the transcriptional regulation for cyclin D l (Tetsu and McCormick, 1999). To determine i f I L K is involved in the regulation of cyclin D l expression through the activation of TCF/LEF-P-catenin complex, a plasmid containing the full length wild-type cyclin D l promoter linked to a luciferase reporter gene, was cotransfected with and without a plasmid encoding I L K kinase-deficient, into APC negative colon cancer cell lines (SW480 and DLD-1). In the presence of catalytically-inactive ILK, the cyclin D l promoter activity is decreased, in comparison to the cells transfected with an empty V5 vector. This suggests that I L K is involved in the regulation of cyclin D l expression in A P C mutant cells (Figure 10). The cyclin D l promoter contains multiple sites for the binding of different transcription factors including AP-1, TCF/LEF, and CREB. In order to demonstrate that cyclin D l is a target of TCF/LEF-P-catenin activity, the activity of the cyclin D l promoter with a mutation in the TCF/LEF binding site, was compared to wild type cyclin D l promoter. The basal activity of cyclin D l promoter with the mutation is significantly less than that of the wild-type cyclin D l promoter, suggesting that the role of TCF-P-catenin is important in cyclin D l expression (Figure 11). This suggests that I L K regulate the expression of cyclin D l through its regulation of the P-catenin/TCF/LEF transcriptional activity. 41 SW-480 DLD-1 Ant i V5 Figure. 10. Inhibition of cyclin D l promoter activity by expression of ILK(KD):V5. SW-480 and DLD-1 cells were co-transfected with 2 mg of ILK(KD) :V5 or empty: V5 and 1 mg of cyclin D l promoter. Forty-eight hours following transfection, cells were assayed for luciferase activity. Transfection efficiency was normalized with pRenilla. Data are mean +/- standard deviation of three independent trials. ILK(KD):V5 expression in SW-480 and DLD-1 cells is shown by Western blot analysis using an anti-V5 antibody. 42 Wild Type TCF mutant SW-480 DLD-1 + - + -- + - + Figure. 11. p-catenin/TCF activity regulates cyclin Dl expression. SW-480 and DLD-1 were transfected with 3 pg of wild-type full length cyclin D l promoter or mutant TCF binding site full length cyclin D l promoter, and 0.02 pg of pRenilla. After 48 hours, the cells were assessed for luciferase activity. Data are mean +/- standard deviation of four independent trials and normalized with pRenilla. 43 3.3.4. Effects of an ILK inhibitor on the regulation of P-catenin/TCF/LEF activity, cyclin Dl expression and E-cadherin expression in APC-/- colon carcinoma cell lines. A small molecule inhibitor of I L K (KPSD-1) has recently been identified (Persad et al., 2000). The effects of KPSD-1 should be similar to those seen with the I L K kinase dead construct, i f it is indeed a specific and selective inhibitor of ILK. Experiments were therefore designed to test whether inhibition of ILK activity using this inhibitor, also resulted in the suppression of P-catenin/TCF activities and cyclin D l expression. 3.3.5. Increasing amounts of KPSD-1 decreases ILK activity and p-catenin/TCF/LEF-1 activity in a dose-dependent manner. After 48 hours of incubation with increasing amounts of I L K inhibitor, DLD-1 and SW480 cells transfected with pTOPFlash showed an inverse correlation with increasing amounts of inhibitor and decreasing TCF/LEF-p-catenin activity. (Figure 12). In addition, the I L K inhibitor decreases the activity of ILK in a dose-dependent manner (Figure 17). 3.3.6. ILK inhibitor decreases cyclin Dl expression in a dose-dependent manner. Culturing the cells for 48 hours with increasing concentrations of I L K inhibitor resulted in a decrease in I L K activity, which correlated with a decrease in the levels of cyclin D l protein in DLD-1 and SW480 cells. There was no observed change in total P-catenin expression or ILK expression. These data further suggest that I L K activity positively regulates cyclin D l protein levels (Figure 13). 44 12 _ ^ r las 8 u_ Q_ TO S 4 CL _ | >> 0 > > o 15 < CD LL on LU — ' 10 _ J LL TC 5 SW-480 DLD-1 0 10' 50 100 K P S D - 1 Concentration (JIM) Figure. 12. KPSD-1 inhibits B-catenin/TCF activiation in a dose-dependnet manner. SW-480 and DLD-1 cells were transfected with 3 pg of pTOPFlash. Cells were incubated in D M E M and 10 % FBS for 6 hours post-transfection. Cells were then incubated for an additional 48 hours in low serum with the indicated concentration of KPSD-1 and vehicle control. Three independent experiments were assessed for luciferase activity and data was normalized with pRenilla. Data are meant +/- standard deviation of 6 independent trials. 45 3.3.7. ILK inhibitor decreases GSK-3 phosphorylation in a dose-dependent manner. GSK-3 normally phosphorylates P-catenin resulting in its ubiquitination-mediated degradation. Since I L K phosphorylates GSK-3 and inactivates its kinase activity, we wanted to determine i f the observed effects of ILK on p-catenin/TCF activity were mediated via GSK-3. Phosphorylated GSK-3 was analyzed using anti-phosphoserine 9/21 antibody as described for the same membranes as above experiments. The inhibition of I L K kinase activity in the presence of the ILK inhibitor corresponds to a decrease in G S K phosphorylation on the serine 9/21 (Figure 13). These data suggest that an APC-independent pathway is operational in these colon cancer cell lines. This pathway involves the inhibition of GSK-3 activity by ILK, leading to increased activity of P-catenin/TCF and cyclin D l . 46 sw-480 DLD-1 0 10 50 100 0 10 50 100 KP-SD-1 (uM) « • • » * • * « < » p-catenin K B C 4 GSK-3 Phospho-serine 9/21 GSK-3 kinase assay I GSK-3 - Cyclin D1 Figure. 13. Cyclin D l expression and inhibition of GSK-3 phosphorylation by KPSD-1 in a dose-dependent manner. SW-480 and DLD-1 cells were incubated with increasing KPSD-1 concentration in D M E M with 2 % serum for 48 hours. Cells were then lysed and equivalent amounts of protein were resolved by 10 % SDS-PAGE. Cyclin D l expression and GSK-3 phosphorylation was analyzed by Western blotting. 47 3.3.8. ILK inhibitor decreases the proliferation of colon carcinoma cell lines in a dose dependent manner. Abnormal levels of p-catenin/TCF/LEF activity may contribute to neoplastic transformation by causing the accumulation of cyclin D l . The absence of cyclin D l should prevent the cells from progressing across the Gl /S phase. To demonstrate that the inhibition of I L K activity results in a decrease in p-catenin-TCF activity and cyclin D l expression, and thereby result in the inhibition of cell growth, cell proliferation rates, colony growth and apoptosis assays were performed. The proliferation rate of cells (Figure 14) and the number of colonies decreases with increasing amounts of the I L K inhibitor (Figure 15). Cell death was not significant until 72 hours after the treatment with the inhibitor (Figure 16). These data demonstrate that the I L K inhibitor causes cell cycle arrest and ultimately cell death in APC-/- colon carcinoma cell lines. This implies that I L K is regulating the cell cycle and preventing apoptosis in SW480 and DLD-1 cells. 48 o o o o 90 60 30 SW-480 T— X c E z O o : o 1 180 120 60 o r 0 DLD-1 • -2 3 D A Y Figure. 14. KPSD-1 decreases the growth rate of SW-480 and DLD-1 ceils in a dose-dependent manner. Equivalent number of SW-480 or DLD-1 cells were incubated in media containing 10 % serum and then treated with KPSD-1 ( O = DMSO, B = 10 p M , 71 = 50 mM, X = 100 p M , diluted in D M E M with 2.5 % serum) for the indicated days. Cells were harvested, stained with Trypan Blue dye and counted. More than 800 cells were counted for each sample. Data are the mean +/- standard deviation of three independent trials. 49 0 10 50 100 Concentration of KPSD-1 (uM) Figure. 15. KPSD-1 decreases the number of colonies formed by SW-480 and DLD-1 in a dose-dependent manner. Colony forming assays were performed on SW-480 and DLD-1 cells as described in "Materials and Methods". KPSD-1 was added to the collagen and cell mixture. Cells were stained with crystal violet after 10 days. The data is the mean +/- standard deviation of six independent trials. Colonies were counted with a colony counter (BioRad). 50 -After 72 Hours of Treatment o a . o C) c o <u c O B D M S O • K P S D - 1 SW-480 DLD-1 Figure. 16. KPSD-1 induces cell apoptosis after 72 hours. A n apoptosis assay was performed as described in "Material and Methods". Annexin V conjugated FITC is bound to phospho serine on the membrane of apoptotic cells. The cells were analyzed by flow cytometry and the results were graphed as percentage of the total cell population. Data are the mean +/- standard deviation of six independent trials. A l l calculations were done using the WinMDI program. 51 3.3.9. ILK inhibitor prevents the nuclear localization of P-catenin in a dose-dependent manner. The overexpression of I L K causes the translocation of B-catenin into the nucleus (Novak et al., 1998). Nuclear lysates of SW-480 cells, incubated with increasing amounts of I L K inhibitor, were prepared to determine if I L K is involved in the translocation of P-catenin in APC mutant cells. Although not complete, the levels of nuclear p-catenin decreased upon inhibition of I L K activity (Figure 17). This suggests that I L K may contribute to the stability of P-catenin and promote it to translocate to the nucleus. 52 S W - 4 8 0 0 10 50 100 uM KP-SD-1 P-Catenin Nuclear p-Catenin Histone E-cadherin LEF-1 ILK ILK kinase assay Figure. 17. Inhibition of I L K activity in SW-480 cells increases the expression of E-cadherin and decreases the nuclear translocation of p-catenin in a dose-dependent manner. SW-480 cells were incubated with increasing concentration of KPSD-1 for 48 hours. Cells were harvested, and divided into two samples. Whole cell lysates were prepared with one sample and nuclear cell lysates were prepared with the other sample. Equivalent amounts of protein were resolved by 10 % SDS-PAGE. Protein levels and post-translational modifications were analyzed by Western blotting. The same lysates were immunoprecipitated for ILK, and I L K kinase activity was measured as described in "Material and Methods". This is a representation of three independent trials. 53 3.4.1. Inhibition of ILK promotes the expression of E-cadherin in a dose-dependent manner. The down regulation of E-cadherin expression is associated with epithelial to mesechymal transformation and with the progression of benign tumors to metastatic cancers (Brache et al. 1996; Huber et al., 1996). Similarly, the expression of E-cadherin protein is lost in I L K overexpressing cells, but is maintained in control transfected cells (Novak et al., 1998). To determine whether I L K activity modulates E-cadherin expression in colon carcinoma, E-cadherin levels were measured in SW480 cells after treatment with the I L K inhibitor. The data show that increasing amounts of I L K inhibitor resulted in an increase in E-cadherin expression (Figure 17). Thus the downregulation of I L K activity correlates with an increase in E-cadherin expression, in a dose-dependent manner. 3.4.2. Inhibition of ILK increases the E-cadherin promoter activity in a dose-dependent manner. Protein levels can be regulated at the level of transcription and translation or post-translationaly. To identify how the E-cadherin molecule is being regulated, the E-cadherin promoter activity was assayed. The E-cadherin promoter contains three E-boxes, which members of the Snail family bind to and inhibit transcription activity (Batlle et al., 2000, Cano et al., 2000). The full-length wild-type E-cadherin promoter was transfected into SW-480 cells, and the cells were incubated with increasing amounts of I L K inhibitor for 48 hours. The results show the E-cadherin promoter activity 54 increased with increasing amounts of ILK inhibitor, suggesting that there was transcriptional regulation (Figure 18). 55 B 3 180 o cn t t | 120H C 0 SW-480 CO 9 = UJ " 60 H KP-SD-1 (pM) 0 10 50 100 Figure. 18. Inhibition of ILK activity increases the E-cadherin promoter activity in a dose-dependent manner. SW-480 cells were transfected with 4 pg of E-cadherin full length wild-type promoter with a luciferase reporter and pRenilla. After a 48 hour incubation period with the indicated levels of KPSD-1, cells were assayed for luciferase activity. Data are mean +/- standard deviation of six independent trials. 56 3.4.3. Inhibition of I L K decreases Snail promoter activity in a dose-dependent manner. Snail regulates the expression of E-cadherin by blocking the activity of constitutivly active transcription factors on the E-cadherin promoter (Batlle et al., 2000, Cano et al., 2000). A n analysis of the effects of the inhibitor on Snail transcription was performed. The full length Snail promoter was transfected into SW480 cells and the cells were incubated with increasing amounts of I L K inhibitor for 48 hours. The Snail promoter activity decreased with increasing amounts of the I L K inhibitor (Figure 19). These results show a decrease in I L K activity correlates with an increase in Snail transcription. 57 Figure. 19. Lack of ILK activity decreases the human Snail promoter activity. SW-480 cells were transfected with 3 pg of full length, wild-type Snail promoter, and treated the same as the cells in the previous experiments. Cells were assessed for luciferase activity after 48 hours. Data are the mean +/- standard deviation of six independent trials. 58 3.4.4. Role of ILK on Snail and E-cadherin expression in stable transfected IEC-18 Specific inhibitors utilized in the study of signal transduction pathways are helpful because they are easy to use and the effect on other pathways is usually minimal. Unfortunately, the effects of the inhibitor may be compromised by cellular activities, such as cellular detoxification and the presence of transmembrane channels that prevent the inhibitor from residing in the cell long enough to have an effect. The inhibitor may have the ability to regulate other presently unknown pathways. To further investigate and understand the role of I L K in regulating E-cadherin expression via Snail, the transcription of E-cadherin and Snail was examined in IEC-18 cells overexpressing ILK. 3.4.5. Inhibition of ILK increases the E-Cadherin promoter activity. It has been documented that I L K stimulates collagen gel invasion of IECs and down regulates E-cadherin expression (Novak et al. 1998). A reporter plasmid containing the full length E-cadherin promoter was transiently transfected into parental IEC-18s, ILK-13 cells, ILK-14 cells, or I L K - K D cells. In concordance with relative protein expression levels, the E-cadherin promoter is more active in cells with low ILK levels and I L K activity, and least active in cells overexpressing ILK (Figure 20). These data demonstrate that I L K decreases the transcription of E-cadherin in epithelial cells. In addition, I L K promotes the transcription of Snail (Figure 21), resulting in a decrease of E-cadherin expression. 59 1200 Parent ILK-14 1 L K - K D 1LK-13 Anti-sense GH31RH A l a 3 Figure. 20. Overexpression of ILK wild-type decreases E-cadherin promoter activity. IEC-18 parental cells, the I L K anti-sense overexpressing stably transfected IEC-18 (ILK-14), the I L K kinase dead overexpressing stably transfected IEC-18 (ILK KD) and I L K wild-type overexpressing stably transfected IEC-18 clone Ala3 (ILK-13) cells were co-transfected with E-cadherin promoter and pRenilla. After 48 hours, cells were assessed for luciferase activity. The data is expressed in R L U and normalized with pRenilla. Data are mean +/- standard deviation of six independent trials. 60 Parent ILK-14 ILK(KD) ILK-13 Anti- GH31RH A1a3 Sense Figure. 21. Functional I L K is involved in enhancing Snail promoter activity. IEC-18 parentals, I L K anti-sense (ILK-14), I L K kinase dead (ILK KD) and I L K overexpressor wild-type clone Ala3 (ILK-13) were co-transfected with the Snail promoter and pRenilla by lipofection. After 48 hours, cells were harvested and assessed for luciferase activity. Activity is expressed as RLTJ and normalized with pRenilla. Data are means +/- standard deviation of six independent trials. 61 3.4.6. Increasing amounts of ILK inhibitor increases the E-cadherin promoter activity in a dose-dependent manner. To further define the activity of the I L K inhibitor in epithelial cells, the effects on I L K overexpressing IEC-18s was examined. ILK overexpressing IEC-18 clones were transiently transfected with the full length wild-type E-cadherin promoter. In agreement with the previous data, the incubation with I L K inhibitor caused the E-cadherin promoter activity to increase (Figure 22). 62 Figure. 22. Dose-dependent activation of E-cadherin promoter activity by KPSD-1. IEC-18 I L K wild-type overexpression clones (Ala3 and A4a) were transfected with 4 pg of E-cadherin full-length wild-type promoter with a luciferase reporter and pRenilla. After a 48 hour incubation period with the indicated levels of KPSD-1, cells were assayed for luciferase activity. Data are mean +/- standard deviation of six independent trials. 63 IV. Discussion Stimulation of integrins by the extracellular matrix results in the activation of various intracellular signaling pathways, that lead to cell survival, cell proliferation, cell migration, and cell differentiation. The Ras/Raf/Mitogen-activated protein kinase signaling pathway, the PI-3 kinase/ PKB/Akt pathway, and the Wnt signaling pathway have all been implicated in integrin signaling (Dedhar, 1999; Dedhar 2000). As previously stated in the introduction, recent evidence suggests an important role for the Integrin Linked Kinase (ILK) in the regulation of these pathways, as overexpression of I L K in epithelial cells has been shown to induce anchorage-independent cell survival as well as an epithelial to mesenchymal transformation (Novak et al., 1998). The molecular basis for the regulation of these phenotypes by I L K is unclear. The data presented here suggest mechanisms for the regulation of I L K activity and the identity of possible downstream effectors. These results also suggest the possible mechanisms for the effect of I L K on gene expression. The data provides evidence that ILK can be stimulated rapidly and transiently by cell attachment to fibronectin, and by growth factors. The stimulation of I L K activity by these agonists is dependent on the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 acts as an intracellular second messenger and therefore, the levels must be maintained within a narrow range for a cell to behave normally. The maintenance of the PIP3 levels is most probably carried out by the lipid phosphatase PTEN that dephosphorylates PIP3 to PIP2. The first two potential effectors of I L K that 64 were analyzed were Akt/PKB and GSK-3 because they are activated in a PI-3 kinase dependent manner. Overexpression of Akt/PKB or inhibition in GSK-3 activity resulted in the same phenotype observed in cells overexpressing ILK. 4.1. The regulation of GSK-3 by ILK kinase activity in Akt/PKB dependent and independent manner. Two downstream effectors of I L K have been identified; the serine-threonine kinases GSK-3 and P K B / A K T , both of which are regulated by PI 3-kinase (Delcommenne et al., 1998) and PTEN (Persad et al., 2000). There is also evidence that I L K regulates downstream components of the WNT-signaling pathways, namely LEF-1 and P-catenin (Novak et al., 1998). Because GSK-3 is known to regulate P-catenin stability, through its ability to phosphorylate P-catenin, it is considered to be a probable target of ILK. The data in figure 1 demonstrate that stable overexpression of kinase-active I L K in IEC-18 cells, as well as in Scp2 cells (data not shown), inhibited the activity of GSK-3, and transient co-transfection of GSK-3 with kinase active I L K in H E K 293 cells resulted in inhibition of GSK-3 activity, whereas cotransfection of kinase-deficient I L K enhanced GSK-3 activity over basal levels. Also, the data suggest that I L K can directly phosphorylate GSK-3 in vitro, yet it is still unclear whether the inhibition of GSK-3 activity by I L K is due to its direct phosphorylation by I L K or via the regulation of P K B / A K T activity. To determine i f ILK is capable of affecting transcription factors, the first trancription factor to be investigated was ILK's effect on c-Jun or the AP-1 D N A binding heterodimer. It is known that c-Jun is regulated by GSK-3 (de Groot et al., 65 1993), and I L K can regulate GSK-3 activity. The hypothesis I tested was that I L K regulates c-Jun and the AP-1 heterodimer in a GSK-3 dependent manner. 4.2. Stimulation of ILK activity by adhesion onto fibronectin is capable of regulating AP-1 transcription activity in a GSK-3 dependent manner. The data presented in this thesis demonstrate a novel signaling pathway for the activation of the AP-1 transcription factor via cell adhesion to FN. Recent publications have shown that adhesion of cells to fibronectin stimulates I L K activity (Delcommenne et al., 1998), and integrin-mediated cell adhesion is known to regulate gene expression via transcription factors such as N F - K B and AP-1 (Baldwin, 1996). Since the regulation of I L K activity is PI-3 kinase dependent (de Groot et al., 1993), a pharmacological PI 3-kinase inhibitor, namely LY294002, was used in adhesion assays to show that adhesion induced and ILK-mediated AP-1 activation are PI 3-kinase dependent. I L K has been shown to inhibit GSK-3 activity in a PKB/Akt dependent and independent manner (Delcommenne et al., 1998). Furthermore, there is evidence that GSK-3 phosphorylates Jun family members and negatively regulates its transactivating potential in intact cells (Nikolakaki et al., 1993). ILK, by inhibiting GSK-3, may prevent GSK-3 dependent C-terminal phosphorylation of c-jun near the DNA-binding domain of c-jun, and thereby, result in promoting the formation of a DNA-protein complex containing c-jun and enhance AP-1 activity (Troussard et al., 1999) (fig. 51). Signal transduction pathways, normally associated with the binding of soluble growth factors to their receptors, are also activated by integrin engagement (Schwartz, 1997). There is evidence that the growth factor-mediated activation of PI 3-kinase 66 effectors such as PKB/Akt depends on cell adhesion (King et al., 1997). Integrin engagement also triggers activation of the R A F - 1 / M E K / M A P K pathway (Lin et al., 1992; Renshaw et al., 1997). However, there is also evidence that I L K regulates AP-1 independently of the RAF-1 / M E K / M A P K pathway (Troussard et al., 1999). Collectively, this suggests that although growth factors and the extracellular matrix can regulate AP-1 via INK activation, the involvement of I L K and GSK-3 also appears to be required for extracellular matrix stimulation of AP-1 activity. Together, these results demonstrate that upon adhesion to fibronectin, I L K activity is stimulated resulting in the inhibition of GSK-3 activity which subsequently, upregulates AP-1 activity. 4.3. Regulation of P-catenin/TCF/LEF activity and E-cadherin expression by Integrin linked kinase in colon carcinomas. When I L K is overexpressed in epithelial cells, Cyclin D l expression increases, the cells grow faster, and do not undergo anoikis (Radeva et al., 1997; Attwell et al., 2000). These cells also have increased levels of nuclear p-catenin, show an increase in P-catenin/TCF/LEF activity and decreased E-cadherin expression (Novak et al., 1997). Similarly, in the presence of a Wnt signal, P-catenin is stabilized and the P-catenin/TCF/LEF complex is active, as well. Human intestinal adenomas that arise as a result of germline mutations in the APC gene also demonstrate an increase in the p-catenin/LEF-l/TCF bipartite complex activity; however, the E-cadherin expression is variable (Ozawa et al., 1998; Stewart and Nelson 1997; Sadot et a l , 1998; Orsulic et al., 1999). Recently, observations were made that I L K expression is upregulated in human 67 colon polyps, and this correlates to the polyp size (Marotta et al., unpublished). The present study demonstrates that the activity of the |3-catenin/TCF/LEF-l transcription factor is significantly decreased when dominant negative I L K is expressed in cell lines that lack A P C function. This result was unexpected, considering the hypothesis that the lack of A P C should abolish any upstream regulation of P-catenin degradation and activity. This finding suggests that ILK may be involved in the regulation of P-catenin pools. Increased P-catenin levels have been previously shown to enhance cell proliferation (Orford et al., 1997; Wong et al., 1998; Zhu et al., 1999), and to induce neoplastic transformation (Kolligs et al. 1999). In support of these findings, the target genes of p-catenin/LEF/TCF, in mammalian cells, include oncogenes such as c-myc and cyclin D l (Shutman et al., 1999, Tetsu and McCormick, 1999). To further address this issue, the promoter activity of cyclin D l , which is a downstream target of the signal transduction pathway(s) and is upregulated by the loss of APC function (Tetsu and McCormick, 1999), was examined in DLD-1 and SW-480 cells. This study demonstrated that the cyclin D l promoter activity decreased in the presence of dominant-negative ILK, indicating that I L K is involved in cyclin D l regulation. Next, the role of P-catenin/TCF/LEF in cyclin D l expression was evaluated by comparing the basal activities of the wild-type promoter and a promoter with a point mutation in the TCF binding site. The promoter activity decreased when the TCF binding site was mutated. This suggests that TCF activity is important in the transcription of cyclin D l promoter. It should be noted however that the mutation did not result in complete abrogation of cyclin D l promoter activity. This indicates that although the TCF binding site may be 68 important, it is not the only site involved in the transcription of cyclin D l expression. As of yet, the importance of the other sites and the ability of I L K to regulate other transcription factors involved remains uncharacterized. 4.4. I L K inhibitor Recently, a small molecule, discovered by high through-put screening, has been shown to specifically decrease ILK activity (Persad et al., 2000; Attwell et al., 2000). The finding that cells exposed to this small molecule resulted in a decrease in (3-catenin/TCF/LEF activity, and in cyclin D l protein levels, further demonstrates the ability of I L K to regulate P-catenin-TCF/LEF activity and its target genes. A P C acts as a scaffolding protein to maintain GSK-3 in the correct orientation to phosphorylate P-catenin. Therefore, the role of GSK-3 is not effective without the presence of APC. Surprisingly, despite the lack of functional A P C in DLD-1 and SW480 cells, it was demonstrated here that the inhibition of ILK, by either exogenous expression of a dominant-negative form of ILK, or by exposure of cells to a small molecule, I L K inhibitor, still resulted in the decrease of the P-catenin/TCF/LEF activity. In contrast to APC having the most important role in targeting P-catenin for degradation, the observations suggest that the activation of GSK-3 plays a more crucial role. I L K has been demonstrated to inhibit the activity of GSK-3, thus this mechanism appears to be important in regulating P-catenin and cyclin D l levels. The activation of GSK-3 is able to promote degradation of cyclin D l and regulate P-catenin levels and location in some cells (Diehl et al., 1998). This decrease in cyclin D l levels supports the finding that decreased I L K activity, as well as an increase in GSK-3 activity in A P C null cells, decreases proliferation, and causes growth arrest and, ultimately, apoptosis. 69 Surprisingly, the presence of the I L K inhibitor caused an increase in E-cadherin protein levels in SW480 cells, which do not normally express E-cadherin. At this stage, the possibility that I L K regulates the expression of Snail was considered. In ILK-overexpressing cells, the Snail promoter was demonstrated to be more active than in cells with lower levels of ILK. In addition, the exposure to the I L K inhibitor resulted in a decrease in Snail promoter activity. A suggested model for this phenomenon is that I L K increases the expression of Snail, which suppresses the expression of E-cadherin. The lack of E-cadherin results in a decrease of p-catenin pools at the membrane and cytoskeletal rearrangement. These results, together with studies investigating the regulation of P-catenin degradation and relocalization, as well as E-cadherin expression and function, are indicative of a model where ILK can regulate p-catenin/TCF/LEF activity and cyclin D l expression by modulating Snail expression, and GSK-3 activity. This model does not rule out the possibility that the expression of LEF-1 may have a role in modulating transcription of cyclin D l . This notion is based upon the observation that LEF-1 expression is decreased when the cells are exposed to the ILK inhibitor, and it has been observed that the overexpression of I L K causes an increase in LEF-1 expression. However, many studies demonstrate that the intranuclear levels of LEF-1 and TCF-4 may be high, yet repress gene expression until P-catenin is translocated to the nucleus and binds to LEF/TCF to form a multiprotein complex (Gianni et al., 2000). The influence of I L K on intestinal/colon/rectal tumor formation and its status as modulator of P-catenin, cyclin D l and E-cadherin expression further emphazies the critical role of I L K in tumorgenesis. 70 VI . Conclusion In epithelial cells, ILK activity is able to regulate two different transcription factors (AP-1 and B-catenin/LEF-l/TCF). Upon stimulation with growth factors or extracellular matirx, I L K is capable of inhibiting the activity of GSK-3 indirectly through Akt/PKB or directly, in a PI-3 kinase dependent manner. In turn, the inhibition of GSK-3 activity prevents the GSK-3 from phosphorylation c-Jun on the D N A binding site, thus promoting the transcriptional activity of the AP-1 heterodimer. In colon carcinoma cell lines lacking functional APC, ILK continues to influence and inhibit GSK-3 activity, leading to the stabilization and translocation of P-catenin to the nucleus, and the promotion of the p-catenin/TCF/LEF-1 transcriptional activity. Thus I L K is able to regulate the transcription of mesenchymal genes, pro-proliferative genes and pro-survival genes by promoting the activity of AP-1 and P-catenin/TCF/LEF-1 transcription factors in a PI-3 kinase dependent manner. 71 References Aberle H , Butz S, Stapper J, Weissig H , Kemler R and H Hoschutzy. 1994. 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