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The role of integrin-linked kinase in tumour angiogenesis and inflammation Tan, Clara Chia-Hua 2006

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T H E R O L E O F I N T E G R i N - L I N K E D K I N A S E I N T U M O U R A N G I O G E N E S I S A N D I N F L A M M A T I O N by C L A R A C H I A - H U A T A N B . S c , M c G i l l University, 1997 M.Sc . , University of British Columbia, 2002 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF C O M B I N E D D O C T O R OF P H I L O S O P H Y A N D D O C T O R OF M E D I C I N E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Biochemistry and Molecular Biology) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A p r i l 2006 ©Clara Chia-Hua Tan, 2006 Abstract Integrins act as transducers of extracellular matrix-mediated cell signaling. A critical signaling component downstream of integrin engagement is integrin-linked kinase (ILK), a serine/threonine protein kinase and adaptor protein, that interacts with the cytoplasmic domains of pi and p3 integrins. ILK couples integrins and receptor tyrosine kinases to the cytoskeleton, thus mediating multiple downstream signaling events that regulate cell adhesion, survival, proliferation, migration and differentiation. Inappropriate ILK activity as a result of deregulated upstream mechanisms or altered transcription, results in epithelial cell transformation that recapitulates malignant cancer cell behaviour. I provide novel data demonstrating that ILK plays an indispensable role in regulating downstream targets that induce nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in murine macrophages, and cyclooxygenase-2 (COX-2), a pro-inflammatory macromolecule, expression in both murine and human macrophages. Cells treated with a highly specific small molecule ILK inhibitor or transfected with a dominant-negative mutant of ILK showed decreased ILK activity resulting in inhibition of lipopolysaccacharide (LPS) stimulated iNOS and NO production and reduction in COX-2 expression in a protein kinase B (Akt/PKB)-dependent manner. I also propose a novel model for induction of N F - K B transcription, where phosphorylation of IKB at the serine 32 position directly or indirectly by ILK or Akt/PKB targets IKB for degradation; consequently N F - K B translocates to the nucleus to initiate transcription of pro-inflammatory molecules. In addition, ILK and Akt/PKB are stimulated in a PI3-kinase dependent manner. I provide evidence for a new paradigm involving ILK in the improper production of vascular endothelial growth factor (VEGF) in normoxic conditions, shedding light on the role of ILK in tumor angiogenesis. Mechanistically, I show that activated ILK stimulates the expression of hypoxia-inducible factor-la (HIF-la) transcription factor, the major inducer of VEGF expression, through the phosphorylation of mammalian target of rapamycin in an Akt/PKB dependent manner in prostate cancer cells. In a positive feedback process, exposure of endothelial cells to VEGF stimulates a transient increase in ILK kinase activity in a PI-3 kinase-dependent manner. Inhibition of ILK activity using small interfering RNA or exposure to the ILK inhibitor uncouples endothelial cell migration and proliferation in response to VEGF. ii TABLE OF CONTENTS A B S T R A C T " T A B L E O F C O N T E N T S i " L I S T OF F I G U R E S - v i i L I S T OF A B B R E V I A T I O N S ix A C K N O W L E D G M E N T S ..... x C H A P T E R 1: I N T R O D U C T I O N 1 1.1 Extracellular Matrix 2 1.2 Integrins 3 1.2.1 Molecular Mechanisms in the Regulation of Integrin Function 5 1.2.2 Focal Adhesions..... 6 1.3 Integrin-Linked Kinase : 8 1.3.1 I L K Structure and Function 8 1.3.2 I L K Chromosomal Localization and Animal Models 11 1.3.3 I L K Interactions 13 1.3.4 ILK/Part icular ly Interesting Novel Cysteine-Histidine rich protein (PINCH) /Parvin (IPP) Complex 13 1.3.5 P I N C H Interactions 15 1.3.6 Parvin Interactions 16 1.3.7 Signals Downstream of IPP Complex. 17 1.3.8 Regulation of I L K 19 1.3.9 I L K as a Downstream Effector of Signaling pathways 21 1.3.10 Role of I L K in Anchorage-Independent Growth and Cel l Cycle Progression :...25 1.3.11 Role of I L K in Cel l Survival and Anoikis 26 1.3.12 Role of I L K in Epithelial and Mesenchymal Transformation ( E M T ) and Nuclear Activation of P-catenin 28 1.3.13 Role of I L K in Migration , Moti l i ty and Invasion 30 1.3.14 Role of I L K in Regulating Myos in Light Chain ( M L C ) 31 1.3.15 Role of I L K in Invasion 32 1.3.16 Role of I L K in Human Disease 32 1.4 Angiogenesis 34 1.4.1 Role of Extracellular Matrix in Angiogenesis 36 1.4.2 Role of Growth Factors and Receptors Associated with Angiogenesis 38 1.4.3 Role of Integrins in Angiogenesis 42 1.4.4 Role of Integrin-linked kinase in Vasculogenesis and Angiogenesis 44 1.5 Role of Integrin-linked kinase in Inflammation 45 1.6 Summary 47 1.7 References 49 C H A P T E R 2: R E G U L A T I O N OF T U M O R A N G I O G E N E S I S B Y I N T E G R I N -L I N K E D K I N A S E ( ILK) 68 2.1 Summary 68 2.2 Introduction 69 2.3 Materials and Methods 72 2.3.1 Ce l l Culture and Transfections 71 2.3.2 Small Interfering R N A (s iRNA) 73 2.3.3 Chemical Inhibitors 73 2.3.4 Western Blotting 74 2.3.5 Quantification of V E G F in Conditioned Media 74 2.3.6 I L K Kinase Assay 74 2.3.7 Invasion and Migration Assay 75 2.3.8 Cel l Viabil i ty and Proliferation Assay 75 2.3.9 Immunohistochemical Staining 76 2.3.10 Endothelial Tube Formation Assay 76 2.3.11 Endothelial Sprouting Assay 76 2.3.12 Chorioallantoic Membrane ( C A M ) of Chick Embryos Assay 77 2.3.13 PC3 Xenograft Tumor Assay 77 2.3.14 Micrographs 78 2.3.15 Plasmids 78 2.4 Results 79 2.4.1 Overexpression of I L K stimulates V E G F expression in a P K B / A k t - and HIF-la-dependent manner 79 2.4.2 Inhibition of I L K expression and activation suppresses P K B / A k t and m T O R / F R A P phosphorylation and inhibits H I F - l a and V E G F expression in prostate cancer cells 82 2.4.3 Pharmacological inhibition of I L K activity results in the inhibition of H I F - l a and V E G F expression in prostate cancer cells ..86 2.4.4 I L K regulates VEGF-mediated endothelial cell migration and blood vessel formation 89 2.4.5 Inhibition of I L K activity inhibits VEGF-stimulated angiogenesis in vivo 96 2.4.6 Inhibition of tumor angiogenesis and suppression of tumor growth in ILK-inhibitor treated PC3 xenograft tumor model 100 2.5 Discussion 102 2.6 References 107 C H A P T E R 3: I N T E G R I N - L I N K E D K I N A S E R E G U L A T E S I N D U C I B L E N I T R I C O X I D E S Y N T H A S E A N D C Y C L O O X Y G E N A S E - 2 E X P R E S S I O N I N A N N F - K B -D E P E N D E N T M A N N E R 112 3.1 Summary 112 3.2 Introduction 113 3.3 Materials and Methods 115 3.3.1 Cel l Lines and Cel l Culture 115 3.3.2 Transfection 116 3.3.3 In Vitro Kinase Assay 117 3.3.4 Western Blot Analysis 117 3.3.5 Luciferase Assays 118 3.3.6 Detection of Nitric Oxide 118 3.3.7 Plasmids 118 3.3.8 Immunohistochemsitry 119 3.4 Results... 120 3.4.1 I L K Up-Regulates N F - K B Activi ty 120 3.4.2 L P S Stimulates N O Production in an ILK-Dependent Manner 123 3.4.3 Inhibition of I L K Suppresses LPS-stimulated i N O S Expression and N O Production in J774 Cells and in Primary Murine Macrophages 125 3.4.4 Inhibition of I L K Suppresses LPS-stimulated N F - K B Expression and NF-KB-dependent i N O S Gene Expression... 130 3.4.5 Inhibition of I L K Suppresses I K B Serine-32 Phosphorylation and Prevents Its Degradation 133 3.4.6 Inhibition of I L K Suppresses LPS-stimulated C O X - 2 Expression in J774 Cells and Human Macrophages 135 3.5 Discussion 138 3.6 References 141 C H A P T E R 4: C O N C L U S I O N A N D F U T U R E D I R E C T I O N S 4.1 Conclusion 147 4.2 Future Directions 151 4.3 References 157 LIST OF FIGURES Figure 1.1 Display of documented occurring a - and P - integrin Combinations 5 Figure 1.2 Schematic diagram of integrin-linked kinase demonstrating the multiple domains and highlighting the conserved sequences 11 Figure 1.3 Schematic diagram of ILK/PINCH/Pa rv in complex found at the focal adhesions 15 Figure 1.4 Schematic diagram of ILK-dependent pathways and phenotypes 22 Figure 2.1 V E G F expression and H I F - l a activity are increased in epithelial cells with a high I L K activity 81 Figure 2.2 V E G F and H I F - l a expression are severely affected by the loss of I L K activity in a P T E N and mTOR/FRAP-dependent manner, in PTEN-nu l l prostate carcinoma cells (PC3) 85 Figure 2.3 Inhibition of I L K activity results in decrease of H I F - l a and V E G F expression in a PI-3 Kinase dependent manner in prostate carcinoma cells 88 Figure 2.4 I L K kinase activity is involved in VEGF-stimulated H U V E C activity 92 Figure 2.5 KP-392 inhibits angiogenesis in matrigel and in vivo 97 Figure 2.6 Inhibition of I L K activity suppresses tumor angiogenesis and tumor growth rate 101 Figure 2.7 Schematic representation of the cell signaling events leading to V E G F production in a prostate carcinoma cell and consequent effects on a neighboring endothelial cell 104 Figure 3.1 I L K upregulates N F - K B activity 121 Figure 3.2 L P S stimulates the production of nitric oxide in J774 cells and up-regulates the N F - K B activity 124 Figure 3.3 Inhibition of I L K activity decreases i N O S expression 126 Figure 3.4 Kinase-dead I L K decreases N F - K B activity in J774 macrophage cells in a dose-dependent manner 132 Figure 3.5 Inhibition of I L K suppresses IKB Serine-32 phosphorylation and prevents its degradation 134 Figure 3.6 Expression of Integrin-Linked Kinase in human alveolar Macrophages 136 Figure 3.7 Inhibition of I L K Suppresses C O X - 2 expression in J774 cells 137 v i i i List of Abbreviations ATP Adenosine trisphosphate B M Basement Membrane BSA Bovine Serum Albumin COX-2 Cyclooxygenase-2 CSF-1 Colony-stimulating factor-1 D M E M Dulbecco's Modified Eagle's Medium DOC Sodium deoxycholate E C L Enhanced chemiluminescent E C M Extracellular Matrix EDTA Ethylenediaminetetraacetic acid GFP Green fluorescent protein FBS Fetal bovine serum Fl/fl floxed/floxed GSK-3 Glycogen synthase kinase-3 IGF-1 Insulin-like growth factor-1 ILK Integrin-linked kinase INOS inducible nitric oxide synthase L O H Loss of heterozygosity M E O H Methanol NGS Normal Goat Serum NO nitric oxide PBS Phosphate Buffered Saline P I 3 - K Phosphatidylinositol 3 kinase P K B Protein Kinase B PMSF Phenylmethanesulfonyl Fluoride PTEN Protein with tensin homology SAGE Serial analysis of gene expression SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis SiRNA small interfering R N A TBS Tris Buffered Saline T M A Tissue Microarray WT Wild Type ix Acknowledgements I would like to take this opportunity to thank my supervisor Dr. Shoukat Dedhar for challenging me to reach far and guiding me through out my graduate career. I have had an ideal environment to carry out my research, having adequate time, appropriate pace and space, using sophisticated cell and molecular biology techniques working in the Dedhar lab, the MD/PhD program and the University of British Columbia Graduate Studies Program, as well, I have gained some experience in different research areas. I would also like to thank my supervisory and advisory committee, Dr. Alice Mui, Dr. David Huntsman, Dr. Aly Karsan, Dr. Anthony Chow and Dr. Lynn Raymond. A special thanks to the past and present members of the Dedhar Lab, Julia Mills, Tim McPhee, Arusha Oloumi, Sarah Attwell, Armelle Troussard, Paul MacDonald, Virginia Gray, Nasrin Mawji, Severine Hennequart-Cruet, Nolan Filipenko, Sujata Persad and Larissa Ivanova. I would also like to thank the faculty, staff and future colleagues/fellow students in the Faculty of Medicine, in particular, Eric Tam, Gayle Pelman, Aruna Somasiri the past and present UBC MD/PhD students Cheng-Han Lee, Paul Yong, Ryan Hung, Jimmy Lee, Michael Rauh, Claire Sheldon, Suvendrini Lena, Amy Webber and Aaron Joe, and the Medical Class of 2006. Finally, I would like to thank my partner Eric Tam, parents, sister and brother-in-law, my close and extended family for their support in this fantastic journey called graduate school. This work was supported by a MD/PhD studentship from the Canadian Institutes of Health Research of Canada and the Canadian Cancer Society. x 1 INTRODUCTION Tumors are a heterogeneous mass consisting of extracellular matrix ( E C M ) , cancer cells and normal cells, such as fibroblasts and infiltrating macrophages. Critical to the study of cancer is to understand cancer cell physiology in the context of c e l l - E C M and cell-cell interactions (Bissell et al., 1982; Bissell et al., 2005; Bissell and Radisky, 2001). Integrins are the key mediators of cell interactions with the extracellular environment (Hynes, 1987; Hynes, 2002). Integrin-linked kinase ( ILK) is a unique adaptor and kinase first discovered to bind to the cytoplasmic domain of integrins (Hannigan et al., 1996). In cancer, deregulated I L K activity is involved in several processes such as anchorage and growth signal independence, evasion of programmed cell death (apoptosis), sustained angiogenesis, and tissue invasion and metastasis (Hannigan et al., 2005) alterations in cell physiology which promotes formation of tumors and the manifestation of disease (Hanahan and Weinberg, 2000). Tumor growth can also be characterized in three phases: initiation, promotion and progression (Hanahan and Weinberg, 2000). Promotion of tumor growth relies on growth signals secreted by inflammatory cells in a paracrine manner to promote the proliferation of malignant cells (Balkwil l and Mantovani, 2001). For tumors to progress, they require an adequate blood supply (Folkman, 1976; Folkman and Cotran, 1976). In this introductory chapter, I w i l l review integrins, the extracellular matrix, and I L K . Furthermore, I w i l l focus on the cellular and molecular mechanisms mediated by I L K in cancer progression, blood vessel formation and inflammation. 1 1.1 Extracellular Matrix During normal embryonic development and in adult life, signaling between extracellular and intracellular components needs to be precisely coordinated and integrated in a spatial-temporal specific manner. The proper regulation of differentiation signals of cell growth and death must be critically balanced to prevent the manifestation of disease such as cancer. Organs are organized into a framework of cells, stroma and extracellular matrix (ECM), a visible network of secreted proteins, glycoproteins and polysaccharides that include collagen, fibronectin, vitronectin and laminin (Boudreau and Jones, 1999). This surrounds all the connective tissues, and underlines all the epithelial and the endothelial sheets of cells (Bissell et al., 2005; Bissell and Radisky, 2001). This provides not only a physical scaffold for the support of tissues and organs, but also serves as the basin for storage, release and presentation of growth factors, and a direct mediator of cell signaling (Bissell et al., 2005). Through interactions with specific transmembrane receptors, the ECM components have profound control over cell survival, proliferation, morphogenesis, migration, polarity and differentiation in a temporal and spatial-specific manner (Bissell et al., 2005). Many transmembrane receptors are responsible for interacting with the ECM, including heparan sulphate proteoglycans, syndecans, hyaluronan binding molecules, and integrins (Zamir and Geiger, 2001). Integrins, a family of cell surface receptors, are the primary mediators of mechanical and chemical signals involved in regulating cytoplasmic kinase activities, growth factor receptors, ion channels, and actin cytoskeleton organization during cell survival, proliferation, differentiation and migration (Hynes, 1987). Indeed, integrins are dynamic, bi-directional interactions that link the intracellular environment to macromolecular 2 extracellular assemblies. They regulate many intracellular signaling pathways through integrin-mediated cell adhesion and integrin clustering (outside-in signaling). These pathways are vital in regulating such diverse processes as embryonic development, cell survival, cell cycle progression, growth, differentiation, motility, and gene expression (Boudreau and Jones, 1999; Clark et a l , 1998; Dedhar and Hannigan, 1996; Giancotti and Mainiero, 1994; Hynes, 1987). A s well , integrins may transform from inactive to active state in response to intracellular signals (inside-out signaling). This regulation can then modulate the adhesive affinity of integrins to the E C M , and can activate E C M -degrading enzymes (Brakebusch et al., 2002). In addition to E C M and cytoplasmic proteins, integrins also associate with several membrane proteins, including I A P (Integrin Associated Protein), caveolin, and the tetraspanins (TM4) (Schwartz and Ginsberg, 2002). It is thought that these proteins function to associate integrins with intracellular signaling proteins and pathways to elicit specific responses to extracellular signals. 1.2 Integrins Integrins can be found in all metazoan species and are composed of non-covalently linked, single span a- and P- subunits, each consisting of a long extracellular domain (up to 1104 residues and 78 residues respectively) and a short intracellular domain (Whittaker and Hynes, 2002). In mammals, 18 a- and 8 P-subunits have been identified (Figure 1.2) (Hynes, 1987; Hynes, 2002) and are known to assemble into 24 a,p functional heterodimeric receptors (see figure 1.1). Integrins are so critical to life that defects in integrin expression and function are responsible for many human diseases, including cancer and autoimmune disease. Targeted knockout studies in various organisms and in tissue culture have underscored the essential roles integrins play in development (Hynes, 3 1987; Hynes, 2002). Integrins are highly selective, specific heterodimers that only bind a specific set of substrates. Many of the integrins, such as a 5 p l , ccvpi and ccvp3 integrins, recognize peptide sequences of E C M components that include the tripeptide arg-gly-asp (RGD) , a sequence shared by several E C M proteins, such as fibronectin and vitronectin (Ruoslahti and Pierschbacher, 1987). Although integrins are capable of binding various E C M components to influence cell behaviour, the cytoplasmic tail itself does not have any known intrinsic catalytic activity, unlike receptor tyrosine kinases; therefore, integrins are dependent on intracellular proteins to relay messages. Likewise, intracellular protein signals are capable of manipulating the avidity and affinity of the extracellular integrin domain for particular ligands. The R G D sequence interacts with integrins to activate several tyrosine kinases and protein kinase signal transduction pathways (Miyamoto et al., 1996). For example a 5 p l and avp3 integrin engagement results in the up-regulation of Bcl-2 and cell survival (Ruoslahti and Pierschbacher, 1987; Ruoslahti et al., 1987; Zheng et al., 1995). 4 Nature Reviews | Immunology Figure 1.1 Display of documented occurring a- and p- integrin combinations (taken from (Kinashi, 2005)adapted from (Hynes, 1987)). 1.2.1 Molecular Mechanisms in the Regulation of Integrin Function The cell surface conformation of integrins is critical to their function. Integrins can be found in three states, the inactive (bent form), primed (extended form) and active state (ligand-bound form) (Legate et al., 2006). The majority of the integrins are in the inactive conformation, characterized by bent extracellular domains that hide the E C M -binding pocket. This conformation is stabilized by interactions between integrin transmembrane domains, membrane-proximal extracellular domains and a salt bridge between the a- and P- integrin cytoplasmic domains (Legate et al., 2006). The present model suggests that phosphatidylinositol phosphate kinase type-ly (P IPKly) and talin, a cytoskeletal protein, complex to interact with the cytoplasmic tail of the P integrins (Critchley, 2004). This causes the a- and P- cytoplasmic tails to dissociate, inducing the 5 integrin extracellular domains to extend and unmask the ligand-binding site to adopt the primed conformation. This conformation allows the integrin to recognize and bind to specific E C M molecules. The separated integrin cytoplasmic domains and talin form a base for the recruitment of other proteins to form focal adhesions. The addition of cytoplasmic and signaling proteins including talin, a-actinin, vinculin and filamin, adapators such as paxillin, Rack-1, ICAP-1 and p3-endonexin and kinases such as Src tyrosine kinase and focal adhesion kinase ( F A K ) (Miyamoto et al., 1996), to the base occurs in a hierarchical and sequential manner that leads to the maturation of focal adhesions, clustering of active, ligand-bound integrins and the assembly of a multiprotein complex that is capable of linking integrins to the actin cytoskeleton and co-ordination of signaling pathways (Zaidel-Bar et al., 2003). These plaques also facilitate cross-talk between integrins and growth factor receptors, which wi l l be further discussed in the next section. This section focuses on the pathways immediately associated with the formation of the focal adhesions. 1 . 2 . 2 Focal adhesions Focal adhesions are sites on the cell where integrins and proteoglycan-mediated adhesions link to the actin cytoskeleton. Focal adhesions are composed of scaffolding molecules, GTPases, and enzymes including kinases, phosphatases, proteases, and lipases. There are several different focal adhesion structures that are defined by their subcellular location, size and composition. Focal complexes are small focal adhesions at the periphery of the spreading or migrating cells. They are regulated by cytoskeletal proteins such as Rac and Cdc42 (Nobes and Hal l , 1995; Nobes et a l , 1995) and precede 6 the larger focal adhesions that are regulated by Rho activity (Chrzanowska-Wodnicka and Burridge, 1996; Ridley and Hal l , 1992). Focal adhesions are structures found at the cell periphery and center. They are associated with the ends of stress fibers in cells cultured on two-dimensional rigid surfaces. Fibrillar adhesions are elongations of focal adhesions that specifically contains a 5 p i integrin and tensin (Pankov et al., 2000). The formation and maturation of the focal adhesion is a hierarchical sequence of integrin clustering and protein aggregation, resulting in a focal platform for signal transduction initiation. Intensive efforts have been made to dissect and distinguish the components of these structures and elucidate their specific roles. A key component of the focal adhesion is the focal adhesion kinase, F A K (Hauck et al., 2002), which mediates signal transduction downstream from integrin molecules that have engaged the matrix. F A K can directly or indirectly interact with the cytoplasmic tail o f in the p integrins. Upon integrin engagement, F A K becomes activated through autophosphorylation, the activated PI-3 kinase which produces secondary phospholipid messengers which, in turn, activates I L K and its downstream signaling components such as A k t / P K B to promote cell proliferation. F A K is a pivotal tyrosine kinase, which also recruits Grb and SOS, and initiates the Ras/ Raf cascade that diverges to promote proliferation and survival. O f particular interest is the formation of the Integrin-linked kinase/PINCH/Parvin (IPP) multiprotein complex that forms a platform at the focal adhesion and recruits more signaling and structural proteins (Legate et al., 2006). Prior to going into more details about the IPP complex, it is worthwhile to briefly discuss another focal adhesion protein. It appears that F A K is upstream of I L K , but new evidence demonstrates that I L K may regulate F A K (Cohen and Guan, 2005). 7 1.3 Integrin-Iinked kinase (ILK) Integrins do not have an intrinsic catalytic activity but are complex mechanochemical sensors and signal transducers of extracellular and intracellular signals. They also have important roles in bringing together the associated proteins required to modulate a myriad of intracellular activities. In fact, it is these associated proteins that define and initiate signals to modulate cell function and transcription, and if not tightly regulated, will lead to profound changes in a cell that confer a selective advantage to progressing to a disease state such as cancer. In cancer research, studies have focused on identifying and elucidating the protein mediators associated with integrins, and the dissection and investigation of the physiological integrin-mediated and growth factor receptor-mediated signals involved in orchestrating the network of pathways that are linked to functional activities in the cell. Therefore, further investigation of integrin-associated proteins will provide more insight into their roles in modulating cell function. To further investigate the multiple integrin-mediated pathways, the Dedhar lab focused their efforts on the study of integrin-associated proteins, specifically the proteins that are or would be associated with the cytoplasmic tail of the p-integrin, in the hope of finding a downstream therapeutic target for the control of cancer progression. This led to the discovery of Integrin-linked kinase (ILK). 1.3.1 ILK Structure and Function Integrin linked kinase (ILK) was identified 10 years ago in a yeast-2 hybrid screen as a protein capable of interacting with the cytoplasmic tails of pi and p3-integrins (Hannigan et al., 1996). Since the discovery of ILK, there has been a large accumulation of 8 evidence, both in vitro and in vivo, from a number of laboratories consistently demonstrating that I L K is an important adaptor protein in regulating actin polymerization and an essential serine-threonine kinase capable of initiating downstream signaling cascades critical for microenvironment-responsive cell signals, normal development and cancer progression (Hannigan et al., 2005). Integrin linked kinase consists of 452 amino acid residues and is arranged into three conserved functional domains (see figure 1.2). The N-terminal domain contains four ankyrin repeats, which mediate protein-protein interactions with L I M domain-containing adapter proteins, P I N C H - 1 , P INCH-2 and Paxil l in (Legate et al., 2006). The ankyrin repeat has been likened to a cupped hand structure in which the palm and the fingers are represented by the oc-helix bundle and the (3-hairpins, respectively (Sedgwick and Smerdon, 1999; Wu , 1999), and facilitates the localization of I L K to focal adhesions, sites where growth factor receptor and integrin-mediated pathways can be bridged to allow cross-talk. Immediately following the ankyrin repeats is a pleckstrin homology motif domain, which can bind phosphoinositol-3,4,5-trisphosphate (PIP3), a secondary messenger and PI-3 kinase product, to augment the kinase activity of I L K . The largest domain is the kinase catalytic domain located at the C-terminus. This domain mediates binding to the cytoplasmic tail o f p i and P3 integrins and has serine-threonine kinase activity (Figure 1.4). It is predicted to fold into a bi-lobated structure that is characteristic of other kinase domains (Dedhar et al., 1999; Wu , 1999). A sequence analysis of the I L K kinase domain reveals divergence at three generally highly conserved subdomains. In the highly conserved G X G X X G sequence of subdomain I, 2 of the 3 critical glycine residues are missing in I L K ( N E N H S G ) . A s well , variance in subdomains V I B ( H R D L ) and VII 9 (DFG) also continues to fuel the debate of whether I L K is a protein kinase, however, the I L K sequence does not show any variance in the crucial subdomain VIII A P E sequence, nor in the critical lysine residue between subdomain I and II which is necessary for the binding of A T P . Furthermore, other proteins lacking all three glycine residues in subdomain I have been identified as true protein kinases. To date, I L K has repeatedly been shown to phosphorylate a peptide representing the P1 integrin cytoplasmic domain (Hannigan et al., 1996), A k t / P K B at serine 473, and G S K - 3 on serine 21/9 in vitro and in cells (Delcommenne et al., 1998; Hannigan et al., 1996; Persad et al., 2000; Persad et al., 2001a). Other physiologically important substrates which I L K can phosphorylate in vitro are myosin light chain, the adaptor protein P-Parvin/Affixin (Deng et al., 2002; Deng et al., 2001; Paralkar et al., 1992; Yamaji et al., 2001), PHI-1, K E P I , CPI-17, G S K 3 p , Myos in phosphatase target subunit 1 and a N A C (reviewed in (Hannigan et al., 2005). The C-terminus of I L K is also critical as it serves to bind to proteins associated with actin cytoskeleton rearrangement such as a-Parvin / C H - I L K B P / actopaxin, P-Parvin/affixin, y-Parvin and Paxil l in (Nikolopoulos and Turner, 2000; Nikolopoulos and Turner, 2001; Nikolopoulos and Turner, 2002), to facilitate diverse signaling pathways. 10 PIP3 binding 2 1 3 363 Nature Reviews | Cancer Figure 1.2 Schematic diagram of integrin-linked kinase demonstrating the multiple domains and highlighting the conserved sequence, (from (Hannigan et al., 2005) 1.3.2 ILK Chromosomal Localization and Animal Models The I L K gene is located on human chromosome 1 lp l5 .5 - pl5 .4 . (Hannigan et al., 1997) and is highly evolutionary conserved with homologues found in C. elegans and drosophila. In the mouse, I L K is located in the 7E1 and 9E1 - 3 region o f chromosome 11 and shares 99 % identity with the human gene (Hannigan et al., 1996; L i et al., 1997; Mackinnon et al., 2002; Nikolopoulos and Turner, 2001; Zervas et al., 2001). The phenotypes of both Drosophila and C. Elegans I L K mutants highlight the importance of I L K in integrin-mediated adhesion. In the C. Elegans pat-4 mutant, embryonic muscle cells were able to form integrin foci, but were unable to recruit vinculin, U N C - 8 9 , actin and myosin filaments, proteins normally found in focal adhesions (Mackinnon et al. 2002). In Drosophila, I L K mutants phenotype was embryonic lethal and embryos had 1 1 defects in muscle attachment secondary to the detachment of actin filaments, similar to the integrin mutants. A s well , clones of cells lacking I L K in the adult wing formed wing blisters as the result of a lack of proper cell adherence (Zervas et al. 2001). I L K deficient mice die at the peri-implantation stage due to a failure in epiblast polarization as a result of abnormal F-actin accumulation at the sites of integrin attachment to the basement membrane and cavitate (Sakai et al., 2003). Mice with chondrocyte-specific disruption of I L K develop chondrodysplasia, characterized by a disorganized growth plate, dwarfism and early death as the result of decreased proliferation, adhesion, spreading and focal adhesion formation (Grashoff et al., 2003; Terpstra et al., 2003). The dependent P K B / A k t and G S K - 3 phosphorylation, and thus the role of I L K as a kinase and signal effector continued to be debatable at that time. Overexpression of I L K in the mammary epithelium under control of the mouse mammary tumor virus promoter ( M M T V ) caused the formation of benign hyperplasia and mammary tumors ranging from papillary adenocarcinoma to spindle cell tumors (White et al., 2001). Moreover, phosphorylation of A k t / P K B at serine 473, and G S K - 3 on 9/21 was observed. This emphasizes the role of I L K as a kinase signaling molecule. Furthermore, recent tissue-specific I L K knock-out models demonstrate the importance of I L K in embryonic development (Sakai et al., 2003) and organogenesis, specifically cortex and cerebellar development, (Mi l l s et al., 2003; M i l l s et al., 2006; Niewmierzycka et al., 2005), and function, specifically in diapedesis (leukocyte migration), and blood vessel formation (Cho et al., 2005; Friedrich et al., 2004; Friedrich et al., 2002; L i et al., 2003a; L i u et al., 2005). 12 1.3.3 Integrin-Iinked kinase Interactions Since its discovery, other groups have also shown that I L K binds directly to the cytoplasmic tails of (31 and (33 integrins (Pasquet et a l , 2002; Plante et al., 2005; Yamaji et al., 2001). I L K also binds paxillin, through a binding site in its kinase domain, linking I L K to F-actin though interactions with a-parvin/CHILK-BP/actopaxin and the actin-binding adaptor molecule vinculin (Nikolopoulos and Turner, 2000; Turner et al., 1999; Wood et al., 1994). MIG2/kindlin-2 binds to the cytoplasmic tail o f (31 and p3 integrins, as well as I L K , and associates with migfilin to create a bridge associating the ILK/Pinch/Parvin (IPP) (discussed in the following section) complex with filamin, F-actin and other integrins (Feng and Walsh, 2004; Tu et al., 2003) (see figure 1.3). These interactions appear to enhance clustering of integrins and provide another link between I L K and the actin cytoskeleton. 1.3.4 ILK/PINCH/Parvin (IPP) Complex Following the priming of the integrin by talin and PIPKIy but prior to ligand binding, I L K combines with parvin and P I N C H in the cytosol to assemble into the IPP complex. I L K is the central component of the IPP complex, binding to P I N C H proteins through the N-terminal ankyrin-repeat domain and parvins at the kinase domain. I L K then links the entire IPP complex to the cytoplasmic tails of p i and p3 integrins. The IPP complex is then immediately recruited to the focal adhesion through interactions with other intracellular proteins, such as paxillin. Simultaneously, other proteins such as vinculin and focal adhesion kinase ( F A K ) are recruited to' the nascent focal complex in a sequential manner (Zaidel-Bar et al., 2003). The stability of the individual IPP 13 components are dependent on complex formation as shown by R N A interference(Fukuda et al., 2003), as the lack of a complex results in an increased degradation of the IPP components. A t least two other factors appear to be required to assemble the IPP complex into focal adhesions; namely, the adaptor molecules paxillin, and MIG/kindl in-2, which both bind directly to the IPP complex through the kinase domain of I L K (Gardner et al., 1996; Mackinnon et al., 2002; Nikolopoulos and Turner, 2000; Nikolopoulos and Turner, 2001; Nikolopoulos and Turner, 2002). The following section wi l l summarize the known binding partners of the IPP complex and how signaling specificity is achieved through differential binding of molecules to P I N C H and Parvin isoforms. 14 Integrins a Nature Reviews | Cancer Figure 1.3 Schematic diagram of ILK/PINCH/Parvin complex found at the focal adhesions (this figure is taken from (Hannigan et al., 2005)). 1.3.5 Particulary Interesting Novel Cysteine-Histidine rich protein (PINCH) Interactions Signaling specificity of the IPP complexes depends on the presence of the different P I N C H or Parvin isoforms. For example, the I L K - P I N C H 1 complex transduces integrin-mediated signals that control cell spreading and migration, whereas ILK-PINCH2 complex cannot, therefore it is thought that accessory proteins which differentially bind to P I N C H isoforms may be responsible for transducing signals that negatively or positively affect this cell behaviour. Ras-suppressor protein RSU1 is an accessory protein that negatively regulates growth factor induced Jun N-terminal kinase ( INK) 15 activity through interactions with P INCH1 (Tsuda et al., 1995; Vasaturo et al., 2000), alternatively, thymosin P4 binds to P I N C H 1 to upregulate I L K activity and positively influence migration and survival of cardiac cells (Bock-Marquette et al., 2004). The P I N C H 1 interaction with N C K 2 , an adaptor protein, is necessary for the localization of P I N C H 1 to the focal adhesion, but the relevance of this is not clear. In addition, studies demonstrate that P I N C H 1 shuttles between the nucleus and the cytoplasm in Schwann cells and may have a role in gene regulation or signaling between the cytoplasm and nucleus (Campana et al., 2003). 1.3.6 Parvin Interactions There are three different isoforms of parvins, a-parvin/CHILK-BP/actopaxin, p-parvin/affixin and y-parvin, all of which have overlapping abilities to bind other partners. The ILK-y-parvin complex is involved initially in integrin signaling and establishing cell polarity during leukocyte migration (Yoshimi et al., 2006). Alpha-parvin/CHILK-BP/actopaxin is known to bind to F-actin directly and indirectly through paxil l in (Attwell et al., 2003; Nikolopoulos and Turner, 2000) and interacts with I L K in a PI3-kinase dependent manner (Attwell et al., 2003). This later study further confirms I L K as a critical kinase and an adaptor necessary for the activation of downstream P K B / A k t and G S K - 3 signaling. HIC5 also binds to a-parvin/CHILK-BP/actopaxin and is capable of shuttling to the nucleus where it modulates the expression of several genes ( K i m -Kaneyama et al., 2002; Shibanuma et al., 2004). A s well , a-parvin/CHILK-BP/actopaxin specifically binds to T E S K 1 , a serine/threonine kinase that phosphorylates and regulates the actin-regulating protein coifin (Edwards et al., 1999). Likewise, studies demonstrate 16 that (3-parvin/affixin binds a-actinin and a -PIX, a guanidine exchange factor, providing a connection between I L K , the IPP complex and actin-regulating GTPases, R a c l and Cdc42 {Filipenko, 2005 #64; (Rosenberger et al., 2003). Alpha-PIX also regulate the actin dynamics and focal adhesion turnover by binding to P A K 1 , a Racl /Cdc42 effector that regulates cytoskeletal dynamics through the L I M kinase-Actin Depolymerizing Factor-Cofilin pathway {Edwards, 1999 #731}. In addition, a - P I X also binds to Calpain-4, the protease which cleaves talin an important step in the disassembly of focal adhesions and in cell migration (Rosenberger et al., 2003). A s well , p-parvin/affixin has been implicated in membrane repair as it binds to the calcium-dependent membrane-repair protein dysferlin at the sarcolemma of skeletal muscle (Matsuda et al., 2005). This IPP complex forms a platform in which I L K mediated downstream signals and the recruitment of other proteins can occur (see Figure 1.3). 1.3.7 Signals Downstream of IPP Complex Integrins not only provide the structural bridge between E C M and intracellular actin cytoskeleton, they are also, as previously described, signal transducers by themselves or in collaboration with other growth factor receptors or G-protein coupled receptors. The cross-talk or synergistic interactions between integrins and growth factor receptors mediates and coordinates intracellular signaling cascades downstream of integrin and growth factor receptor engagement, including the R a s / R a f - l / M E K / E r k l / 2 pathway, Rho family GTPases, PI-3-Kinase, ribosomal S6 Kinase (RSK) , Jun amino-terminal kinase (JNK) , F A K and Paxil l in, and p l 3 0 C A S (Comoglio et al., 2003; Giancotti and Tarone, 2003; Lee and Juliano, 2004; Schwartz and Ginsberg, 2002). Proliferation and migration 17 are examples of cell processes which are dependent on these interactions for the initiation of growth-factor mediated signaling cascades. Although transmembrane integrin and growth factor receptors are induced to cluster by matrix proteins, intracellular signaling mediated by growth factor receptors, depends on binding of the growth factor. In epithelial cells, proliferation signals from mitogens, such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) , have to collaborate with signals from integrins for progression of the cell cycle (Danen and Yamada, 2001; Pardee, 1989). In endothelial cells, basic FGF/av(53 and VEGF/av(35 signaling pathways are important for cell survival, but differentially activate Ras-Raf-ERK signaling (Alavi et al., 2003). Hood et al. proposed that these two signals independently account for protection of endothelial cells from distinct mediators of apoptosis in an E R K activated background (Hood and Cheresh, 2002). The av(53/bFGF pathway promotes an ERK-independent survival mechanism preventing stress-mediated death based on coupling to the mitochondria, whereas the a v p 5 / V E G F pathway prevents integrin receptor-mediated death in an ERK-dependent manner. Together these two signals result in angiogenesis, but the differential activation of these signaling pathways impacts the distinct biological response of b F G F and V E G F . Some integrins are able to cross-talk with more than one type of growth factor receptor, such as av[33 integrin, which associates with growth factor receptors and modulates cell cycle progression in response to IGF-1 , P D G F , and V E G F (reviewed by (ffrench-Constant and Colognato, 2004). Expression of integrins and growth factor receptors change as the cell responds to the environment. For example, to promote cell survival appropriate intracellular signals are initiated and emphasized. oc5pi integrin and epidermal growth factor receptor 18 (EGFR) expression is upregulated in normal rat intestinal epithelial cells during serum deprivation to differentially activate the P K B / A k t , but not E r k l / 2 (Lee and Juliano, 2004), as cell survival becomes more important than cell proliferation. The interconnected networks of integrin and growth factor signaling are tightly regulated, therefore, any disruptions in either network could lead to a complete shut down of signaling or the propagation of signals that are no longer responsive to the primary stimulus. Both scenarios could result in autonomous growth and transformation. 1.3.8 Regulation of ILK I L K is regulated by integrin engagement and secondary messenger products of the PI3 kinase. I L K clustering has been observed within adhesion sites in epithelial cells suggesting that I L K binds to primed and activated integrins (L i et al., 1999), and that I L K localization is concomitant with integrin activation (Attwell et al., 2003). Studied more extensively for its role in cell survival, cell cycle progression and the regulation of transcription factors is the PI3 kinase signaling pathway. The class I A subgroup of the PI3 kinases are composed of a regulatory 85 kDa subunit and a 110 kDa catalytic subunit, and are activated by growth factor receptor tyrosine kinases. Binding of the p85 regulatory subunit to stimulated growth factor receptor tyrosine kinases occurs via interactions with SH2 domain or association with adaptor proteins. This results in the activation of the p i 10 catalytic subunit to phosphorylate membrane-bound PtdIns-4 (PIP) and PtdIns(4,5)P2 (PIP2), converting them to PIP2 and PtdIns(3,4,5)P3 (PIP3), respectively. The production of PIP2 and PIP3 are tightly regulated, but upon stimulation with growth/survival factors such as P D G F , nerve growth factor (NGF) , insulin, or 19 insulin-like growth factor (IGF), PI3 kinase is activated and phosphoinositides are transiently produced for the duration of the stimulus (Cantley and Neel, 1999). It is thought that the sudden downregulation of this signal is due mostly to the activity of a phosphatase called phosphatase and tensin homolog deleted on chromosome 10, also termed M M A C 1 or TEP1 (PTEN) , which decreases the levels of PIP2 and PIP3 by dephosphorylating the 3 position (Stambolic et al., 1998). A s well , P T E N has been shown to de-phosphorylate proteins such as F A K , although the in vivo significance of this finding requires further investigation (Vivanco and Sawyers, 2002). ILK-dependent phosphorylation is regulated in a PI3 kinase-dependent manner. Addition of PIP3 or overexpression of PI3 kinase p i 10 catalytic a-subunit increases I L K -dependent kinase activity in vitro and cell culture studies, respectively (Delcommenne et al., 1998). In cells that lack P T E N , such as prostate cancer cells, I L K is constitutively active (Persad et al., 2000; Persad et al., 2001a; Persad et al., 2001b). The expression of thymosin-p4 in cardiomyocytes has been demonstrated to increase the activity of I L K , as measured by phosphorylation of A k t / P K B on serine 473 residue (Bock-Marquette et al., 2004). Thymosin-p4 binds to L I M domains-4 and -5 of P I N C H 1 , and upregulates I L K activity, positively influencing migration and survival of cardiac myocytes. On the other hand, the catalytic activity of I L K is negatively regulated by the phosphatase I L K -associated protein ( I L K A P ) , a serine/threonine phosphatase, which has been shown to reduce the kinase activity of I L K in vitro and the phosphorylation of G S K - 3 P in vivo, but not the phosphorylation of A k t / P K B (Kumar et al., 2004; Leung-Hagesteijn et al., 2001). Decrease in I L K A P expression and an increase in I L K activity have been thought to correlate with poor patient outcome with melanoma (Dai et al., 2003). D A B 2 / D O C 2 , a 20 tumour suppressor that is markedly downregulated in ovarian and colorectal carcinoma (Kleeff et al., 2002; M o k et al., 1998), was shown to inhibit proliferation and tumorigenesis, and induce anoikis or apoptosis with the lack of integrin stimulation, with a concomitant downregulation of I L K activity in xenograft ovarian tumors and breast cancer cells, respectively (Mok et al., 1998). Another protein phosphatase, stomach-cancer-associated tyrosine phosphatase-1 (SAP-1), induces apoptosis by inhibiting I L K kinase activity and Ak t / P K B activation, ,though this phosphatase is thought to inhibit I L K activity indirectly by disrupting the focal adhesion (Takada et al., 2002). Work done by Mongroo et al. indicates that p-parvin/affixin inhibits I L K signaling downstream of receptor tyrosine kinases in breast cancer cells (Mongroo et al., 2004). Early studies demonstrate that I L K autophosphorylates in vitro, but this function in vivo requires further investigation (Zervas et a l , 2001). 1.3.9 ILK as a Downstream Effector of Signaling Pathways Molecular and genetic approaches have converged to confirm that I L K serves as a physical link between integrins and cytoskeleton, and is a key effector of signaling pathways during development. However, overexpression of I L K in epithelial cells results in anchorage-independent growth and cell-cycle progression, increased cell survival, epithelial-mesenchymal transformation and nuclear activation of P-catenin, migration, motility and invasion. A brief description of the known primary I L K effectors w i l l be given prior to incorporating them into the context of the cognate pathways. The two main substrates of I L K are A k t / P K B and G S K - 3 (see figure 1.4). 21 PTEN Growth factors/ chemokines PIP2 4 - PIP3 PI3K Actin .... Integrins 1 a Cytoskeleton p-Parvin RAC-GTP/ CDC42-GTP | Cell spreading Migration Nature Reviews | Cancer Figure 1.4 Schematic Diagram of ILK - dependent pathways and phenotypes. Taken from (Hannigan et al., 2005). There are three isoforms of A k t / P K B : a which is the most abundant, p and y. A l l three are composed of an N-terminal P H domain, a central kinase domain, and a C -terminal hydrophobic regulatory domain (Hi l l and Hemmings, 2002). A k t / P K B is regulated by upstream secondary messengers and secondary activating kinases. PIP2 and PIP3 both bind and localize A k t / P K B to the plasma membrane. This also induces a conformational change to expose the activation loop of A k t / P K B , which is subsequently phosphorylated at the threonine 308 site by 3-phosphoinositide-dependent kinase-1 (PDK-1) (Alessi et al., 1997a; Alessi et al., 1997b). PDK-1 also phosphorylates the 22 activation loop of other A G C family kinases such as 70 kDa ribosomal S6 kinase (p70 s 6 k), 90 kDa ribosomal S6 kinase (p90 R S K ) , serum and glucocorticoid-induced protein kinase (SGK) , and protein kinase C isoforms (Alessi, 2001). For full activation, A k t / P K B a requires subsequent phosphorylation at serine 473 in the C-terminal hydrophobic regulatory domain (Alessi et al., 1997a) by a kinase termed P D K 2 or hydrophobic motif kinase ( H M K ) . There are multiple candidates for P D K 2 / H M K , including I L K , M A P K A P kinase-2, P K C isoforms, DNA-dependent protein kinase, A T M , P D K - 1 and A k t / P K B , itself, and now mTOR/rictor (Bayascas and Alessi , 2005). A k t / P K B is a potent inhibitor of apoptosis, or programmed cell death-an observation that can be partially explained by its regulation of the targets such as B A D , caspase-9, C R E B , I K K and the forkhead transcription factors. B A D is a pro-apoptotic member of the Bcl-2 family that initiates an apoptotic cascade by binding to B c l - X L on the mitochondrial membrane, and opening a channel to cause the release of cytochrome c into the cytosol (Datta et al., 1997). A k t / P K B blocks this cascade by phosphorylating B A D at serine 136, which promotes the association of B A D with 14-3-3, thus sequestering it in the cytosol. In addition, A k t / P K B inactivates the forkhead family of transcription factors by phosphorylation at multiple serine/threonine residues, rendering the transcription factors unable to upregulate a subset of pro-apoptotic factors such as p27 and Fas (Brunet et al., 1999; Burgering and Kops, 2002). Alternatively, C R E B transcription factor is activated by A k t / P K B phosphorylation to increase the expression of the anti-apoptotic genes Bcl-2 and Mcl-1 (Du and Montminy, 1998). A k t / P K B also indirectly regulates N F - K B by phosphorylating and activating I K K - a , which, in turn, phosphorylates and targets I K B for degradation, allowing the release and translocation of 23 N F - K B to the nucleus to upregulate prosurvival factors, such as B c l - X L , caspase inhibitors, and c-Myb (Barkett and Gilmore, 1999; Lauder et al., 2001). A k t / P K B can phosphorylate M D M 2 , which then binds to p53 and enhances its degradation, resulting in the inhibition of the p53 mediated cell cycle arrest, apoptosis and D N A (Mayo and Dormer, 2001). A k t / P K B also inactivates glycogen synthase kinase -3 (GSK-3) by phosphorylation to promote cell cycle entry, and regulate glucose metabolism. A s well , A k t / P K B phosphorylates m T O R to promote growth and translation of proteins (Granville et al., 2006). G S K - 3 was the first substrate of A k t / P K B identified (Cross et al., 1995). G S K - 3 regulates both glucose metabolism and cell proliferation. A k t / P K B phosphorylates G S K -3 on serine 9, which results in its inactivation. This then results in the activation of several proteins, including cyclin D I , P-catenin, and the activator protein (AP-1) transcription factor. G S K - 3 phosphorylates P-catenin at multiple sites, targeting it for ubiquitination and subsequent degradation. Therefore, when G S K - 3 is inactivated (through Wnt signaling), this results in the stabilization and nuclear localization of P-catenin, which then associates with lymphoid enhancer factor/T-cell factor ( L E F / T C F ) transcription factor to promote the expression of cell cycle progression genes such as cyclin D I , c-myc and matrilysin (Hi l l and Hemmings, 2002). G S K - 3 also affects cyclin D I directly by phosphorylating it at threonine 286, targeting it for ubiquitination and subsequent degradation. Inactivation of G S K - 3 by P K B / A k t prevents this phosphorylation, thereby increasing cyclin D I levels (Diehl et al., 1998). G S K - 3 also phosphorylates c-Jun, a component of the AP-1 transcription factor, rendering it inactive 24 and unable to promote the expression of a wide variety of genes, including cyclin D I and V E G F . 1.3.10 Role of ILK in Anchorage-Independent Growth and Cell Cycle-Progression Proliferation is a tightly regulated complex cellular function. Normal cells pass through a limited number of cell divisions before they undergo cell death. When epithelia are placed on a surface, they proliferate until the surface of the dish is covered by a monolayer of cells just touching each other. Then mitosis ceases. This phenomenon, called contact inhibition results in growth arrest and occurs upon engagement of cadherins, homophilic cell-to-cell receptors, and integrins. Cancer cells are refractory to contact inhibition growth through abnormal expression of proteins that mediate cell cycle progression Overexpression of I L K increases the expression of several key components of the cell cycle machinery, including cyclin A , cyclin D I , and C D K 4 proteins, and reduced the inhibitory activity of p27 k i p l (Radeva et al., 1997). Several lines of evidence demonstrate that the inhibition of G S K 3 as a result of ILK-mediated phosphorylation leads to activation of the transcription factor C R E B (cyclic-AMP-responsive-element-binding protein) or P-catenin / T C F , resulting in the stimulation of cyclin D I expression, and the subsequent promotion of transition from G l to S phase (D'Amico et al., 2000; Persad and Dedhar, 2003; Persad et al., 2001b; Troussard et al., 1999). Two other studies further reinforce the role of I L K in promoting cell cycle progression through stimulation of cyclin D I expression. The inhibition of P T E N suppresses the nuclear translocation of p-catenin, and the presence of I L K A P also decreases G S K 3 P phosphorylation and nuclear 25 translocation of P-catenin (Kumar et al., 2004; Leung-Hagesteijn et al., 2001). The regulation of the transcription factor AP-1 involves a GSK-dependent and -independent mechanisms. The inhibition of G S K 3 prevents the phosphorylation of c-Jun, an AP-1 transcription factor component, allowing AP-1 to bind D N A , thus stimulating the expression of cyclin D I . Alternatively, the direct phosphorylation of Jun transcriptional co-activator a - N A C by I L K also promotes AP-1 transcription factor activation (Quelo et al., 2004). 1.3.11 Ro le of ILK in Ce l l Surv iva l and A n o i k i s Integrin mediated c e l l - E C M anchorage promotes cell survival. If cells are prevented from establishing E C M attachment, this often induces a specific form of apoptosis (Kerr, Wyll ie , Currie et al. 1972) referred to as anoikis (Frisch and Francis, 1994; Frisch and Screaton, 2001). The mechanisms regulating adhesion dependent cell survival occurs at the level of cytoplasm and in the nucleus. A t the cytoplasmic level, it is thought that the underlying mechanism involves the recruitment of the apoptosis initiator, caspase-8, to the plasma membrane by unligated integrins or integrin P subunit cytoplasmic domain, where it becomes activated in a death receptor independent manner. When integrin ligation occurs, the integrin-caspase complex is disrupted and the cells can survive (Frisch and Screaton, 2001). The engagement of integrins induces the clustering of proteins at the focal adhesion to initiate cascades, such as the M A P kinase cascade, PI3 kinase / A k t / P K B pathway to modulate gene expression. This coordination of signals is important in cell migration and the ability for cells to survive without adhesion is advantageous for cancer cell propagation (Frisch and Screaton, 2001). 26 Molecular and genetic approaches have converged to confirm that I L K is a key effector of integrin function regulating ECM-dependent cell adhesion (Grashoff et al., 2004; Hannigan et al., 2005). The promotion of cell survival by I L K has been studied extensively and compiling evidence demonstrate that this regulation is primarily due to the ability of I L K to promote the phosphorylation and full activation of anti-apoptotic A k t / P K B on serine 473. Upon phosphorylation, A k t / P K B stimulates downstream anti-apoptotic pathways, such as activation of N F - K B , inhibition of forkhead transcription factors ( F K H R / A F X / F O X ) and inactivation of pro-apoptotic proteins such as B A D (Nicholson and Anderson, 2002). It is interesting to note that both I L K and N F - K B are highly activated in ERBB2-overexpressing breast cancer cells (Makino et al., 2004). Recent studies demonstrate that I L K regulates N F - K B activity in macrophages in an Akt/PKB-dependent manner, thus it is possible that I L K may be involved in the prosurvival functions of N F - K B . It is thought that by an uncharacterized mechanism, I L K might indirectly phosphorylate A k t / P K B indirectly through recruitment of another serine 473 kinase (Hi l l and Hemmings, 2002) or through a-parvin-mediated targeting of A k t / P K B to l ipid rafts (Fukuda et al., 2003). The observation that the lack of P T E N in human cancers, such as prostate, breast and glioblastoma, with concomitant A k t / P K B phosphorylation further validates the critical role I L K plays in cell survival. Exposure of cells to the I L K specific small molecule inhibitor results in a decrease in A k t / P K B phosphorylation and subsequent sensitivity to apoptosis. In addition, A k t / P K B was recently shown to promote survival by transcriptional upregulation of P P A R p , and this control was found to be due, at least in part, to the transcriptional upregulation of I L K (Di-Poi et al., 2005a; D i -Po i et al., 2004; Di -Po i et al., 2005b). 27 1.3.12 Role of ILK in Epithelial-Mesenchymal Transformation (EMT) and Nuclear Activation of p-catenin The overexpression of I L K in epithelial cells results in a morphological transformation into a fibroblastic state accompanied by a down-regulation of E-cadherin, increased production of fibronectin (Wu et al., 1998), nuclear translocation of P-catenin (Novak and Dedhar, 1999; Novak et al., 1998), downregulation of the epithelial markers cytokeratinl8 and M U C 1 , and the upregulation of the mesenchymal markers L E F 1 and vimentin (Barbera et al., 2004; Bravou et al., 2003; Bravou et al., 2006; Guaita et al., 2002; Hannigan et a l , 1996; Marotta et al., 2003; Marotta et al., 2001; Novak and Dedhar, 1999; Novak et al., 1998; Persad et al., 2001b; Somasiri et al., 2001; Tan et al., 2001; White et al., 2001; W u et a l , 1998; X i e et al., 2004; X i e et al., 1998). This transformation is reminiscent of epithelial-mesenchymal transformation ( E M T ) , a prominent hallmark of metastatic cancer cells (Bissell and Radisky, 2001; Thiery, 2002). Presently, the mechanisms underlying morphological change can be partially explained by the classical Wnt signaling pathway. In the absence of Wnt signaling, P-catenin is complexed with axin, A P C , and GSK3-P and targeted for degradation following constant phosphorylation by G S K 3 - p (Nelson and Nusse, 2004). In the presence of Wnt signaling, p-catenin is uncoupled from the degradation complex and translocates to the nucleus, where it binds Lef /TCF transcription factor, thus activating genes that direct cell fate, polarity, and proliferation of tumor cells (reviewed in (Cavallaro, 2004; Cavallaro and Christofori, 2004)), such as cyclin D . A primary event that governs E M T is the disruption of the E-cadherin-mediated stable cell-cell interactions (Conacci-Sorrell et al., 2003; Conacci-Sorrell et al., 2002), ultimately leading to the mesenchymal phenotype. 28 The intracellular domain of E-cadherin interacts with a - ,P-, y- and pl20-catenin, to link to the cytoplasmic actin filaments (Cavallaro and Christofori, 2004; Imai et al., 2004) and stabilize cell-cell interactions. The upregulation of the transcription factor S N A I L , a zinc finger protein that represses E-cadherins, by binding to the E-boxes present in E-cadherin promoter (Thiery, 2002), is thought to be the main mechanism that regulates E-cadherin expression (Batlle et al., 2000). The mechanism of regulation of S N A I L expression is still unclear, however decreased G S K - 3 p activity and the stimulation of the PI3-kinase/Akt/PKB pathway and activation of N F - K B have recently been implicated in regulating its transcription (Bachelder et al., 2005; Gril le et al., 2004; Zhou et al., 2004). To date, the mechanism underlying how I L K regulates S N A I L is unclear but recent studies suggest that I L K may promote the expression of S N A I L repressor through regulation of N F - K B transcription factor activation in an A k t / P K B dependent manner (Barbera et al. 2004; Rosano et al. 2005). Earlier studies propose Zeb-1 as the candidate for a transcriptional repressor of E-cadherin that is upregulated in cells overexpressing I L K , although this activation also appears to be indirect (Guaita et al., 2002). This pattern of the role of I L K in E M T has been validated with histopathology specimens (Bravou et al., 2003; Bravou et al., 2006; Marotta et al., 2003; Marotta et a l , 2001). To date the molecular mechanisms of I L K in E M T are unclear and are the focus of future studies. I L K is also a signaling intermediate in transforming growth factor-P (TGF-p)-mediated E M T (Lee et al., 2004; L i et al., 2003a), a crucial event in kidney fibrotic disease and in the progression towards metastasis (Beck et al., 2001). Recent data suggests that PI3K-I L K - A k t pathway is independent of the TGFp-induced Smad pathway that requires TGFp-mediated epithelial to mesenchymal transition (Lee et al., 2004). 29 1.3.13 Role of ILK in Migration, Motility and Invasion ILK-mediated E M T is accompanied by increased cell migration and invasion (Somasiri et al., 2001). It is well established that the R H O family of GTPases can control cell spreading and motility (Clark et al., 1998). The Rho family of proteins consists of about 20 genes that encode signaling molecules containing a small GTPase domain, and the family is divided into 5 groups: Rho-like, Rnd, CDC42- l ike , Rac-like and R h o B T B . These proteins regulate numerous cellular activities, including cell growth, differentiation, proliferation and tumor cell invasion. The Rho-like subfamily is necessary for the formation of stress fibers and focal adhesions in cells. Specifically, RhoC has been shown to promote tumor invasion (Clark et al., 1998) and the overexpression and constitutive activation of RhoC upregulate genes that enhance invasion in breast carcinoma cells (Wu et al., 2004). To support this, inhibition of RhoC results in inhibition of melanoma cell spreading (Collisson et al., 2003). The Rac-like subfamily induces membrane-ruffling, lamellipodia generation, and other membrane structures important for cell motility, such as filopodia. The Rac proteins can also stimulate growth transformation, Jun N-terminal kinase ( JNK) activation, and promote cell survival. Filipenko et al. (2005) recently showed a critical role for I L K in the early events of cell attachment and spreading resulting from a dramatic reorganization of the actin cytoskeleton (Filipenko et al., 2005). They demonstrated that I L K is a key player in this cytoskeletal reorganization by activation of the small GTPases, Rac and C D C 42 via the guanine nucleotide exchange activity of a -PIX. The absence of I L K activity and 30 association with a - P I X results in a delayed or decreased ability of the cell to adhere to extracellular matrix and spread ( L i et al., 2003b; Turner et al., 1999). 1.3.14 Role of ILK in Regulating Myosin Light Chain (MLC) The contraction and relaxation of the smooth muscle is regulated by transient reversible phosphorylation of the myosin light chains by calcium/calmodulin-dependent myosin light-chain kinase ( M L C K ) and dephosphorylation by myosin light chain phosphatase (MP). Inhibition of M P is thought to be responsible for calcium sensitization of smooth-muscle contraction. I L K promotes contraction by phosphorylating and inactivating the myosin phosphatase target subunit in the absence of calcium. This suggests that I L K may modify the myosin phosphatase (MP) activity (Muranyi et al., 2002), and regulate calcium-sensitization and -independent contraction of smooth muscles by directly phosphorylating myosin light chain or inhibiting M P . I L K is also capable of phosphorylating the myosin light chain phosphatase inhibitor proteins CPI-17 and PH-1 , thus inhibiting myosin light chain phosphatase ( M L C P ) activity bound to myosin. In addition, I L K phosphorylates K E P I , a phosphorylation-dependent type-1 protein phosphatase inhibitor at threonine 73, resulting in an increase in inhibition of the phosphatases PP1C and myosin phosphatase M P H (Erdodi et al., 2003). This presents another possible way I L K may mediate cell migration and morphology, and a novel means by which I L K affects signaling pathways by inhibiting protein phospatases (Deng et al., 2002). Moreover, Huang et al. showed that I L K activity and localization to focal adhesions is induced in response to the induction of myogenic differentiation, and is required in myogenic differentiation and myotube formation (Huang et al., 2000). 31 1.3.15 Role of ILK Invasion Invasion is a process coordinated between malignant and normal cells, the E C M and stroma. Invasion is dependent, in part, on the E C M , ECM-receptors, including integrins and a balance of ECM-degrading enzymes, such as matrix metalloproteinases ( M M P s ) and their inhibitors, known as tissue inhibitors of metalloproteinases (TIMPs). With the use of genetic cell studies, enzymology and the highly specific small molecule I L K inhibitor Troussard et al (2000) show that I L K stimulates AP-1 activity that results in an increase in M M P - 9 (matrix metalloproteinase-9) expression (Troussard et al., 2000) and subsequent ease in invasion into matrigel by intestinal and mammary cells overexpressing I L K . Similarly, this was also shown by inhibiting of I L K activity in glioblastoma cells (Koul et al., 2005). More recently, Zhiyong et al. demonstrate that I L K regulates osteopontin-dependent matrix metalloproteinase-2 ( M M P - 2 ) and urokinase-type plasminogen activator (uPA) expression to convey metastatic invasion in murine mammary epithelial cancer cells (Zhiyong et al. 2006). 1.3.16 Role of ILK in Human Disease To date, I L K has been implicated in multiple cancers and non-cancer diseases and has been shown to be expressed in diseased tumors, specifically cancer and scar contractures (Levinson et al., 2004). I L K expression and possible activity are altered in a variety of human cancers. B y immunohistochemistry and immunoblotting, elevated I L K expression has been detected in the following human cancers: prostate (Graff et al., 2001; Kieffer et a l , 2005; Levinson et al., 2004; Q i et al., 2005), familial and sporadic colon 32 (Bravou et a l , 2003; Bravou et a l , 2006; Marotta et al., 2003; Marotta et al., 2001), gastric (Ito et al., 2003), ovarian (Ahmed et al., 2004; Ahmed et al., 2003), breast cancer (reviewed in (Hannigan et al., 2005)), primitive ectodermal tumors and Ewing's sarcoma (Chung et al., 1998), pancreatic (Sawai et a l , 2006), anaplastic thyroid (Younes et al., 2005), and melanoma (Dai et al., 2003). In addition, mounting evidence suggests that the treatments which target constitutively active I L K in P T E N lacking glioblastomas may be promising (Koul et a l , 2005; Morimoto et a l , 2000; Nadjar et al., 2005; X i e et al., 2004). In melanomas, gastric and colon cancers, increased I L K expression correlates with a higher-grade of disease and with metastases. Specifically, in human colon cancer, I L K overexpression results in activation of P-catenin, down-regulation of E-cadherin and activation of the A k t - F K H R pathway (Bravou et al., 2006). Moreover, I L K activity is associated with interleukin-la-induced cancer progression of pancreatic cancer and poor patient survival (Sawai et al., 2006). In prostate cancer, I L K expression has also been demonstrated to correlate positively with tumor grade and inversely with patient survival rates. Interestingly, I L K upregulation has been observed in response to ionizing radiation of lung cancer cells, hypoxia of hepatocellular carcinoma cells and hyperthermia of prostate cancer cells (Cordes, 2004; Zhang et al., 2003). In cancer cells, deregulation of I L K can occur at the transcriptional level. Although, the mechanisms responsible for the increase in I L K expression are not well delineated, recent studies demonstrate that I L K transcription is stimulated in keratinocytes by the nuclear receptor peroxisome-proliferatro-activated receptor-p/6 (PPARp/6) (Di-Poi et al., 2005a; D i -Po i et al., 2004). This transcription factor is 33 regulated by A P C , a tumor suppressor, in colon cancer cells and promotes survival of keratinocytes by activating phosphorylating and activating A k t / P K B . Studies by L i et al. suggest that kidney cells stimulated by TGF(3 increase I L K expression through the activation of S M A D 2 transcription factor ( L i et al., 2003a). A s well , I L K has been demonstrated to be upregulated in different stages of asbestos-induced carcinogenesis in rats (Sandhu et al., 2000) and the I L K pathway was upregulated in hexachlorobenzene-induced gender-specific rat hepatocarcinogenesis (Plante et al., 2005). 1.4 Angiogenesis Angiogenesis is the formation of blood vessels from pre-existing blood vessels. This is a major mechanism that underlies physiology, such as wound repair, and pathological neovascularization in adults, rheumatoid arthritis, post myocardial infarction, diabetic retinopathy and tumor progression (Carmeliet, 2005a; Hanahan and Folkman, 1996). Neovascularization is important for embryonic development and also encompasses arteriogenesis, venogenesis and lymphangiogenesis. Vasculogenesis and angiogenesis are coordinated and complex processes involving E C M and vascular endothelial cells and is regulated by various angiogeneic factors including vascular endothelial growth factor ( V E G F ) , fibroblast growth factor-2 (FGF-2), interleukin-8 (IL-8), transforming growth factor-ct (TGF-a) (Vinals and Pouyssegur, 2001). These factors promote cell proliferation, chemotactic migration, and capillary/tube-like network formation of vascular endothelial cells in vitro and in vivo. The endothelial-specific mitogen V E G F has been shown to be a key positive regulator of tumor angiogenesis including glioblastoma, colon and breast (reviewed in (Carmeliet, 2005a; Carmeliet, 34 2005b). The interactions between E C M and the cells are required for new blood vessel formation. Integrins cooperate with the V E G F receptors to promote activation of an in vitro angiogenic program in vascular endothelial cells. Cel l response to oxygen deprivation drives the expression of many genes important for angiogenesis, red cell production, and glycolysis. These include key vascular and hematopoetic growth factors, such as V E G F , erythropoietin, platelet-derived growth factor (PDGF) , and TGF-oc (Semenza, 2000). A major V E G F transcription factor is H I F - l a . The transcription of H I F - l a is regulated through the regulation of mammalian target of rapamycin (mTOR), in turn mediating 4E-BP1 (Semenza, 2003). Briefly, mammalian T O R (mTOR) is a large protein kinase that exists in two distinct complexes within cells. One complex consists of mTOR, G(3L and Raptor (regulatory-associated protein of mTOR) ( K i m et al., 2002; K i m et al., 2003) and the other complex is comprised of mTOR, G p L and Rictor ( K i m et al., 2002; K i m et al., 2003). The first complex is sensitive to rapamycin and regulates cell growth by phosphorylating S6K1. The latter complex is not sensitive to rapamycin and its cellular function is unclear (Sarbassov et al., 2004). Studies demonstrate that upon activation of m T O R by A k t / P K B phosphorylation, m T O R then forms a complex with G p L and raptor, which increases m R N A translation via activation of S6-kinase and inhibition of eIF4E binding protein (4E-BP) (Hay, 2005). Transcription of HIF subunits is constitutive, however, H I F - l a proteins undergo propyl hydroxylation and are targeted for ubiquitination by the von Hippel-Lindau ( V H L ) protein and degraded by a proteasome (Ivan and Kaelin, 2001). Under hypoxic or growth factor-induced conditions, unhydroxylated H I F - l a subunits avoid posttranslational 35 modification, become stabilized and complex with HIF-P subunits to drive transcription of hypoxia-induced promoters (reviewed in (Semenza, 2003). Loss of V H L function results in constitutive HIF stabilization and predisposes to particular tumors, such as renal clear cell carcinomas, cerebellar hemangioblastomas, retinal angiomata and pheochromocytomas, all of which have a major vascular component (Hughson et al., 2003; Semenza, 2003). Tumor growth and metastasis depends upon angiogenesis, a process by which quiescent vasculature is induced to sprout new capillaries. Avascular tumors cannot expand in size because of a lack of blood supply bringing nutrients and oxygen, and removing waste, but their ability to switch on the production of angiogenic factors explains how they can trigger neovascularization, maintain oxygen-dependent A T P production, expand, and metastasize (Folkman, 1976; Folkman and Cotran, 1976). This cellular activity requires several well orchestrated cell-matrix, cell-cell and signaling pathways that lead to the coordination of physiological cellular activities including proliferation, survival, invasion and migration. 1.4.1 Role of Extracellular Matrix in Angiogenesis The conformation and abundance of the different components of the E C M regulate and direct cellular processes involved in angiogenesis. Signals from the E C M to the cytoskeleton are transduced through specific integrins. The major integrins responsible for these interactions are a 2 p i , a i p i and avP3, a 5 p i , which are collagen and fibrin/fibronectin receptors, respectively (Davis and Senger, 2005). 36 The E C M components that are encountered by endothelial cells during sprouting angiogenesis, include interstitial fibrin and collagen, which themselves are capable of supporting chemotactic migration alone (Davis and Senger, 2005; Dejana et al., 1985). Endothelial cell proliferation and survival are highly dependent on adhesion to the E C M by integrins {Davis, 2005), which activates the p44/p42 (Erkl /Erk2) mitogen-activated protein kinase ( M A P K ) signal transduction cascade and suppresses Fas-induced apoptosis (Aoudjit and Vuor i , 2001). Interstitial collagen, the fibrin matrix, and basement membrane laminin differentially regulate various stages of angiogenesis. It has been proposed that laminin-1 rich basement membranes induce persistent integrin-dependent activation of GTPase Rac in endothelial cells which results in the expression of matrix degradative enzymes such as M M P s and A D A M s . Upon degradation of the basement membrane, endothelial cells are exposed to interstitial collagens, which activate signaling pathways that drive cytoskeletal reorganization and sprouting morphogenesis. Cel l adhesion to interstitial collagen I results in the activation of both Src and Rho and suppression of protein kinase A ( P K A ) . This promotes the formation of prominent actin stress fibers which mediates endothelial cell retraction and capillary morphogenesis. In addition, collagen I mediated activation of Src disrupts VE-cadherin from cell junctions and promotes disruption of cell-cell contacts, ultimately transforming the cell to have invasion and migration capabilities. During vessel maturation, laminin continues to play an intimate role in directing endothelial cell morphology. In particular, Cdc42 and R a c l are activated for lumen formation while proliferative pathways such as R a s - M A P K and N F - K B suppressed. (Klein et al., 2002; Mettouchi et al., 2001). The E C M can further regulate angiogenesis 3 7 by binding various angiogenic cytokines such as fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor ( V E G F ) (Brader and Eccles, 2004). This acts as a reservoir for coordinating the spatial and temporal specific release of proangiogenic factors. 1.4.2 Role of Growth Factors and Receptors Associated with Angiogenesis Numerous inducers of angiogenesis have been identified, including the members of the V E G F family, angiopoietins, transforming growth factor-a and - p (TGF-a and-P), platelet-derived growth factor (PDGF) , tumor necrosis factor-a (TNF-a) , interleukins, chemokines, and a member of the F G F family. These soluble factors crosstalk with integrin-mediated signaling to promote or inhibit blood vessel formation. This particular section of this chapter w i l l focus on two of the above angiogenesis inducers, F G F and V E G F . The F G F family consists of at least 22 distinct family members, which have been identified in a variety of organisms (Gospodarowicz, 1974; Ornitz and Marie, 2002) and are involved in the entire program for the acquisition of the angiogenic phenotype (reviewed by (Presta et al., 2005). During embryonic development, FGFs play a critical role in morphogenesis, and in adults, these proteins control the nervous system, tissue repair, wound healing and tumor angiogenesis (Eswarakumar et al., 2005). Unlike other growth factors, F G F s act in concert with heparin or heparin sulfate proteoglycans (HSPGs) to activate F G F receptors. These receptors are expressed primarily in skeletal tissues and are overexpressed in a variety of human diseases such as lymphoma, prostate and breast cancer. In response to F G F stimulation, a variety of 38 signaling proteins are phosphorylated including Src homology (She) proteins, phospholipase-cy, Signal Transducers and Activators of Transcription 1 (STAT1) , Grb2-associated binder -1 (Gabl) , and F G F R substrate 2 a (FRS2a). Together, these phosphorylation events stimulate intracellular signaling pathways that control cell proliferation, differentiation, migration, survival and morphology (Holgado-Madruga et al., 1996; Holgado-Madruga et al., 1997) (Schaeper et a l , 2000) (Maroun et al., 2000) (Kouhara et al., 1997). Pivotal in mediating downstream events is the tyrosine phosphorylation of the docking proteins F R S 2 a and FRS2(3 and the subsequent recruitment of Grb to form a FRS2a-Grb platform which is responsible for assembling positive and negative signaling proteins to mediate F G F signal translation (Holgado-Madruga et al., 1996; Holgado-Madruga et al., 1997) (Schaeper et al., 2000) (Maroun et al., 2000). Recruitment of Sos or Gab l results in the activation of the R a s / M A P kinase signaling pathway and the PI3 kinase/Akt/PKB pathway, respectively (Holgado-Madruga et al 1996, Kouhara et al 1997, Wong et al. 2002). These signals continue until Grb-2-mediated recruitment of Cb l promotes ubiquitination of F G F receptor resulting in a downregulation of the F G F mediated signals (Kaabeche et al., 2004). A n intimate cross-talk exists among basic fibroblastic growth factor (bFGF) and the different members of the V E G F family during angiogenesis, lymphangiogenesis and vasculogenesis (reviewed in (Presta et al., 2005). Studies demonstrate that F G F and V E G F may exert a synergistic effect in different angiogenesis models (Xue and Greisler, 2002), and F G F may require V E G F / V E G F R activation for promoting angiogenesis (Castellon et al., 2002; Jih et al., 2001). 39 The V E G F family comprises six members, including placental growth factor (PIGF), V E G F - A , V E G F - B , V E G F - C and V E G F - D , that interact differently with three cell surface tyrosine kinase V E G F receptors. V E G F gene expression is up-regulated by a variety of factors such as hypoxia, acidosis and stimulation by growth factors including F G F , T N F , E G F , T G F - 0 and IL-1 . To date, V E G F - A / V E G F receptor 2 ( V E G F R - 2 ) interaction appears to play a major role in blood vessel angiogenesis where as V E G F - C and - D are mainly involved in lymphangiogenesis through their interaction with V E G F R - 3 , which is expressed on lymphatic endothelia (reviewed in (Ferrara et al., 2003). V E G F R - 2 is the predominant tyrosine kinase receptor in angiogenic signaling and is involved in regulating endothelial cell migration, proliferation, differentiation and survival as well as vessel permeability and dilatation. Upon dimerization, V E G F R - 2 undergoes tyrosine phosphorylation at multiple sites that serve as binding sites for adaptor molecules such as Nek, Grb-10, Grb-2, Sck, human cellular protein tyrosine phosphates A ( H C T P A ) and the regulatory p85 subunit of PI3-kinase (reviewed in (Cebe-Suarez et al., 2006). In particular, phosphorylation of V E G F R at tyrosine 1175 has been implicated in activation of many phospholipase Cy-1 (PLOy-Independent signaling pathways (Takahashi et al., 2001). V E G F R - 2 is inactivated by internalization into endocytic vesicles and direct dephosphorylation by SHP-1 and SHP-2. V E G F R - 2 dependent mitogenesis is thought to occur through the activation of the classical Ras-dependent signaling cascade, and possibly through the S 6 / A k t / P K B pathway. Interestingly, c-Src and nitric oxide (NO) (Cebe-Suarez et al., 2006) have been implicated as intracellular mediators of V E G F signaling. In addition as well as 40 components of the E C M such as heparan sulfate (Holmqvist et al., 2004) and fibronectin (Wijelath et al., 2004) can also modulate mitogenic signaling. VEGFA-dependent regulation of cytoskeletal organization and cell migration occurs through recruitment of the adaptor protein Shb and activation of PI3 kinase resulting in the regulation of F A K phosphorylation and leading to the recruitment of paxillin, talin or vinculin to the focal adhesions (Kanno et al., 2000). A s well , the recruitment of Src, PI-3 kinase and PLCy-1 also plays a role in cytoskeletal rearrangement (Zeng et al., 2002a; Zeng et al., 2002b). Finally, activation of SAPK/p38 pathways and the small GTPases Rho, Rac and Cdc42 also contributes to actin dynamics and cell contraction in endothelial cell migration. Ce l l survival is dependent on V E G F R - 2 signaling. Activation of the PI3 kinase/Akt/PKB pathway induces the expression of anti-apoptotic molecules including Bcl-2 , A l and I A P (inhibitor of apoptosis) family of proteins. Unfortunately, this survival signal is dependent on stable adherens junctions that contains a transient tetramer composed of V E G F R - 2 , PI3-kinase, VE-cadherin and P-catenin (Grazia Lampugnani et al., 2003). Disruption of this complex results in apoptosis. V E G F mediated signaling is complicated because the V E G F receptors form multiprotein complexes with various coreceptors such as neuropilins, heparan sulphate, integrins and cadherins. These associations allow further coordination of signal amplitude, timing and specificity with extracellular cues from soluble ligands, cell-cell and c e l l - E C M interactions. 41 1.4.3 Role of Integrins in Angiogenesis Specific combinations of integrins and their ligands have been identified on the surface of endothelial cells (Stupack and Cheresh, 2004). These include a 5 p l integrin which binds fibronectin (Koivunen et al., 1993; Koivunen et al., 1994), a i p i and a2pi integrins which are receptors for laminins and collagens, and the a v integrins (Hynes, 1987): avP3 and avp5 which recognize R G D sequences in various E C M proteins namely vitronectin, fibronectin, thrombospondins and von Willebrand factor (Eliceiri and Cheresh, 1999; Eliceiri and Cheresh, 2001). In addition, avP3 integrins have also been reported to bind to various fragments of collagens, laminins and other matrix proteins (Brooks et al., 1994a; Brooks et al., 1994b). Fibronectin (FN), a widely distributed E C M component, is expressed in embryonic vascular networks as a component of the basement membrane (Risau and Lemmon 1988; Francis et al., 2002). Mouse knock-out models of a5 or fibronectin produce embryonic lethal phenotypes. Although the studies demonstrate that a5 and fibronectin are not required in endothelial cell development from their angioblast precursors, a5 is critical for tubulogenesis and in its absence, vasculogenesis fails to occur in the yolk sac and embryo (George et la., 1993; Yang et la., 1993). In pathological states, both fibronectin and a 5 p i are upregulated by the pro-angiogenic growth factor, bFGF ( K i m et al., 2000). This further emphasizes the critical role of a 5 p i integrin in coupling fibronectin with intracellular events that are necessary for vasculogenesis and angiogenesis (Koivunen et al., 1993; Koivunen et a l , 1994). Unlike the a 5 p i integrins, the a i p i and a 2 p l integrins are less critical for organism development, and instead appear to play a role in vascular remodeling and 42 pathological angiogenesis. Expressed in endothelial cells, these two integrins are receptors for collagens and laminins and are up-regulated in response to V E G F (Senger et al., 2002). Downregulation of a i p i and a 2 p i activity using blocking antibodies inhibits both VEGF-induced endothelial migration on collagen and angiogenesis (Senger et al., 2002) . Similarly, tumor growth and angiogenesis are significantly reduced in a l - and a2-null mice (Chen et al., 2002; Gardner et al., 1996). There are five integrins that contain a common a v subunit. The av-null mice have a lethal phenotype and demonstrate extensive blood vessel hemorrhaging. Although avp3 and avp5 integrins have been shown to be upregulated by angiogenic growth factors, and promote endothelial cell adhesion and migration on vitronectin and other E C M molecules (reviewed in (Stupack and Cheresh, 2004), mice lacking the P subunit (PI, P3, P5, P6 and P8) did not show changes in angiogenesis (Hodivala-Dilke et al., 2003) et al. 1999; Huang et al. 2000). Furthermore, Reynolds et al. (2002) demonstrate that mice lacking avp3 and/or avP5 integrins undergo enhanced pathological angiogenesis (Reynolds et al., 2002). On the contrary, antagonists, such as antibodies and peptidomimetics, against avp3 and avP5 integrin activity decreases VEGF-induced angiogenesis and tumor progression. This suggests that the lack of avp3 and avp5 integrin engagement with the appropriate E C M molecule results in the inhibition of angiogenesis (Stupack and Cheresh, 2004). Clearly, the role of avp3 and avP5 integrins in angiogenesis requires further study. 43 1.4.4 Role of Integrin-linked kinase in Vasculogenesis and Angiogenesis Recent evidence demonstrates the importance of I L K in angiogenesis. The I L K knockout mouse is peri-implantation lethal (Friedrich et al., 2004). Moreover, EC-targeted knockout of I L K in endothelial cells results in embryonic lethality with severe defects in placental and embryonic vascularization (Friedrich et al., 2004) secondary to detachment and apoptosis as the result of the inhibition of cell survival pathways due to the lack of integrin activation or signal transduction through the integrin. Furthermore deletion of I L K in zebrafish using antisense morpholino oligonucleotides results in marked patterning abnormalities of the vasculature and is similarly lethal (Friedrich et al., 2004). E x vivo deletion of I L K from purified endothelial cells of adult mice resulted in the downregulation of the active-conformation of pi-integrins concomitant with a reduction in A k t / P K B phosphorylation and caspase 9 activation ultimately leading to apoptosis. Re-introduction of activated A k t / P K B to cells lacking I L K did not completely rescue the cells from apoptosis which suggests that I L K activates other pro-survival signals in addition to A k t / P K B (Friedrich et al., 2004). I L K also regulates V E G F induced vascular morphogenesis in a PI3 kinase-dependent manner (Kanesaki et al., 2005). Kaneko et al. reported that I L K is critical in the chemotactic migration of endothelial cells, as well as cell proliferation due to the decrease in cell survival in the absence of I L K kinase activity (Kaneko et al., 2004). B y comparing capillary length, number and relative capillary area per field between V E G F stimulated endothelial cells and cells containing a dominant-negative, kinase deficient I L K , Watanabe et al. demonstrated that I L K is required in mediating capillary network formation and tubulogenesis (Watanabe et al., 2005) in vitro. Cho et al. demonstrated that I L K protects 44 endothelial cells from anoikis and nutrient-deprived stress induced apoptosis (Cho et al., 2005). Vouret-Craviari et al. proposed that I L K is critical in organizing cell-matrix adhesions that cluster a 5 p i integrins on fibronectin and recruit paxill in to the focal adhesion site to induce cytoskeletal changes (Vouret-Craviari et al., 2004). Because of mounting evidence demonstrating the importance of I L K in vasculogenesis, H I F - l a expression has been demonstrated to be regulated by A k t / P K B activity, and I L K is a major regulator of I L K activity, we hypothesized that I L K plays a role as a V E G F mediator and effector of tumor angiogenesis. 1.5 Role of Integrin-linked kinase in Inflammation Inflammation is a localized protective reaction of tissue to irritation, injury, or infection, characterized by pain, redness, swelling, and sometimes loss of function. It involves the cooperation of many cells and macromolecules. The transcription factor N F - K B is known to be of major importance in the production of pro-inflammatory molecules. N F -KB is widely expressed and important in the regulation of genes involved in mammalian immune and inflammatory responses, apoptosis, and cell proliferation and differentiation (Ghosh and Karin, 2002), and has been associated with cancer and neurodegenerative processes. The N F - K B family is a group of related and inducible transcription factors including RelA/p65, Re lB , c-Rel, p50 and p52 (reviewed in (Ghosh and Karin, 2002). In unstimulated cells, N F - K B is sequestered in the cytoplasm and bound to inhibitors of N F -KB (IKB) (Ghosh and Karin, 2002). Upon activation, I K B is degrades and N F - K B translocates to the nucleus to bind D N A . Stimulation of cell with cytokines, bacterial lipopolysaccharides (LPS) , phorbo esters, or potent oxidants leads to rapid 45 phosphorylation and subsequent ubiquitination and proteosome degradation of IicBa (Karin and Ben-Neriah, 2000). The N F - K B heterodimer translocates to the nucleus and binds to a specific KB consensus sequence and associates with transcriptional coactivators to express genes necessary for promoting cell survival and the inflammatory response in a timely and potent manner. There are two pathways, classical and alternate, that lead to the activation of different N F - K B transcription factors and the biological consequences of these pathways. Depending on the stimulus and the specific cell, NF-KB-dependent transcription leads to the expression of proteins involved in inflammation, innate immunity and survival (Karin and Greten, 2005) (Figure 1.9). The classical pathway involves the stimulation of receptors by pro-inflammatory stimuli and genotoxic stress such as pathogenic components and cytokines. This initiates a cascade of events that involve the phosphorylation-dependent degradation of IKBS by I K K - P and y and the release and nuclear translocation of N F - K B heterodimer (p65 and p50). This is the majority of the heterodimer combinations involved in transcription. The alternate pathway involves stimulation by certain tumor necrosis factor (TNF)-family members and leads to IKK-cc dependent phosphorylation of p i 00, a precursor of p52. The p i 00 is proteolytically degraded to p52, which complex to Re lB to form a p52/RelB heterodimer(reviewed in (Karin and Greten, 2005). I L K has also been shown to promote cell survival in an Akt/PKB-dependent manner (Hannigan et al., 2005). Based on these observations I hypothesized that I L K is able to activate N F - K B activity through activation of I K K and the degradation of I K B . TO test this, I determined i f the lack of I L K activity would result in a decrease in N F - K B activity and whether the expression of N F - K B enhanced or induced gene expression of 46 inducible nitric oxide ( iNOS) and cyclooxygenase-2 (COX-2) two genes known to be regulated by N F - K B activity. More recently, Douglas et al (2006) observed nuclear P-catenin in fibroproliferative response to acute lung injury and this was accompanied with increased expression of the cotranscriptional regulator T C F - 1 , resulting in increase cyclin D expression. This suggests that I L K may regulate proinflammatory processes through this pathway as well . Because mediators of inflammation, cytokines, chemokines, proteases and inhibitors of apoptosis are regulated by N F - K B activity, it has been thought that this nuclear protein might provide the link from inflammation to tumour promotion and progression. 1.6 Summary Many of the events that occur during the normal progression of vascular development in the embryo are recapitulated during situations of neoangiogenesis in the adult (Carmeliet 2003). Most notably, many tumors promote their own growth and dispersion to form metastases by recruiting host blood vessels to form in the vicinity of the tumor. While there is an abundance of molecular and genetic evidence demonstrating that I L K plays a critical role in vasculogenesis, the role of I L K in tumor angiogenesis remains unclear. We therefore examined whether I L K is involved in the stimulation of V E G F expression in tumor cells, and secondly i f I L K is required for VEGF-mediated endothelial cell migration and formation of blood vessels. We found that I L K is involved in both the expression of V E G F in tumor cells, and the cellular mechanisms that promote V E G F -mediated cell activity. Specifically, the present model implicates I L K in the indirect phosphorylation of m T O R by ILK-dependent A k t / P K B , consequently resulting in the 47 phosphorylation and inactivation of 4E-BP1 , a translation inhibitor, by mTOR. In turn, leading to the expression of H I F - l a and ultimately leading to the expression of V E G F . Secondly, we demonstrate that the I L K kinase and adaptor functions are necessary for the VEGF-stimulated endothelial cell invasion, migration and proliferation, however the exact molecular events remain elusive. I L K is serine/threonine protein kinase and adaptor protein that interacts with the cytoplasmic domain of the (31 and |33 integrin to mediate cytoskeletal reorganization and intracellular signal cascades (Dedhar, 2000; Hannigan, 2005; Zervas, 2001). I L K has been demonstrated to regulate the activity of transcription factors such as (3-catenin-TCF/Lef -1 , A P - 1 , and C R E B and other kinases such as A k t / P K B through its kinase activity. I L K activity in turn is regulated in a PI3 -kinase dependent manner (Delcommenne et al., 1998; Persad et al., 2000). Since the transcription factor, N F - K B has been shown to be activated by A k t / P K B , which is known to lead to activation of iN O S in mice (Xie et al 1994), we wanted to determine whether I L K could also regulate N F - K B activity. We examined the role of I L K in lipopolysaccharide (LPS) stimulated expression of i NOS and N O , a physiologically relevant system for studying the regulation of N F - K B . 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Here we show that I L K is essential for H I F - l a and V E G F expression in prostate cancer cells, and that it is also essential for VEGF-stimulated endothelial cell migration, tube formation and tumor angiogenesis. Consequently, I L K plays important roles in two key aspects of tumor angiogenesis: V E G F expression by tumor cells, and V E G F stimulated blood vessel formation. Our findings suggest that I L K is a promising therapeutic target for the inhibition of tumor angiogenesis. We show that Integrin-linked kinase ( ILK) stimulates the expression of V E G F by stimulating H I F - l a protein expression in a P K B / A k t and m T O R / F R A P dependent manner. In human prostate cancer cells, knock-down of I L K expression with s i R N A , or inhibition of I L K activity, results in significant inhibition of H I F - l a and V E G F expression. In endothelial cells, V E G F stimulates I L K activity, and inhibition of I L K expression or activity results in the inhibition of VEGF-mediated endothelial cell migration, capillary formation in vitro, and angiogenesis in vivo. Inhibition of I L K activity also inhibits prostate tumor angiogenesis and suppresses tumor growth. These 1 Tan C, Cruet-Hennequart S, Troussard A, Fazli L, Costello P, Sutton K, Wheeler J, Gleave M, Sanghera J and Dedhar S. (2004) Regulation of tumor angiogenesis by integrin-linked kinase (ILK). Cancer Cell. Vol 5 p79-90. All the work in this chapter was carried out by me except for figures 2.5 B and C. 68 data demonstrate an important and essential role of I L K in two key aspects of tumor angiogenesis: V E G F expression by tumor cells, and V E G F stimulated blood vessel formation. ' 2 . 2 Introduction Angiogenesis plays a critical role in cancer progression (Hanahan and Weinberg, 2000). Tumor growth and metastasis have been shown to be dependent on angiogenesis, and inhibition of tumor angiogenesis, by selectively inhibiting the growth, survival and migration of endothelial cells, is perceived as an attractive, non-toxic means of regulating tumor progression (Kerbel, 1991). A variety of proteins have been identified as potential targets of anti-angiogenesis therapy, and despite poor results in clinical trials of some of the anti-angiogenic strategies (Kerbel and Folkman, 2002), the potential of anti-angiogenic therapy continues to be an attractive means of cancer control. One of the key mediators of angiogenesis is vascular endothelial cell growth factor ( V E G F ) , which can promote the proliferation, survival, and migration of endothelial cells and is essential for blood vessel formation (Ferrara, 2002). V E G F is expressed by activated endothelial cells, but more importantly for tumor angiogenesis, V E G F expression and secretion is stimulated in tumor cells by activation of oncogenes such as Ras (Rak et al., 2000), as well as by the activation of the PI-3 Kinase pathway (Jiang et al., 2001; Fukuda et a l , 2002), which has inherent oncogenic properties. The PI-3 Kinase pathway can be constitutively activated via autocrine growth factors, by constitutively activated growth factor receptors such as Erb-B2, by activating mutations 69 in PI-3 Kinase or its downstream effector, P K B / A k t , or by the mutational inactivation, or loss of the tumor suppressor, P T E N (Cantley and Neel, 1999). The constitutive upregulation of expression of V E G F by tumor cells is felt to be a major contributor to tumor angiogenesis (Ferrara, 2002). V E G F expression is regulated at the level of transcription, by a variety of transcription factors which include, A P - 1 , N F - K B , and hypoxia inducible factor-la (HIF-l a ) (Huang et al., 2000; Ryan et a l , 1998; Damert et al., 1997). The major physiological stimulus for V E G F expression is hypoxia, resulting in the transcriptional induction of the V E G F gene by H I F - l a (Forsythe et al., 1996; Carmeliet et a l , 1998; Ryan et a l , 1998), which is a heterodimeric transcription factor composed of H I F - l a and H I F - i p subunits (Jiang et al., 1996). The hypoxia-mediated stimulation of H I F - l a expression is regulated by the inhibition of ubiquitin-mediated degradation and consequent stabilization of the H I F - l a subunit under hypoxic conditions. A s a result H I F - l a accumulates, 'dimerizes with H I F - i p , and activates transcription of target genes, including V E G F (reviewed in Harris, 2002). Recently, however, the expression of V E G F via the activation of the PI-3 Kinase pathway has also been shown to be mediated by H I F - l a (Jiang et al., 2001; Fukuda et al., 2002). Signaling via receptor tyrosine kinases induces H I F - l a expression by an independent mechanism involving the stimulation of increased rates of H I F - l a protein synthesis via PI-3 Kinase dependent stimulation of P K B / A k t and m T O R / F R A P which activates the translational regulatory protein eIF-4E binding protein 1 (4E-BP1) and p70 S6 kinase. (Fukuda et al., 2002; Laughner et al., 2001; Gingras et al., 2001; Peterson et al., 1999). These findings indicate that H I F - l a regulates both hypoxia- and growth factor-induced V E G F expression. 70 One of the components of the PI-3 Kinase pathway, immediately upstream of P K B / A k t is integrin linked-kinase ( ILK) (Hannigan et al., 1996; W u and Dedhar, 2002; Troussard et al., 2003). I L K can interact with the cytoplasmic domain of p integrin subunits and is activated by both integrin activation as well as growth factors (Wu and Dedhar, 2002). I L K is a PI-3 Kinase-dependent kinase (Persad et al., 2001; Delcommenne et al., 1998), and is an upstream regulator of the phosphorylation of P K B / A k t on serine-473 (Troussard et a l , 2003; Persad et a l , 2001; Persad et al., 2000; Lynch et al., 1999; Delcommenne et a l , 1998), one of the two phosphorylation sites required for the full activation of P K B / A k t . Overexpression of I L K induces anchorage independent cell growth and suppression of anoikis, and promotes hyperplasia and tumor formation in vivo (Wu and Dedhar, 2002). I L K activity is also constitutively activated in PTEN-nu l l cancer cells, and the constitutive activation of P K B / A k t in such cells is inhibited upon inhibition of I L K activity (Persad et al., 2000). I L K also promotes cell migration and invasion (Persad and Dedhar, 2003). Because of these oncogenic properties of I L K , we decided to explore the potential role of I L K in promoting tumor angiogenesis. We wanted to determine whether I L K is involved in the stimulation of expression of V E G F in tumor cells, and secondly whether I L K is required for V E G F -mediated endothelial cell migration and formation of blood vessels. In this paper we report that overexpression of I L K stimulates V E G F expression in a P K B / A k t and HIF-la-dependent manner, and that inhibition of I L K expression or activity in V E G F expressing prostate cancer cells (DU145 and PC3), results in dramatic inhibition of V E G F expression and secretion via inhibition of P K B / A k t activity and HIF-l a expression. Furthermore, inhibition of I L K activity or expression in V E G F -71 stimulated endothelial cells results in the inhibition of endothelial cell migration and blood vessel formation in vitro and in vivo. A small molecule I L K inhibitor suppresses tumour angiogenesis and tumour growth in a PC-3 xenograft tumor model. Our results demonstrate an important and essential role of I L K in two key aspects of tumor angiogenesis: V E G F expression by tumor cells, and V E G F stimulated blood vessel formation, and suggest that I L K may be a promising therapeutic target for the inhibition of tumor angiogenesis. 2.3 Materials and Methods 2.3.1 Cell Culture and Transfections Prostate carcinoma cell lines positive and null for P T E N (DU145 and PC3 respectively) ( A T C C ) were cultured as suggested by A T C C . PC3 cells were transiently transfected with V5-tagged ILK-dominant negative ( I L K - D N : V 5 ) , Empty vector, GFP-tagged P T E N (PTEN:GFP) , HIF- la-dominant negative, HIF-1 response element conjugated to G F P reporter (HRE:GFP) , and/or Renilla for transfection control using 2 - 3 |ag of c D N A with 6 ul of Lipofectin reagent (Life Technologies Inc.), according to the manufacturer's guidelines. DU145 cells were transfected with 4 ul of Fugene reagent (Roche Molecular Biochemicals). Intestinal epithelial cells (IEC-18) were stably transfected with I L K wi ld-type (ILK-13 A l a 3 ) , I L K dominant-negative ( I L K - D N GH31RH) or I L K anti-sense (ILK-14 Antisense) (Hannigan et al., 1996). The parental IEC-18 cells were used as the control. These cells were cultured as previously described (Delcommenne et al., 1998). Human umbilical vein endothelial cells ( H U V E C ) ( A T C C ) were cultured as suggested by 72 A T C C . ILK-13 A l a 3 and H U V E C cells were transfected with 6 u.1 of Lipofectamine 2000 reagent (Life Technologies Inc.), according to the manufacturer's guidelines. 2.3.2 Small Interfering RNA (siRNA) PC3 cells were transfected with small interfering R N A (s iRNA) that specifically targets the I L K gene ( I L K - H or I L K - A ) or non-silencing control using 6 ul of Lipofectin in O P T I - M E M (GibcoBRL) overnight. The cells were passaged 36 hours after incubation in complete media and harvested 36 to 48 hours later as previously described (Troussard et al., 2003). s i R N A duplexes were synthesized by Xeragon Inc., M D . Sequences of the human I L K gene specifically targeting the p H domain ( ILK-H) (Troussard et al., 2003) and the integrin binding domain ( I L K - A ) were chosen. The sequence of the D N A target of I L K - A is 5' - G A C G C T C A G C A G A C A T G T G G A - 3 ' . A non-silencing s i R N A (16-base overlap with Thermotoga maritimia) was used as the control. 2.3.3 Chemical Inhibitors Cells were exposed to the highly specific small molecule inhibitor (KP-392; Kinetek Pharmaceuticals) (Cruet-Hennequart S et al., 2003; M i l l s J et al., 2003; Tan C et al., 2002; Persad S et al., 2001; Persad S et al., 2001; Tan C. et al., 2001; Persad S et al., 2000) and equivalent amounts of D M S O vehicle. PI-3 Kinase inhibitor LY294002 (Calbiochem) and M E K 1 inhibitor PD98059 (Cell Signaling Technology) were dissolved in D M S O as the vehicle. 73 2.3.4 Western Blotting Cel l lyses and western blotting were carried out as previously described by us (Troussard et a l , 2003; Cruet-Hennequart et al., 2002; Tan et al., 2001; Persad et al., 2001). The following antibodies were used in this study: anti-phosphoserine-473 P K B / A k t , anti-P K B / A k t , and anti-phosphoserine-21/9-Glycogen Synthase Kinase-3 (GSK-3) antibodies from N e w England Bio labs, anti-V5 antibody from Invitrogen, anti-ILK and an t i -HIF- la antibodies from B D Transduction, anti-phosphoserine-2448-mTOR/FRAP and anti-m T O R / F R A P antibodies from Cel l Signaling, an t i -VEGF antibody from Oncogene Research Products, anti-green fluorescent protein (GFP) antibody from Boerhinger Mannheim, and anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase from Jackson Laboratories. 2.3.5 Quantification of VEGF in Conditioned Media Secreted V E G F was quantified by using an enzyme linked immunoassay (ELISA) kit for human V E G F (Oncogene Research Products) according to the manufacturer's instructions. 2.3.6 ILK Kinase Assay H U V E C s were starved for 24 hours prior to the experiment. The cells were exposed to increasing amounts of V E G F for 1 or 24 hours, followed by lysis with NP-40 lysis buffer. Equivalent amounts (250 ug) of lysate were immunoprecipitate overnight with 3 ug of mouse monoclonal anti-ILK antibody (Upstate Biotechnologies Institute) at 4 ° C . The immune complexes were isolated with protein A / G plus agarose beads (Santa Cruz Biotechnology), and washed 3 times with NP-40 lysis buffer and three times with last 74 wash buffer (50 m M H E P E S p H 7, 2 m M M g C l 2 , 2 m M M n C l 2 , 200 u M N a 3 V 0 4 , protease inhibitors). The kinase assay was performed using 2 u.g of G S K - 3 fusion protein (New England Biolabs) as a substrate, 200 u M A T P in the reaction buffer (50 m M H E P E S p H 7, 2 m M M g C l 2 , 2 m M M n C l 2 , 200 u M N a 3 V 0 4 , 200 u M NaF) for 30 minutes at 30°C. Phosphorylation of the substrate was detected by Western blot with anti-GSK-3 serine-21/9 antibody. 2.3.7 Invasion and Migration Assay Invasion and migration was analyzed with a modified Boyden chamber assay (cell culture inserts with a polycarbonate-filter (PVP, 8 urn pore size; Corning Incorporated, N Y ) covered with growth factor reduced Matrigel (Becton Dickinson, M A ) ) . 200 ul of cell suspension (5x10 4 cells) were added to the upper wells and allowed to attach for 2 hours at 37°C. Increasing amounts of KP-392 and equivalent amounts of D M S O diluted in 100 ul were added to the upper well for a complete volume of 300 ul . The lower compartment was filled with F-12K medium containing 0 or 20 ng/ml V E G F . Chambers were incubated for 16 hours in a 5% C 0 2 , 99% humidity and 37°C atmosphere. Cells on the under side of the filter were quantified by staining the cells with crystal violet, followed by counting the number of cells per magnified field of view (6 fields /membrane). 2.3.8 Cell Viability and Proliferation Assay The metabolic activity of cells was determined in vitro using the colorimetric cell proliferation / tetrazolium salt WST-1 reagent/Electro Coupling Solution (WST-1/ECS) assay kit (Chemicon International, C A ) according to manufacturers instructions. The 75 plate was incubated with the reagent for an additional 30 minutes to 1 hour and quantified by spectrophotometry (OD = 450 nm). A l l experiments were performed in triplicate. 2.3.9 Immunohistochemical Staining. Tissues were frozen in compound-embedding medium (OCT; Miles Inc. IN), and 10-um sections were collected on positively charged slides (Wax-It Histology Services Inc., B . C . Canada). Sections were fixed with cold acetone and blocked with 3 % hydrogen peroxidase, followed by 1 % bovine serum albumin and normal rabbit serum. Sections were then incubated with the anti-mouse CD31 antibody ( B D Pharmingen) overnight at 4°C, followed by incubation with a horseradish peroxidase conjugated anti-rat secondary antibody (Jackson Laboratories) for 1 hour at room temperature. The tissue was washed 3 times with P B S in between each step. The antibody localization was visualized with N O V A R E D substrate kit (Vector Laboratories, C A ) , used as directed by manufacturer and the slides were counterstained with hematoxylin. 2.3.10 Endothelial Tube Formation Assay The endothelial tube formation assay was performed as previously describe with minor modification (Maeshima et al., 2000). A suspension of H U V E C in medium was seeded in triplicate into Matrigel-precoated 24 well plates in the presence of 50 u M KP-392. 2.3.11 Endothelial Sprouting Assay Microcarrier beads coated with denatured collagen (Cytodex3; Sigma) were seeded with H U V E C s . Fibrin gels were made by dissolving 2.5 ug/ul bovine fibrinogen (Sigma), 0.05 mg/ml Aprotinin (Sigma) in F-12K medium followed by passing the solution through a 0.22 um pore size filter to sterilize and mixed with a fraction of H U V E C -76 coated bead. This mixture was transferred gently to 96-well plates together with HUVEC-coa ted beads at a density of 25 beads/well with a wide mouth pipette tip. Clotting was induced by the adding 1.2 units/ml thrombin. After clotting was complete, F-12K medium containing the indicated inhibitors, equivalent amounts of D M S O vehicle, 0 or 20 ng/ml V E G F and 1 % F B S was carefully applied onto the gel. After 3 days of incubation with daily changes of the medium, the number of capillary-like tubes formed/microcarrier bead (sprouts/bead) was counted by microscopy, monitoring at least 150 beads for each treatment. Only sprouts greater than 150 um in length and comprised of atlleast 3 endothelial cells were counted. 2.3.12 Chorioallantoic Membrane (CAM) of Chick Embryos Assay White Leghorn chicken eggs were fertilized and incubated at 37°C under conditions of constant humidity. The developing C A M was separated from the shell by opening a window at the broad end of the egg above the air sac on embryonic day 6. The opening was sealed with Parafilm (American National Can, IL) and the eggs were returned to the incubator. On embryonic day 8, 30 ng/ml V E G F and 50 u M of KP-392 in D M S O or equivalent amounts of D M S O vehicle were loaded onto 2 mm gelatin sponges (Pharmacia Upjohn) and placed on the surface of the developing C A M . Eggs were resealed and returned to the incubator for 10 days as previously described (Roskelley et al., 2001). 2.3.13 PC3 xenograft tumor assay 6 to 8 week old male nude mice (Harlan Sprague Dawley, Inc., IN , U S A ) were allowed to acclimatize for 1 week in the Jack Be l l Research Centre animal facility. Procedures 77 involving animals and their care conform to institutional guidelines (University of British Columbia Animal Care Committee). 100 ul of a PC3 cell suspension (RPMI media (GibcoBRL) ; 5% F B S ; 2 X 1 0 7 cells/ml) was injected subcutaneously into the left and right flank regions of the nude mice via a 27-gauge needle under halothane anaesthesia. 10 mice comprised each experimental group. 1 week post inoculation, animals were dosed daily with the I L K inhibitor (KP-307-2) by intraperitoneal injection of 100 mg/kg of I L K inhibitor (at a concentration of 10 mg/ml in 5% Tween80 in saline). Control mice received equivalent volumes of vehicle (5% Tween 80 in saline). 1 tumor was removed lweek post treatment for angiogenesis marker analysis. The Image-Proplus from Media Cybernetics (Carlsbad, C A ) was used to measure the CD31 positive immunostained endothelial lining (red objects) of neovasculature within the tumors. Tumor growth was monitored 3 times a week by measuring the height, length and width of each tumor with a caliper. The tumor volumes were calculated from a formula (axbxc/2) that was derived from the formula for an ellipsoid (7td 3/6). Data were calculated as the percentage of original (day 1) tumor volume and graphed as fractional tumor volume ± S E M . Mice were sacrificed by day 28 after KP-392 or vehicle administration. 2.3.14 Micrographs A l l images were generated using the Nikon Eclipse T E 200 microscope and N i k o n Cool P I X 950 digital camera. 2.3.15 Plasmid We would like to thank Dr. Peggy Olive for the H R E : G F P . 78 2.4 Results 2.4.1 Overexpression of ILK stimulates VEGF expression in a PKB/Akt-and HIF-1a-dependent manner We have previously demonstrated that overexpression of I L K in IEC-18 rat intestinal epithelial cells results in anchorage-independent cell cycle progression, tumorigenicity in nude mice, activation of P K B / A k t , inhibition of G S K - 3 , and stimulation of A P - 1 , N F - K B and P-catenin/LEF transcription factors (reviewed in W u and Dedhar, 2002). Overexpression of a kinase-deficient mutant of I L K , or I L K anti-sense c D N A , did not result in the stimulation of these pathways or phenotypes. Because activation of other oncogenes such as Ras, or the PI-3 Kinase pathway have been shown to stimulate V E G F expression in tumor cells (Rak et al., 2000), we wanted to determine whether I L K overexpression also resulted in the stimulation of V E G F expression. A s shown in Fig . 2.1 A , the expression of both isoforms of V E G F is markedly stimulated in the I L K overexpressing clone of IEC-18 cells ( ILK-13, A l a 3 ) , as compared to control clones expressing the E359K kinase-deficient ILK-dominant negative ( I L K - D N , G H 3 1 R H ) , or antisense-ILK (ILK-14) (Novak et al., 1998; Hannigan et al., 1996). In addition the data in Figure 2.1 A , also show markedly increased phosphorylation of P K B / A k t on serine-473 in the absence of any changes in P K B / A k t expression. Since one of the major transcriptional regulator of the V E G F gene is hypoxia inducible factor-la (HIF - l a ) , we transfected the different IEC-18 clones described above with a HIF-1 response element fused to a green fluorescence protein (GFP) reporter ( H R E : G F P ) (Ruan and Deen, 2001). A s shown in Figure 3.1 A , this reporter is only active in the I L K overexpressing clone, suggesting that the stimulation of V E G F expression in these cells is likely mediated by the upregulation or activation of the HIF-1 transcription factor. We were unable to 79 directly analyze H I F - l a protein expression in these clones because of the lack of availability of suitable anti-rat H I F - l a antibodies. Inhibition of I L K expression in the A l a 3 I L K overexpressing cells with I L K s i R N A , resulted in the suppression of V E G F expression (Figure 2. IB) , showing that I L K is indeed responsible for the stimulation of V E G F expression in these cells. We have previously shown that the I L K overexpressing clones have constitutive high-level expression of cyclin D I (Radeva et al., 1998), and inhibition of I L K expression by s i R N A inhibits cyclin D I expression (Troussard et al., 2003). A s shown in Figure 2 . IB, I L K s i R N A also results in cyclin D I expression in the I L K overexpressing cells. These data demonstrate that overexpression of kinase-active I L K results in the stimulation of V E G F expression via the activation of P K B / A k t and the HIF-1 transcription factor. 80 B - * * i i " i o ^. ^ ^ LU —I —I —I • » V E G F « • » Akt/PKB phosphoserine-473 %p wm Akt/PKB HRE:GFP p-Actin 25 50 s iRNA (nM) Contro II K A ILI\-A Contro ILK-A m ILK m HIF-1a VEGF ***** Cyclin D1 p-Actin Figure 2.1 VEGF expression and HIF-la activity are increased in epithelial cells with a high ILK activity. (A) Immunoblot analysis with the indicated antibodies of N P -40 cell lysates of the indicated cell lines transfected with 3 ug H R E : G F P and pRenilla (transfection efficiency control), and exposed (24 hrs) to 1% F B S media. I L K 13 A l a 3 clones overexpress I L K , I L K - D N clones express dominant negative I L K (Hannigan et al., 1996). (B) Immunoblot analysis with the indicated antibodies of the R I P A IEC-18 I L K 13 A l a 3 cell lysates 4 days post-transfection with the indicated type and amount of s i R N A . I L K - A was I L K specific s i R N A and control was non silencing s i R N A from Thermotoga maritimia (Troussard et al., 2003) A l l figures are a representation of 3 trials. 8 1 2.4.2 Inhibition of ILK expression and activation suppresses PKB/Akt and mTOR/FRAP phosphorylation and inhibits HIF-1a and VEGF expression in prostate cancer cells In order to analyze in more detail the ILK-mediated signaling pathway leading to the stimulation of V E G F , and to assess the relevance of I L K in V E G F expression in cancer cells, we decided to inactivate I L K expression or activity in human prostate cancer cells which express V E G F . The PI-3 Kinase pathway is constitutively activated in many cancer cells lines. In certain human prostate cancer cell lines (PC3 and L n C A P ) the PI-3 Kinase pathway is constitutively activated due to the loss of expression of the tumor suppressor P T E N (Davies et al., 1999; Stambolic et al., 1998). We have previously shown that I L K activity is also constitutively activated in these cells, and that inhibition of I L K activity suppresses P K B / A k t activity in these PTEN-nu l l cells (Persad et a l , 2000). V E G F expression has been shown to be constitutively elevated in PC3 cells (Jiang et al., 2001). Because we had found that I L K stimulated V E G F expression (Figure 2.1), we wanted to determine whether inhibition of I L K activity in PC3 cells resulted in the inhibition of V E G F expression. A s shown in Figure 2.2A, inhibition of I L K activity by transfection of kinase-deficient, I L K - D N (E359K) (Persad et a l , 2000), or wild-type P T E N , resulted in inhibition of V E G F expression at the protein level as determined by Western blotting. In addition, the expression of H I F - l a protein is also substantially inhibited by D N - I L K and P T E N (Figure 2.2A). A s expected, D N - I L K also inhibited phosphorylation of P K B / A k t on serine-473 (Figure 2.2A). Kinase-deficient ILK-dominant negative and P T E N also inhibited the activity of the HIF-1 response element (HRE) , as shown in Figure 2.2C, 82 suggesting that the upregulation of V E G F expression in these cells is likely due to the ILK-mediated upregulation of H I F - l a expression. We have recently utilized double stranded R N A interference ( s iRNA) to knock down I L K protein expression (Troussard et al., 2003). Furthermore, we have shown that I L K knockdown by s i R N A results in significant inhibition of P K B / A k t Serine-473 phosphorylation and activation (Troussard et al., 2003). We therefore exposed PC-3 cells to increasing concentrations of I L K specific s i R N A (Troussard et al., 2003). A s shown in Figure 2.2B, I L K s i R N A resulted in the complete depletion of I L K expression in PC-3 cells. This was associated with a suppression of phosphorylation of P K B / A k t on serine-473. Expression of P K B / A k t was not affected (Figure 2.2B). Furthermore I L K s i R N A mediated knockdown of I L K also resulted in significant inhibition of expression of both H I F - l a and V E G F protein (Figure 2.2B). It has been recently shown that P K B / A k t can regulate the expression of H I F - l a protein at the translational level by stimulating the phosphorylation of m T O R / F R A P , which is a regulator of protein synthesis (Gingras et al., 1999; Peterson et al., 1999). We therefore wanted to determine whether the I L K -mediated expression of H I F - l a and V E G F also involved m T O R / F R A P . A s shown in Figure 2.2B, s i R N A mediated knockdown of I L K resulted in the inhibition of m T O R / F R A P phosphorylation on serine-2448, concomitant with the inhibition of the P K B / A k t phosphorylation. The expression of m T O R / F R A P protein was not affected by the knockdown of I L K . These data suggest that in the PC3 cells, the constitutive activation of I L K drives V E G F expression most likely via H I F - l a through the activation of P K B / A k t and m T O R / F R A P , resulting in increased translation of H I F - l a protein. This is further substantiated by the observation that transfection of a dominant-negative HIF-83 l a construct into PC3 cells almost completely inhibits V E G F expression as well H R E activity (Figure 2.2C). 84 A § Q HRE:GFP > S£? Q- z 5 9 I S § I ^ . - 7 E ^ H — « — z iB =! E I Q- — LU LU 1 2 CL V E G F HIF-1a % HRE:GFP ILK(DN):V5 Ak t /PKB phosphoserin-473 ~* PTEN:GFP A k t / P K B VEGF V5-tag for ILK-DN :V5 _W_mmM f3-Actin G F P - T a g for P T E N : G F P (3-Actin B _5 5 P 1 0 0 s iRNA (nM) 1 1 1 x i i § _ § _ § _ O - O — O -m m m ILK m - m* m phosphoserine-Akt/PKB Ak t /PKB —• - — phophoserine-mTOR HIF-1a V E G F (3-Actin Figure 2.2 V E G F and H I F - l a expression are severely affected by the loss of I L K activity in a P T E N and mTOR/FRAP-dependent manner, in PTEN-null prostate carcinoma cells (PC3). (A) Immunoblot analysis with the indicated antibodies of the R I P A lysate of PC3 cells transfected with 1 - 2 u.g of Empty:V5, I L K - D N : V 5 or P T E N : G F P vector. (B) Immunoblot analysis with the indicated antibodies of the R I P A lysate of PC3 cells transfected with the indicated type and amount of s i R N A . (C) Immunoblot analysis with the indicated antibodies of NP-40 lysate of PC3 cells co-transfected with the indicated constructs and pRenilla after 48 hours. Cells were noted not to be dying by cell viability assay. A l l figures are a representation of 3 independent trials. 85 2.4.3 Pharmacological inhibition of ILK activity results in the inhibition of HIF-1a and VEGF expression in prostate cancer cells We have identified highly selective small molecule inhibitors of I L K activity. These A T P competitive inhibitors have been extensively characterized, and shown to inhibit I L K activity and the activation of all of the downstream effectors of I L K (Cruet-Hennequart et al., 2003; M i l l s et al., 2003; Tan et al., 2002; Persad et al. 2001; Tan et al., 2001; Persad et al., 2000, Troussard et al., 2000). The inhibitors are equally effective and specific as I L K inhibition by dominant-negative I L K and I L K s i R N A (Persad et al., 2001; Troussard et al, 2003). We therefore wanted to determine whether exposure of human prostate cancer cells to the I L K inhibitor would also inhibit the expression of H I F - l a and the expression and synthesis of V E G F . A s shown in Figure 3.3 A , exposure of both PC3 and DU145 prostate cancer cells to the I L K inhibitor KP-392 (Persad et al., 2001; Persad et al., 2001) resulted in the inhibition of both H I F - l a and V E G F expression in a dose-dependent manner. Despite poor cellular permeability of this inhibitor, resulting in the exposure of cells to relatively high concentrations, it can be seen that there is significant inhibition of both H I F - l a and V E G F expression at 25 u M KP-392, especially in PC3 cells. A s well , it can clearly be seen that KP-392 also suppresses the phosphorylation of P K B / A k t on serine-473 in a dose-dependent manner. Again there is no effect on the expression of P K B / A k t . In addition, as shown in Figure 3.3B, not only V E G F cellular expression but also its secretion, as determined by an enzyme-linked immunosorbant assay (ELISA) of the conditioned cell media, is inhibited by the I L K inhibitor KP-392. In contrast to PC3 cells, V E G F expression is not completely inhibited by the I L K inhibitor in DU145 cells, despite substantial inhibition of H I F - l a expression. This suggests cell type differences in the regulation of V E G F expression. 86 In Figure 3.3C, we demonstrate that H I F - l a expression is stimulated by serum in serum starved PC3 cells, and that inhibition of I L K as well as PI-3 Kinase with the respective pharmacological inhibitors KP-392 and LY294002, inhibits H I F - l a expression. Collectively, the data shown in Figure 3.2 and 3.3 demonstrate that I L K is a critical component of the constitutively activated PI-3 Kinase -PKB/Akt signaling pathway resulting in the stimulation of H I F - l a and V E G F . Inhibition of I L K expression or activity can result in substantial inhibition of the expression of both H I F - l a and V E G F , suggesting that I L K may be an important therapeutic target for the inhibition of expression of the angiogenic factor, V E G F . 87 P C 3 cells 0 25 50 100 DU145 cells 0 25 50 100 KP-392 (uM) V E G F *""» HIF-1oc B Ak t /PKB phosphoserine-473 Ak t /PKB 3-Actin 0 10 25 50100 KP-392 (uM) — — phosphoserine-473 Akt/PKB — Akt/PKB * • » V E G F (3-Actin V E G F in M e d i a (ng/ml) Relative Decrease of V E G F in Media 0 _ 2 0.00 -1 1 1 1 1 0 1 1 0 0 25 0 0 0 mmm 1 1 1 50 100 0 0 0 20 F B S (%) KP-392 ( L I M ) LY294002 (u.M) HIF-1a Figure 2.3 Inhibition of I L K activity results in decrease of H I F - l a and V E G F expression in a PI-3 Kinase dependent manner in prostate carcinoma cells. (A) Immunoblot analysis with the indicated antibodies of RIPA lysate of PC3 and DU145 cells starved (20 hrs), and then exposed (24 hrs) to the indicated amounts of KP-392, equivalent amounts of DMSO vehicle and 1 % FBS. (B) Quantification by ELISA of the level of secreted VEGF protein in conditioned media. The X-axis represents increasing concentrations of KP-392 as shown in the above immunoblot. The first graph represents a single experiment. The second graph is the accumulation of 3 independent trials. (C) Immunoblot analysis of RIPA lysates of DU145 cells cultured under the indicated conditions (24 hrs). The treatment of these cells with KP 392 at the time points shown here caused little to no cell death as assessed by cell viability assay. All figures are a representation of 3 trials. 88 2.4.4 ILK regulates VEGF-mediated endothelial cell migration and blood vessel formation V E G F stimulates endothelial cell survival and migration, and promotes the formation of new blood vessels (Ferrara, 2002). Since the activity of I L K is stimulated by various growth factors and chemokines (Wu and Dedhar, 2002; Freidrich et al, 2002), and I L K also promotes cell migration and invasion (Persad and Dedhar, 2003), we wanted to determine whether I L K also played a role in VEGF-mediated endothelial cell migration and vascular morphogenesis. A s shown in Figure 2.4A, V E G F stimulates I L K kinase activity in a dose-dependent manner in quiescent human umbilical vein endothelial cells ( H U V E C ) . The stimulation of I L K activity by V E G F is dependent on PI-3 Kinase activity since the V E G F stimulation of I L K activity is inhibited in the presence of the PI-3 Kinase inhibitor, LY294002. These data support previous studies showing I L K to be a PI-3 Kinase dependent kinase (Delcommenne et al., 1999; Lynch et al., 1998) and demonstrate that V E G F stimulates I L K activity in a PI-3 Kinase dependent manner. We next determined whether I L K was required for the stimulation of V E G F -mediated cell migration of H U V E C cells. A s shown in Figure 2.4B, V E G F stimulates the migration of H U V E C cells, and inhibition of I L K activity with the pharmacological I L K inhibitor, KP-392, results in a dose-dependent inhibition of VEGF-mediated H U V E C cell migration. Furthermore, inhibition of I L K expression in H U V E C cells by I L K s i R N A also inhibited V E G F stimulated H U V E C cell migration (Figure 2.4C), demonstrating an essential role for I L K in the stimulation of endothelial cell migration by V E G F . We also noted inhibition of Cycl in D I expression in the I L K s i R N A transfected H U V E C cells (Figure 2.4D), indicating that inhibiting I L K may also inhibit V E G F stimulated H U V E C cell proliferation. A s shown in Figure 2.4D, H U V E C cell 89 proliferation in response to V E G F is inhibited in the I L K s i R N A transfected cells. Cel l viability in the I L K s i R N A transfected cells was not significantly altered (data not shown). These data demonstrate an essential role of I L K in VEGF-media ted H U V E C cell migration and proliferation. It has been demonstrated that V E G F promotes its own expression in endothelial cells via a positive autocrine loop involving H I F - l a expression and activity (Stoeltzing et al., 2003; Zhong et al., 2000). It is therefore interesting to note that V E G F stimulated H I F - l a expression in H U V E C cells is inhibited by I L K s i R N A (Figure 2.4D), suggesting that I L K is a component of this positive feedback loop. We next wanted to determine whether I L K is required for VEGF-mediated blood vessel formation. To evaluate this we initially utilized an in vitro endothelial cell sprouting assay. A s shown in Figure 2.5A, V E G F significantly stimulated H U V E C capillary sprouting, which was quantified as described in the Experimental Procedures. Both the KP-392 I L K inhibitor and the PI-3 Kinase inhibitor LY294002 inhibited cell sprouting. Exposure of cells to 50 u M KP-392 and 20 u M LY294002 completely inhibited V E G F induced H U V E C sprouting. In contrast, the M E K inhibitor, PD98059, did not have any significant inhibitory effect in this assay. The I L K inhibitor had only minor effects on H U V E C cell viability, and only at very high concentrations (Figure 2.5A). These data demonstrate that PI-3 Kinase and I L K activities are required for VEGF-mediated vascular morphogenesis in vitro. Another assay that is frequently used for the demonstration of angiogenesis in vitro is the endothelial tube formation assay in which endothelial cells placed on matrigel in the presence of angiogenic factors results in the endothelial cells forming tube like structures morphologically similar to capillaries. This tube formation represents the 90 contribution of cell survival, migration and proliferation (Folkman and Haudenschild, 1980). A s shown in Figure 2.5B, H U V E C cells cultured on matrigel in the presence of V E G F formed tube structures that were completely inhibited by the I L K inhibitor, K P -392. A t the same KP-392 concentrations, significant inhibition of H U V E C cell migration and capillary sprout formation was observed (Figure 2.4B and 2.5A). These three different in vitro assays, demonstrate that the inhibition of I L K activity has a dramatic effect on endothelial cell function in response to V E G F suggesting an essential role of I L K in blood vessel formation. 91 Figure 2.4 I L K kinase activity is involved in VEGF-stimulated H U V E C activity (A) I L K kinase activity is stimulated by V E G F in H U V E C . Cells were starved (24 hrs), and exposed to the indicated amounts of V E G F and LY294002. The I L K kinase assay was carried out as described in the Experimental Procedures (EP). This is a representation of 3 independent trials. (B) Decrease in I L K activity reduces H U V E C invasion and migration towards V E G F , and inhibits endothelial cell sprouting in vitro. 2 hours after H U V E C were seeded on the upper chamber, indicated amounts of KP-392 were added to this chamber, and the migration assay was performed and analyzed as described in EP . This graph represents the mean of three experiments ± SD. 93 E CD-CD r Q . C 2 i CD w o 70 60 50 40 30 20 10 0 p<0. , 25 nM SiRNA Control ILK-A Control ILK VEGF (ng/ml) 0 0 20 20 D 0 20 VEGF (ng/ml) I < 2 < siRNA § * § * (25 nM) ** ILK HIF-1oc m Cyclin D1 p-Actin CD O c CO -Q o < E c o 10 25 nM SiRNA Control ILK-A Control ILK VEGF (ng/ml) 0 0 20 2( Figure 2.4 Figure 2.4 (C) Knockdown of I L K expression reduces H U V E C invasion and migration towards V E G F . Equal number of H U V E C transfected for 3 days with the indicated s i R N A (25 nM) were seeded in the upper chamber. The experiment was performed as described in EP . This graph represents the mean of 3 experiments ± SD. (D) Immunoblot analysis with the indicated antibodies of RIPA lysate of transfected H U V E C (25 n M indicated siRNA) that were starved (24 hrs), then exposed (24 hrs) to V E G F (0 or 20 ng/ml) 2 days post-transfection. . The graph represents relative H U V E C growth after above conditions, measured by W S T 95 2.4.5 Inhibition of ILK activity inhibits VEGF stimulated angiogenesis in Vivo We next wanted to determine whether inhibiting I L K activity resulted in the inhibition of VEGF-stimulated angiogenesis in vivo. We utilized a well-established assay for angiogenesis, the chicken chorioallantoic membrane ( C A M ) assay (Auerbach et al, 1975), to determine the effects of the I L K inhibitor, KP-392. A s shown in Figure 2.5C, inhibition of I L K activity had a significant effect on V E G F stimulated blood vessel formation in vivo. In the C A M assay (Figure 2.5C), the incorporation of KP-392, compared to vehicle alone together with V E G F resulted in the complete blockage of growth of blood vessels towards V E G F . It is interesting to note that the blood vessels are not lysed in the presence of KP-392, but rather they fail to grow towards V E G F and seem to be repelled away from V E G F , demonstrating that the inhibition of I L K predominantly inhibits the migration of endothelial cells and blood vessels towards V E G F . 96 < o o 7 o n " Absorbance (450 nm) o o o o o p p ^ Ko co en b> N3 M o o o n o o co 0 1 0 ( 0 C 3 (D M M % 3. 2 2 H O ->• M « O l -»• o o o o Number of Sprouts / Bead NJ NJ NJ NJ o o o o o fO o 05 NJ co NJ NJ O Ol O O O O O O O Figure 2.5 KP-392 inhibits angiogenesis in matrigel and in vivo. These are three assays used to assess angiogenesis in vitro and in vivo. (A) V E G F stimulates the early stages of vessel tube formation. HUVEC-coa ted beads imbedded in fibrin were incubated in the indicated conditions ( V E G F (0 or 20 ng/ml); KP-392 (uM)). Micrographs of a typical bead were taken at 72 hours. Number of capillary-like tubes formed per microcarrier bead (sprouts/bead) were counted and analyzed at described in EP. The graph represents the mean of three experiments ± SD. The graph below represents relative cell viability after exposure (24 hrs) to the indicated amounts of K P -392, LY294002 and V E G F , measured by WST-1/ECS assay. Results represent mean absorbance ± SD. 98 Control KP-392 (50 uM) Control KP-392 (50 uM) Figure 2.5 (B) This assay shows the formation of endothelial cell-cell interactions in a 3-dimensional culture is disregulated by KP-392. H U V E C suspension in Matrigel were incubated with V E G F and D M S O (Control) or 50 u M KP-392. Micrographs of a typical field were taken to illustrate H U V E C tube formation. (C) Photographs of developing chick chorioallantoic membranes incubated with 30 ng/ml V E G F and D M S O (Control) or 50 u M KP-392. Dashed circle outlines the area covered by the gelatin sponges. Arrows show blood vessels migrating away from the area containing KP-392. A l l figures are a representation of 3 independent experiments. 99 2.4.6 Inhibition of tumor angiogenesis and suppression of tumor growth in ILK-inhibitor treated PC-3 xenograft tumor model The data presented above suggest that inhibition of I L K activity or expression should inhibit tumor angiogenesis and i f PC3 tumor growth in vivo is dependent on tumor vascularization then I L K inhibition should also induce tumor growth inhibition. To determine whether inhibition of I L K affected tumor angiogenesis and tumor growth in vivo, we established PC3 tumors in nude mice (Figure 2.6), and treated mice with established tumors with the I L K inhibitor KP-307-2, an analog of KP-392. A s shown in Figure 2.6A, there was a statistically significant effect on tumor vascularization as determined by micro-vessel density in anti-CD31 stained KP-307-2 treated and control tumor sections. In addition there was statistically significant tumor growth suppression in the I L K inhibitor treated mice over a 28 day dosing regimen (Figure 2.6B). The inhibitor was well tolerated with no obvious side effects or weight loss (data not shown). These data indicate that I L K is a mediator of prostate tumor angiogenesis, and therefore a target for anti-angiogenic therapy. 100 co 80 I 70 | g 60 c j o 50 3 CD | % 40 ^ 2 30 8 o 10 0 Immunoreactivity of CD31 - PC3 Xenografts **p=0.008824 CONTROL TREATED B CD 3000-E 2500-p > 2000-CD 1500-D) C 1000-CO Ch 500-o--Control Treated 15 20 25 30 Day Figure 2.6 Inhibition of I L K activity suppresses tumor angiogenesis and tumor growth rate. Nude mice with PC3 flank tumors were treated with daily i.p. injection of 100 mg/kg I L K inhibitor or vehicle for 28 days. (A) 7 days after dosing, 1 tumor from each mouse was harvested and tumor vascular density was analyzed as described in EP. The bar graph shows the means of neovascular densities / field for each group ± SD. Shown are representative photographs of neovasculature in the PC3 tumors. (B) Relative change in tumor volumes in 8 mice in the control group and 10 mice in the treatment group ± S E M . **: p<0.01; *: p<0.05 101 2.5 Discussion Angiogenesis is important in cancer progression and is one of the hallmarks of tumor metastasis (Hanahan and Weinberg, 2000). A principal mediator of tumor angiogenesis is V E G F and a major transcriptional activator of the V E G F gene is H I F - l a (Harris 2001). It has been reported that the PI-3 Kinase/Akt signaling pathway mediates angiogenesis and the expression of V E G F in cells, by elevating the levels of H I F - l a protein in cells independent of hypoxic condition (Semenza 2002). This hypoxia-independent stimulation of H I F - l a and V E G F in cancer cells can be mediated by autocrine or chronic stimulation by growth factors such as IGF-1 , constitutive activation of PI-3 Kinase, or the constitutive activation of P K B / A k t due to the inactivation of the tumor suppressor, P T E N (Brazil et al., 2002; Galetic et al., 1999). Because I L K is PI-3 Kinase dependent and an upstream target of A k t / P K B , and because an increase in I L K expression is positively correlated with prostate carcinoma grade (Graff et al., 2001), I L K was a likely candidate to be involved in the regulation of V E G F and H I F - l a expression through A k t / P K B activity regulation. In addition, the regulation of H I F - l a translational rate has been shown to be through the regulation of m T O R / F R A P , a downstream target of A k t / P K B (Fukuda et al., 2002). In this paper we have shown that in human prostate cancer cells, I L K is essential for the regulation of H I F - l a expression and the consequent production of V E G F . Functional inactivation of I L K by exposure to a highly selective chemical inhibitor, or stable or transient transfection of the ILK-dominant negative construct into cell models with high I L K activity, result in a decrease in H I F - l a protein levels and V E G F expression. Furthermore, depletion of I L K protein by s i R N A in PC3 cells effectively 102 decreases A k t / P K B and m T O R / F R A P phosphorylation, H I F - l a levels and V E G F expression. These data suggest that in certain cancer cells, such as prostate carcinoma cells, I L K plays a crucial role in H I F - l a and V E G F expression via activation of P K B / A k t and phosphorylation of m T O R / F R A P (Figure 2.7). V E G F gene transcription can also be stimulated by the transcription factors AP-1 and N F - K B (Harris, 2002). Since I L K has also been shown to regulate the activities of both of these transcription factors (Troussard et al, 2000; Tan et al, 2002), it is possible that in certain cell types I L K could regulate V E G F expression via signaling pathways that activate these other transcription factors. Although in this study we have not ruled out the contribution of AP-1 and N F - K B in the ILK-mediated regulation of V E G F expression in the prostate cancer cell lines examined, the data presented here support a significant role of H I F - l a in the I L K regulation of V E G F expression. This is particularly true for the PC3 cells in which inhibition of I L K expression or activity results in almost complete suppression of both H I F - l a and V E G F expression. Furthermore, in the PTEN-nu l l PC3 cells in which the PI-3 Kinase pathway and I L K are constitutively upregulated, transfection of dominant-negative H I F - l a results in substantial inhibition of V E G F expression It is interesting to note that I L K m R N A has been shown to be upregulated by hypoxia (Scandurro et al, 2001; Grimshaw et al, 2001), suggesting that I L K may also play a role in hypoxia induced V E G F expression. Thus the role of I L K in hypoxic tumors with constitutive activation of PI-3 Kinase may be quite substantial. 103 Prostate Cancer Cell Integrin Growth Factor ILK * < * I Akt/PKB-P-serine473 \ mTOR/FRAP-P-serine-2448 \ HIF-1 oc ^ Protein HIF-1 p VEGF Protein , t VEGF Gene Endothelial Cell • •VEGF i ILK Activity t \ Migration Invasion Tubulogenesis Angiogenesis Figure 2.7 Schematic representation of the cell signaling events leading to V E G F production in a prostate carcinoma cell and consequent effects on a neighboring endothelial cell. Shown is our model for the production of V E G F in prostate carcinoma cell and the effects of V E G F on endothelial cell function. Phosphorylation of serine-473 of A k t / P K B by activated I L K in prostate cancer cells results in the full activation of P K B / A k t that promotes the phosphorylation of serine-2448 of m T O R / F R A P . This activates m T O R / F R A P , which increases the levels of H I F - l a protein translation. H I F - l a protein combines with HIF-1 p to form an active transcription factor. This heterodimer binds to the V E G F promoter and activates V E G F transcription, expression and secretion. V E G F binds to its receptor and stimulates I L K activity. I L K regulates downstream targets involved in cell survival, proliferation, invasion and migration. 104 We have also shown here that I L K plays an essential role in VEGF-stimulated endothelial cell-mediated blood vessel formation in vitro and in vivo (Figure 2.6). Migration and proliferation of human endothelial cells in response to V E G F is inhibited upon inhibition of I L K activity or expression. Furthermore the ability of V E G F stimulated endothelial cells to form capillary-like structures in vitro is also severely inhibited by inhibiting I L K activity. This inhibition appears to be due primarily due to inhibition of cell migration and proliferation, both of which can be regulated by I L K in response to growth factors or engagement of integrins (Wu and Dedhar, 2001; Cruet-Hennequart et al, 2003). The inhibition of angiogenesis in vivo in the C A M assay by inhibition of I L K suggests that the primary effect of I L K inhibition is on endothelial cell migration and ability to form vessels, as there did not appear to be any obvious cell lysis in these assays. This agrees with our finding that H U V E C cell survival appears not to be affected as significantly as cell migration and proliferation upon I L K inhibition. Recent evidence from the systemic and targeted knockout of I L K suggests an important role of I L K in cell adhesion and actin accumulation (Sakai et al, 2003), processes crucial for cell morphogenesis and migration, as well as in cell proliferation (Tepstra et al, 2003) We have also shown here that inhibition of I L K with a highly selective I L K inhibitor results in the statistically significant suppression of tumor angiogenesis as well as tumor growth in a mouse xenograft model of PC3 tumor growth in SCID mice. These data suggest that inhibitors of I L K activity may be considered as angiogenesis inhibitors effective for the suppression of tumor angiogenesis. The integrins avp3 , avPs and OC5P1 have also been shown to be crucial regulators of endothelial cell function during angiogenesis (Hood and Cheresh, 2002; Eliceir i and 105 Cheresh, 2001; Friedlander et al., 1995). Furthermore, angiogenesis inhibitors such as endostatin and tumstatin have been shown to function by inhibiting integrin function and signalling (Maeshima et al, 2002). Tumstatin has been shown to inhibit endothelial cell survival by binding to otvp3 and inhibiting ctvP3-metiated signalling to P K B / A k t (Maeshima et a l , 2002). Since I L K is also regulated by integrins and since I L K is involved in avp3-regualed cell proliferation (Cruet-Hennequart et al., 2003), as well as in regulating anoikis (Attwell et al., 2000), it is likely that I L K also plays an important role in integrin-mediated endothelial cell function during angiogenesis. We have recently created transgenic mice in which the I L K gene is flanked by Lox-P sites, and have demonstrated that cells from these mice can be used to conditionally knockout I L K expression in cells isolated from these mice (Troussard et al., 2003). 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Chem. 277,3109-3116. Troussard, A . A . , Costello, P., Yoganathan, T . N . , Kumagai, S., Roskelley, C D . , and Dedhar S. (2000). The integrin linked kinase ( ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 ( M M P -9). Oncogene 79,5444-5452. Troussard, A . A . , Mawji , N . M . , Ong, C , M u i , A . , St. Arnaud, R., and Dedhar, S. (2003). Conditional knock-out of integrin-linked kinase ( ILK) demonstrates and essential role in P K B / A k t activation. J. B i o l . Chem. 278, 22374-22378. Wu, C , and Dedhar, S. (2001). Integrin-linked kinase ( ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. J. Cel l . B i o l . 155, 505-510. Zhong, H . , Chile, K . , Feldser, D . , Laughner, E . , Hanranhan, C , Georgescu, M . M . , Simons, J.W., and Semenza, G . L . (2000). Modulation of hypoxia-inducible factor 1 alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/ P T E N / A K T / F R A P pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 60, 1541-1545. Ill 3 Integrin-linked kinase regulates inducible nitric oxide synthase and cyclooxygenase-2 expression in an NF-xB-dependent manner2 3.1 Summary Nitric oxide (NO) and prostaglandins are produced as a result of the stimulation of inducible nitric oxide synthase ( iNOS) and cyclooxygenase (COX-2) respectively in response to cytokines or lipopolysaccharide (LPS). We demonstrate that the activity of integrin-linked kinase ( ILK) is stimulated by L P S activation in J774 macrophages. Inhibition of I L K activity by dominant-negative I L K or a highly selective small molecule I L K inhibitor, in epithelial cells or L P S stimulated J774 cells and murine macrophages, resulted in inhibition of i N O S expression and N O synthesis. L P S stimulates the phosphorylation of I K B on serine-32 and promotes its degradation. Inhibition of I L K suppressed this L P S stimulated I K B phosphorylation and degradation. Similarly, I L K inhibition suppressed the L P S stimulated iNOS promoter activity. Mutation of the N F - K B sites in the i N O S promoter abolished L P S and I L K mediated regulation of i N O S promoter activity. Overexpression of I L K stimulated N F - K B activity, and inhibition of I L K or protein kinase B (PKB/Ak t ) suppressed this activation. We conclude that I L K can regulate N O production in macrophages by regulating i N O S expression through a pathway involving P K B / A k t and N F - K B . Furthermore we also demonstrate that I L K activity is required for L P S stimulated C O X - 2 expression in murine and human 2 Tan C, Mui A and Dedhar S. (2002) Integrin-linked kinase regulates Inducible Nitric Oxide Synthase and Cycooxygenase-2 expression in an NF-kB-dependent Manner. J. Biol Chem. 277 (5), pp. 3109 -3116. All the work in this chapter was carried out by me. 112 macrophages. These findings implicate I L K is a potential target for anti-inflammatory applications. 3.2 Introductions Mouse macrophages express an inducible form of nitric oxide synthase ( iNOS), which catalyzes the production of nitric oxide (NO) from L-arginine. Macrophage-derived N O is important for host defense, microbial and tumour cell ki l l ing (reviewed in MacMick ing et al., 1997). Activating stimuli such as lipopolysaccharide (LPS) (Alley et al., 1995) other bacterial cell wall products (Brightbill et al., 1999) and cytokines such as interferon (IFN-gamma) (Gao et al., 1997) all stimulate i N O S expression in induced macrophages. However since excess production of N O results in inappropriate tissue injury and septic shock, i N O S expression is subject to stringent regulatory control. The mouse i NOS promoter has been extensively studied and consists of two clusters of regulatory elements (Xie et al., 1994; Lowenstein et al., 1993). A proximal region (region I or RI, -48 to -209) functions as the basal promoter containing an octamer element and a N F - K B binding site, which mediates responsiveness to L P S . The distal region (RII, -913 to -1029) functions as an enhancer element and responds to L P S and IFN-gamma through N F - K B , IRF-1, ISRE, and G A S binding sites. The N F - K B sites are essential for LPS-mediated N O production (Xie et al., 1994). Protein kinase B , (PKB/Akt ) has been shown to phosphorylate and activate the I K B kinase ( I K K ) (Ozes et al., 1999; Romashkova and Makarov, 1999), which in turn phosphorylates I K B . Phosphorylated I K B is targeted for ubiquitin-mediated degradation, 113 thus releasing active N F - K B and allowing its translocation into the nucleus (Baeuerle and Baltimore, 1996). Integrin-linked kinase ( ILK) is an ankyrin-repeat containing serine/threonine protein kinase that can interact with the cytoplasmic domain of the p i integrin and regulates integrin dependent functions (Dedhar, 2000; Zervas et al., 2001). It has been demonstrated to regulate the activity of transcription factors such as P -catenin-TCF/LEF-1 (Novak et al., 1998; Tan et a l , 2001), AP-1 (Troussard et al., 1999), and C R E B (D 'Amico et al., 2000). I L K activity is regulated in a PI 3-Kinase dependent manner (Delcommenne et al., 1998; Persad et a l , 2000; Morimoto et al., 2000; Persad et al., 2001) and I L K can regulate the phosphorylation and activation of P K B / A k t (Delcommenne et al., 1998; Persad et al., 2000; Morimoto et a l , 2000; Persad et al., 2001). Since the transcription factor, N F - K B has been shown to be activated by P K B / A k t , which is known to lead to activation of i N O S in mice (Xie et al., 1994), we wanted to determine whether I L K could also regulate N F - K B activity. In order to examine a physiologically relevant system for the regulation of N F - K B by I L K , we examined the role of I L K in lipopolysaccharide (LPS) stimulated expression of i N O S and N O . We found that I L K is an upstream regulator of LPS-mediated phosphorylation of I K B , and of NF-KB-dependent expression of i N O S . Mouse and human macrophages have different i N O S promoters (Zhang et al., 1996). In order to determine a similar role of N F - K B in human macrophages, we analyzed the expression of cyclooxygenase-2 (COX-2) , a protein that regulates the production of proinflammatory prostaglandins by catalyzing arachidonic acid into prostaglandins (Smith et al, 2000; Will iams et al., 1999). There are two isoforms of 114 C O X , C O X - 1 and C O X - 2 , which are products of two different genes. C O X - 1 is constitutively expressed in most tissues and is a housekeeping gene (Funk et al, 1991). C O X - 2 is not detectable in most normal tissues or resting immune cells, but cytokines, growth factors and endotoxins can induce its expression (Hempel et al., 1994; Riese et al., 1994; Mestre et al., 2001). The role of N F - K B has been demonstrated to be important in mouse and human macrophage/monocytic cells in the induction of C O X - 2 (Mestre et al., 2001; Lee et al., 2001; Chen et al., 2000; Chen et al., 2001; Allport et al., 2001; Allport et al., 2000; L u k i w et al.,1998). The AP-1 and C R E B transcription factors, in addition to N F - K B , have been demonstrated to be important in the regulation of C O X - 2 expression (Von Knethen et al., 1999; Ogasawara et al., 2001). We demonstrate here that in addition to regulating i N O S gene expression in an NF-KB-dependent manner, I L K activity is also required for LPS-mediated C O X - 2 expression in murine and human macrophages. 3.3 Materials and Methods 3.3.1 Cell Lines and Cell Culture Rat intestinal epithelial cells, IEC-18 were obtained from American Type Culture Collections ( A T C C ) . IEC-18 cell clones (ILK-13 Ala3 andAlC3) stably overexpressing wild-type sense I L K c D N A , ILK-14 cells stably expressing antisense I L K c D N A , J L K -K D , kinase-deficient c D N A were all prepared as described previously (Novak et al., 1998; Hannigan et al., 1996). IEC-18 cells and stably transfected cell clones were routinely cultured in a-minimal essential medium ( a - M E M ) (Gibco B R L ) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco B R L ) , glucose (3.6 mg/ml) 115 and insulin (10 u,g/ml). Stably transfected derivatives were grown in the presence of G418 (80 u-g/mL) to maintain selection pressure. Mouse monocyte-macrophage cells J774.1 ( A T C C ) were cultured in Dulbecco's modified Eagle's medium ( D M E M ) supplemented with 10% heat-inactivated F B S . Primary mouse macrophages were a gift from Dr. Urs Steinbrecher's lab (University of British Columbia, Vancouver, Canada), and they were isolated as described by Hamilton et al. (38-40). Human monocyte-derived macrophages were a gift from Dr. Anthony Chow's lab (University of British Columbia, Vancouver, Canada), and they were isolated as described by Sly et al. (Sly et al., 2001; L i u et al., 1994). A l l cells were grown at 37 °C, in a 99 % humidified atmosphere of 5 % CO2 in air. Bone marrow cells were isolated from femurs and plated in tissue culture plates for 2 hours at 37 ° C in 100 ng/ml CSF-1 in 10 % FBS/Iscove's modified Dulbecco's media supplemented with 30 % conditioned media from cells producing the pZIP-Tex virus (Brunet et al., 1999). The nonadherent cells were then removed and placed in fresh tissue culture plates and allowed to differentiate into macrophages. After several passages over weeks, 100 % of the cells were Mac-1-positive. 3.3.2 Transfection Cells were seeded into 6-well dishes 24 hours prior to transfection such that they would be approximately 60 % confluent on the day of transfection. Transfection of IEC-18 cells were carried out by using Lipofectin (Gibco B R L ) according to the manufacturer's guidelines, and 2 to 3 u.g of plasmid per well o f a 6-well dish. Transfection of J774 cells was carried out by using L ipofec tAMINE (Gibco B R L ) according to manufacturers instructions, and 2 to 3 u.g of plasmid per well as described by Pierce et al., 1996. 116 Transfections were carried out for 4 to 6 hours in O p t i - M E M (Gibco B R L ) medium. The transfection medium was then replaced by serum-containing medium for 6 hours prior to the beginning of an experiment. 3.3.3 In Vitro Kinase assay I L K kinase assays were carried out as previously described (Delcommenne et al., 1998; Hannigan et al., 1996). The precipitates were washed and the reactions were carried as described by Hannigan et al.,1996. Mye l in basic protein ( M B P ) was used as a substrate for I L K kinase activity. Phosphorylated M B P was resolved by 15% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( S D S - P A G E ) , and visualized by autoradiography. The I L K inhibitor, KP-392, was obtained from Kinetek Pharmaceuticals and was used as previously described (Tan et al., 2001; Persad et al., 2000; Persad et a l , 2001; Troussard et a l , 2000; Persad et al., 2001). A l l experiments were done with equivalent amounts of vehicle, D M S O . 3.3.4 Western Blot analysis Cells were harvested in Nonident P-40 lysis buffer and stored at -70°C. Protein concentrations were measured using a BioRad Bradford protein assay kit. Equivalent amounts of protein were resolved in S D S - P A G E , transferred on to P V D F (Immobilion-P Millipore) membranes and probed with antibody. The protein of interest was visualized with enhanced chemiluminescent ( E C L ) (Amersham) reagents. The following antibodies were used in the experiments: anti-ILK (Upstate Biotechnology Inc), anti-lKB and anti-phosphoserine-32 I K B (New England Biolabs), anti-Akt and anti-phosphoserine-473 Ak t 117 (New England Biolabs), anti-iNOS (New England Biolab), ant i-COX-2 (Transduction Labs) and anti-(3-Actin (Transduction Labs). 3.3.5 Luciferase Assays 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 a modified luciferase activity (pRenilla; dual luciferase; Promega). Triplicate samples were assayed for each trial o f each condition in these experiments. 3.3.6 Detection of Nitric Oxide Lipopolysaccharide (LPS) (Escherichia Co l i 055:b5) (Difco (Detroit, M l ) ) was added at the concentrations indicated or at 500 ng/ml for 24 hour experiments and at 1 ug/ml for 1 hour experiments. Supernatants were removed after incubation times of 24 hours, and nitric oxide concentrations were determined as described by O'Farrell et al (46, 47). Triplicate samples were assayed for each trial of each condition in these experiment. 3.3-7 Plasmids The I L K wild-type and dominant negative plasmids are described in the following articles (Delcommenne et al., 1998; Persad et a l , 2000; Persad et al., 2001; Persad et al., 2000). The N F - K B response element promoter conjugated to a luciferase reporter was a kind gift from Dr. Nathan Yoganathan's lab (Kinetek Pharmaceuticals, Vancouver, Canada). The i NOS promoters were a kind gift from Dr. W .J . Murphy's lab (University of Kansas Medical Center, Kansas City, U S A ) . 118 3.3.8 Immunohistochemistry Human biopsy specimens were fixed in paraformaldehyde and paraffin (Sigma). 5 inn sections were prepared and the sections were placed on Silane (Sigma) coated slides. Conventional deparaffinization and rehydration techniques were employed. The sections were heat-retrieved treated in a p H 5 acetate buffer at 95°C for 10 minutes. They were treated with hydrogen peroxide and Triton X-100 buffer (Sigma), before blocking with 5 % milk. The sections were then incubated with I L K antibody (1/250 dilution) (Upstate Biotechnologies) in 5 % milk overnight at 4°C. The control staining was visualized with strepavidin secondary antibody (Jackson) and D A B (Sigma). Haematoxylin (Sigma) counterstaining was done by soaking the slides in the dye for 2 minutes, then washing with distilled water. Slides were mounted with Permount (Sigma). Micrographs were generated using the N i k o n Eclipse TE300 microscope and N i k o n D I digital camera. 119 3.4 Results 3.4.1 I L K upregulates N F - K B activity In order to determine i f I L K regulates N F - K B activity, reporter assays were performed. These experiments were carried out by transfecting the N F - K B response-element promoter coupled to a luciferase reporter, into IEC-18 intestinal epithelial cells and stably transfected clones overexpressing sense or anti-sense I L K previously characterized (Novak et al., 1998; Delcommenne et al., 1998). Overexpression of wi ld type I L K , but not kinase-deficient mutant or anti-sense I L K , stimulates N F - K B response-element promoter activity. A s shown in Figure 3.1 A , N F - K B activity is substantially higher in two independent I L K overexpressing cell lines, relative to the control cell lines. Transfection of kinase deficient-dominant negative I L K c D N A (Figure 3.IB), as well as incubation with highly selective, small molecule inhibitor of I L K (KP-392) (Tan et al., 2001; Persad et al., 2000; Persad et al., 2001; Troussard et al., 2000; Persad et al., 2000) (Figure 2.1D), both inhibit N F - K B response element promoter activity in the I L K overexpressing IEC-18 clone, in a dose-dependent manner. This indicates that the observed stimulation of N F -K B reporter activity in this cell line is ILK-dependent. Furthermore, transfection of the I L K overexpressing cells with a potent dominant-negative P K B / A k t c D N A ( P K B - A A A ) (Delcommenne et al., 1998; Persad et al., 2000), also resulted in the inhibition of N F - K B activity, suggesting that the I L K induced stimulation of N F - K B in these cells involved P K B / A k t (Figure 3.IC). 120 CD NF-KB Activity Relative RLU (%) o cn o o o o ro o < NF-KB Activity Relative RLU (%) _ o 00 o o > 3 o NF-KB Activity Relative RLU (%) 00 o ro NF-KB Activity Relative RLU (%) 7 ro 5T cn o W CD 3 cn CD > 3 CD X CO 3D m CO w C/> „ m -* -a p 3 CO co CO o < CD O < ro o o cn O ro o o ro cn o CO o o CO A cn o o o ft cn O O O D Figure 3.1 (A) ILK upregulates N F - K B activity. IEC-18 cells and stable clones were transfected with the N F - K B response element (pGL3NF-KB response element reporter with a luciferase reporter (^)) or a control plasmid (pGL3Basic, no promoter with a luciferase reporter (•)). 48 hours post-transfection, cells were harvested and assessed for luciferase activity. Samples were normalized with pRenilla and activity is expressed as relative RLU. Data are mean +/- standard deviation of six independent trials. (B) Kinase-dead ILK decreases N F - K B activity in epithelial cells in a dose-dependent manner. ILK wild-type, overexpressing IEC-18 cells (clone Ala3 ILK-13) were co-transfected with increasing amounts of ILK(KD):V5, and the total amount of plasmid was kept constant by supplementing with appropriate amounts of Empty:V5, and 2 ug of pGL3NF-KB response element promoter. After 48 hours, cells were assayed for luciferase activity. All samples were normalized with pRenilla. Data are mean +/- standard deviation of four independent trials. Increased expression of ILK (KD):V5 was monitored by Western blot using anti-V5 antibody. ( C ) Transfection of dominant-negative PKB/Akt inhibits N F - K B activity in epithelial cells in a dose-dependent manner. ILK wild-type, overexpressing IEC-18 cells (clone Ala3 ILK-13) were co-transfected with increasing amounts of dominant-negative PKB/Akt (AAA):HA and pGL3NF-KB response element promoter. After 48 hours, cells were assayed for luciferase activity. All samples were normalized with pRenilla. Data are mean +/- standard deviation of four independent trials. Increasing PKB/Akt (AAA):HA protein expression was monitored with Western blot using an anti-HA anitbody. (D) Pharmacological inhibition of ILK with ILK inhibitor KP-392 decreases N F - K B activity in a dose-dependent manner. ILK-13 IEC-18 cells were transfected with equivalent amounts of either pGL3NF-KB or pGL3Basic and pRenilla. Cells were incubated with complete media for 6 hours post-transfection. Cells were then treated with increasing concentrations of KP-392 for an additional 24 hours. Cells assessed for luciferase activity. All samples were normalized with pRenilla. Data are mean +/- standard deviation of three independent trials. 122 3.4.2 LPS stimulates NO production in an ILK dependent manner We next wanted to determine the physiological relevance of the ILK-mediated stimulation of N F - K B . Since L P S is known to stimulate iNOS expression and N O production in murine macrophages in an NF-KB-dependent manner, we examined the role of I L K in this pathway. A s shown in Figure 3.2A, L P S stimulates both N O production and N F - K B activity in the J774 macrophage cell line. We next wanted to examine whether L P S had any effect on I L K kinase activity in the cell line. A s shown in Figure 3.2B, I L K kinase activity is rapidly and transiently stimulated by L P S , as is the phosphorylation of A k t / P K B on serine-473 and IKB on serine-32. The subsequent degradation of IKB as shown in Figure 2.2B allows release and translocation of N F - K B to translocate to the nucleus. There is no significant change in I L K and A k t / P K B protein levels, within the time course of activation of I L K and A k t / P K B phosphorylation (1 hour). 123 B 0 10 20 30 60 L P S Exposure (min) I L K kinase activity w w « * w w I L K ^ mmm mm —- — Akt Phospho-473 - — — I — - I K B Phospho-32 * - « • . I K B Figure 3.2 (A) L P S stimulates the production of nitric oxide in J774 cells and upregulates the N F - K B activity. J774 cells were transfected with equivalent amounts of pGL3NF-KB ( H ) and pRenilla. After transfection, cells were incubated in complete media for 6 hours. Cells were then stimulated with increasing amounts of LPS in complete media for an additional 24 hours. After 24 hours, nitric oxide production was measured according to the Greiss method and cells were harvested, lysed and assessed for luciferase activity. Data are mean +/- standard deviation of six independent samples. ( B ) L P S stimulates I L K activity and I K B serine-32 phosphorylation. Cells were exposed to 1 Lig/ml of LPS for 60 minutes. ILK kinase activity was determined in J774. AKT serine-473 and IKB serine-32. 124 3.4.3 Inhibition of ILK suppresses LPS stimulated iNOS expression and NO production in J774 cells and in primary murine macrophages In order to determine whether the L P S stimulated N O production is I L K -dependent, we exposed J774 cells to L P S and increasing doses of a recently identified, highly selective ILK-inhibitor, KP-392 (Tan et al., 2001; Persad et al., 2000; Troussard et al., 2000; Persad et al., 2001). We observed a parallel dose-dependent inhibition of N O production and i NOS expression (Figure 3.3A). A s can be seen in Figure 3.3A, incubation with the KP-392 also inhibits LPS-stimulated I L K activity in these cells in a dose-dependent manner. To determine i f this effect of I L K inhibitor on LPS-stimulated N O production could be demonstrated in primary murine macrophages, murine monocytes were differentiated into macrophages using macrophage-colony stimulating factor ( M - C S F ) as described in "Materials and Methods". The macrophages were maintained for 24 hours without M - C S F prior to the experiment. The macrophages were then exposed to L P S in the presence of increasing amounts of I L K inhibitor (KP-392). A s shown in Figure 3.3B, the I L K inhibitor decreased LPS-stimulated N O production, in a similar manner to that observed in the J774 cell line after 24 hours. 125 Figure 3.3 126 Figure 3.3 (A) Inhibition of I L K kinase activity decreases iNOS expression, J774 cells were incubated with increasing concentrations of KP-392 and 500 ng/ml of L P S for 24 hours in 5 % serum. Nitric oxide production was measured according to the Greiss method. Cells were harvested and inducible nitric oxide synthase ( iNOS) expression was measured by Western blotting. Parallel experiments of I L K kinase activity was measured as described in materials and methods. Data are mean +/- standard deviation of four independent trials. (B) Inhibition of I L K kinase activity decreases iNOS expression. J774 cell clones were created and incubated with 500ng/ml of L P S . After 24 hours, nitric oxide production was assessed by the Greiss method. Data are mean +/- standard deviation of three independent trials. 127 ^^5^  t^ lCJ J^ t^ J^ ' '•^ (H^ -. ^fJ/K^ffi Serum + + + + + LPS - + + + + KP-392 (|uM) 0 0 10 50 100 Primary Mouse Macrophage Figure 3.3 128 Figure 3.3 (C) Inhibition of I L K kinase activity decreases iNOS expression. (J774 cells were incubated with increasing concentrations of KP-392 and 500 ng/ml of L P S for 24 hours in 5 % serum. Nitric oxide production was measured according to the Greiss method. Cells were harvested and inducible nitric oxide synthase ( iNOS) expression was measured by Western blotting. Parallel experiments of I L K kinase activity was measured as described in materials and methods. Data are mean +/- standard deviation of four independent trials. (D) Inhibition of LPS-stimulated NO production in primary mouse macrophages by KP-392. Primary mouse macrophages were incubated with 500ng/ml of L P S and increasing amounts of KP-392 as indicated. After 24 hours, nitric oxide production was assessed by the Greiss method. Data are mean +/- standard deviation of three independent trials. 129 3.4.4 Inhibition of ILK s u p p r e s s e s L P S st imulated N F - K B e x p r e s s i o n a n d N F - K B d e p e n d e n t i N O S g e n e e x p r e s s i o n In order to examine the mechanism of the effect of I L K on N O production, we first determined whether inhibition of I L K would also suppress LPS-stimulated N F - K B transcription. J774 cells were co-transfected with increasing amounts of the dominant-negative I L K - K D : V 5 plasmid and the N F - K B response element promoter. A s can be seen from Figure 3.4A, N F - K B activity is inhibited in a dose-dependent manner by increased expression of the dominant negative I L K . It is well known that macrophages have extremely low transfection efficiency (Mack et al., 1998; Stacey et al., 1993; Pierce et a l , 1996; Feng et al., 2000; Mijarovic et al., 1997), therefore, we were unable to detect the levels of I L K - K D expression on a western blot. Therefore, J774 cells were transfected with the N F - K B response element promoter-reporter constructs and reporter activity was measured in L P S stimulated J774 cells exposed to increasing concentrations of KP-392 I L K inhibitor. A s shown in Figure 3.4B, inhibition of I L K decreases L P S -stimulated N F - K B response element promoter activity. The mouse i N O S promoter possesses two N F - K B sites, which have been shown to be essential for LPS-mediated N O production. To determine whether LPS-stimulated i N O S expression could also be inhibited by the I L K inhibitor, and whether this I L K dependent iNOS expression was dependent on N F - K B , the J774 cells were transfected with either the full length, wi ld type iN O S promoter (Xie et al., 1994), or the i N O S promoter with point mutations in the N F -K B sites (Xie et al., 1994). The cells were then stimulated with L P S and treated with increasing concentrations of KP-392 I L K inhibitor. L P S stimulated the full length iNOS promoter but only showed minimal stimulation of i N O S promoter activity containing 130 point mutations in the N F - K B sites (Figure 3.4C). In addition, the KP-392 I L K inhibitor inhibited the LPS-stimulated i NOS promoter activity in a dose-dependent manner, similar to that observed with the N F - K B response element promoter (Figure 3.4C). 131 St D > _J o CC < 0 CO | y ra LL CD B III LPS - + + + + + ILK(KD):V5 (ug) 0 0 0.5 1.0 1.5 0 Empty:V5(ug) 1.5 1.5 1.0 0.5 0 1.5 N F - K B response promoter + + + + + -pGL3Basic - - - - - + 6 n > - 1 i < ® CO ~ X _ -7 EC LPS -KP-392 (uM) 0 hi + + + + 0 10 50 100 0 L P S - + + + + + iNOS promoter full length + - + + + + iNOS promoter mutant - + - -KP-392 (nM) 0 0 0 10 50 100 Figure 3.4 (A) Kinase-dead I L K decreases N F - K B activity in J774 macrophage ceils in a dose-dependent manner. J774 macrophages cells were co-transfected with increasing amounts of I L K ( K D ) : V 5 , and the total amount of plasmid was kept constant by supplementing with appropriate amounts of Empty:V5, and 2 ug of p G L 3 N F - K B response element promoter. Cells were incubated with 500ng/ml of L P S and assayed for luciferase activity 24 hours later. A l l samples were normalized with pRenilla. Data are mean +/- standard deviation of four independent trials. ( B ) KP-392 decreases N F - K B activity in a dose-dependent manner. Cells were transfected with equivalent amounts of p G L 3 N F - K B and pRenilla. After transfection, cells were incubated with 500 ng/ml of L P S and increasing amounts of KP-392 and total mount of drug vehicle was kept constant by the relevant supplementary addition of D M S O for 24 hours, diluted in D M E M with 5% F B S . Cells were harvested and assessed for luciferase activity. Data are mean +/- standard deviation of six independent trials. (C) I L K regulates iNOS transcription in an N F - K B dependent manner. J774 cells were transfected with the indicated plasmids (full-length wild-type murine iNOS promoter or double mutant N F -K B binding sites murine iNOS promoter). Cells were then incubated with 500 ng/ml of L P S and increasing concentrations of KP-392. After 24 hours, cells were harvested and assessed for luciferase activity. Data are mean +/- standard deviation of four independent trials. 132 3.4.5 Inhibition of I L K suppresses IKB serine 3 2 phosphorylation and prevents its degradation To gain further insight into the mechanism of the I L K induced regulation of N F -K B , we examined the effects of inhibition of I L K activity on I K B phosphorylation and degradation. A s shown in Figure 3.5, L P S treatment of J774 cells results in a stimulation of phosphorylation Of I K B on serine-32, the site for ubiquitin-mediated degradation of I K B (Baeuerle and Baltimore, 1996). A s can be seen in Figure 3.5, L P S treatment also leads to degradation of I K B , which correlates with its phosphorylation on serine-32. Exposure of cells to 50 um K P - 3 9 2 I L K inhibitor for 1 hour prior to L P S stimulation markedly inhibits I K B phosphorylation, thus stabilizing and preventing its degradation (Figure 3.5), and preventing subsequent N F - K B activation by retaining N F - K B in the cytoplasm. 133 LPS only KP-392 and LPS Minutes 5 10 30 60 5 10 30 60 I K B Phospho-32 ILK mm mmm ^, mm mm mm mm mm «BP ww0* ^PPP** Figure 3.5 Inhibition of I L K suppresses I K B serine-32 phosphorylation and prevents its degradation. J774 cells were treated with L P S alone or with L P S and K P -392. I K B and I K B serine-32 phosphorylation and protein levels were determined by Western blot analysis in J774 cells exposed to KP-392 (50 uM) for one hour prior to exposure to L P S (1 u,g/ml) and KP-392 (50 u M ) for the indicated times. 134 3.4.6 Inhibition of ILK suppresses LPS stimulated COX-2 expression in J774 cells and human macrophages The regulation of i NOS production differs between human and mouse macrophages, in large part due to the differences between the human and murine i N O S promoters. It is known that the induction of another pro-inflammatory protein, C O X - 2 , by L P S stimulation in mouse macrophages also involves the activation of N F - K B (D'Acquisto et al., 1997; Abate et al., 1998; DAcquis to et al., 2000; Paik et al., 2000). Recent publications have also identified N F - K B as a major regulator of C O X - 2 expression in humans (Chen et al., 2000; Macao et al., 2000; Koj ima et al., 2000). Therefore, to investigate whether the I L K inhibitor is effective in decreasing inflammatory responses and regulating N F - K B activity in humans, we analysed LPS-stimulated cyclooxygenase-2 (COX-2) expression. We first determined i f I L K was present in human peripheral macrophages. A s can be observed in Figure 3.6, I L K expression is readily detectable in human alveolar macrophages. We next incubated J774 cells and human monocyte derived macrophages with L P S and increasing amounts of KP-392. A s shown in Figure 3.7A and 3.7B, L P S stimulated C O X - 2 is inhibited in a dose-dependent manner with increasing amounts of I L K inhibitor, in both mouse and human macrophages. 135 Figure 3.6 Expression of Integrin Linked kinase in alveolar macrophages. (A) Rabbit IgG control staining on lung biopsy. Human lung biopsy were fixed with paraformaldehyde and imbedded in paraffin. The paraffin blocks were sliced into 5 urn thick sections and stained with haematoxylin and rabbit IgG antibody at the same concentration as the I L K antibody. Staining was carried out as indicated in "Materials and Methods". (B) I L K staining on lung biopsy. Section from the same block of tissue was stained with I L K (Upstate Biotechnologies) antibody. 136 A - + + + + + L P S 0 0 10 25 50 100 KP-392 (JLIM) Mouse C O X - 2 Mouse (3-Actin ILK B - + + L P S 0 0 100 K P - 3 9 2 (LIM) Human C O X - 2 Human C O X - 1 Human p-Actin ILK Figure 3.7 (A) Inhibition of I L K suppresses C O X - 2 expression in J774 cells. Cells were incubated with L P S (500 ng/mL) and indicated amounts of KP-392 for 24 hours. C O X - 2 expression was measured by Western blot. (B) Inhibition of I L K suppresses C O X - 2 expression in human peripheral macrophages. Cells were incubated with L P S (500 ng/mL) and indicated amounts o f KP-392 for 24 hours. C O X - 2 expression was measured by Western blot. 137 3.5 Discussion The expression of the inducible form of nitric oxide synthase ( iNOS), which catalyzes the production of nitric oxide (NO) from L-Arginine, is regulated by the transcription factor N F - K B in murine macrophages in response to L P S and cytokines (MacMicking et al., 1997). Macrophage-derived N O is an important host defense, microbial and tumour cell ki l l ing agent (MacMicking et al., 1997), as well as a regulator of pro-inflammatory genes in vivo. The ability to modulate i N O S expression could potentially control chronic and acute inflammatory diseases; therefore, it is important to understand the regulation of i N O S . In this paper we have provided novel data indicating that the integrin-linked kinase ( ILK) , which couples integrins and growth factors to downstream signalling pathways (Dedhar, 2000), can regulate i N O S expression and N O production in murine macrophages, and regulate the expression of a pro-inflammatory protein, C O X - 2 , in both murine and human macrophages. I L K is a PI 3 kinase-dependent kinase (Delcommenne et al., 1998; Persad et al., 2000; Morimoto et al., 2000; Wangle et al., 1999) capable of regulating the phosphorylation and activation of P K B / A k t , which has recently been shown to regulate N F - K B activation by activating I K B kinase (Ozes et al., 1999; Romashkova and Makarov, 1999). Here we have shown that I L K can also regulate N F -K B activation in a physiologically relevant system. Our data indicates that I L K activity is rapidly stimulated in response to L P S in murine macrophages, and results in the phosphorylation and degradation of I K B . Importantly, we have shown here that inhibition of I L K using a specific small molecule inhibitor or dominant-negative form of I L K , results in the inhibition of I K B phosphorylation on serine 32 and the prevention of 138 its degradation. In addition, I L K inhibition suppresses L P S stimulated N F - K B promoter activity as well as i N O S promoter activity and N O production. The inhibition of I L K also inhibits i N O S protein expression. The precise mechanism involved in the regulation of I K B phosphorylation by I L K is not yet clear and is under investigation. A possible mechanism is that I L K is a critical upstream mediator of N F - K B activation through its capacity to regulate P K B / A k t kinase activity by phosphorylating serine-473 (Persad et al., 2000). In this paper we demonstrated that ILK-stimulated N F - K B activity is inhibited by dominant-negative P K B / A k t . This implicates P K B / A k t in I L K ' s regulation of N F - K B . Our data further suggest that I L K may play a pivotal role in the regulation of N O production by coupling integrin and L P S signalling. It has been shown that N O production is significantly stimulated in the presence of integrin binding R G D peptide (Muller et al., 1997; Attur et a l , 2000). Furthermore, ligation of a5(31 integrin with a specific antibody stimulates N O production (Muller et al., 1997; Attur et al., 2000; Schwartz et al., 2001). Since I L K can interact directly with the cytoplasmic domains of integrin p i and P3 subunits, and can couple integrins to the actin cytoskeleton and downstream signalling components such as A k t / P K B (Dedhar, 2000), it is likely that the integrin-mediated stimulation of N O involves I L K . Thus I L K appears to be an important mediator of N O production by i N O S in macrophages and may play a role in other cell types such as endothelial cells, chondrocytes and osteoblasts. Since human and mouse i NOS promoters are very different, we have also demonstrated the role of I L K on inflammatory molecule expression in human macrophages, by assessing the expression of C O X - 2 . The LPS-inducible expression of C O X - 2 , also a 139 proinflammatory enzyme, is regulated by N F - K B in both mice and human promoters (Lee et al., 2001; von Knethen et al., 1999; Muller et al., 1997). A s shown by our results, I L K plays a role in regulating the expression of C O X - 2 in human macrophages. Since I L K has been shown to regulate transcription factors such as AP-1 and C R E B (Troussard et al., 1999; D ' A m i c o et al., 2000), it is probable that I L K could regulate the transcription of C O X - 2 through an N F - K B , A P - 1 , and/or C R E B dependent manner. Investigations are currently underway to determine the identity of the transcription factors involved in I L K regulated transcription activity of the C O X - 2 promoter. 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Leukoc B i o l . 59, 575-585. 146 4 Conclusion and Future Directions 4.1 Conclusions A tumor is a heterogeneous composition of malignant and normal cells, and extracellular matrix ( E C M ) . Within this dynamic microenvironment, growth factors and E C M components interact with each other physically and in a paracrine manner to facilitate and select for malignant cells (Ferrara et al., 1993; Wiseman and Werb, 2002). These malignant cells display increased cell proliferation, survival and migration (Hanahan and Weinberg, 2000), characteristics which are recapitulated in epithelial cells overexpressing I L K . Indeed, molecular and genetic approaches have converged to confirm that I L K is a key effector of integrin and tyrosine kinase receptor cross-talk, as well as an important regulator of cell-cell and c e l l - E C M mediated intracellular signaling (Delcommenne et al., 1998; Hannigan et al., 2005; Persad et al., 2000; Persad and Dedhar, 2003). Deregulation of I L K activity during these processes may lead to tumorgenesis. In this thesis, I have described three novel roles for I L K in macrophages, cancer cells and endothelial cells. In addition, I have reinforced the kinase function of I L K as a P D K 2 candidate in the phosphorylation of A k t / P K B on serine-473. Angiogenesis is a process involving the coordination of multiple cell signals and mechanisms that result in proliferation, cell survival and migration. These mechanisms have been demonstrated to be PI3 -kinase dependent and are critical to blood vessel formation. Hamada et al. demonstrated in an in vivo model that PI3-kinase-Akt/PKB-P T E N pathway in murine endothelial cells is required for normal cardiac and vascular development (Hamada et al., 2005). A s well , they demonstrated that the loss of P T E N -mediated control of this pathway contributes both to susceptibility to new tumorgenic 147 mutations and accelerated tumor growth, secondary to enhanced tumor angiogenesis. Moreover, the loss of P T E N also results in an increased expression of Ang-2, V E G F - A , V E G F R 1 and V E G F R 2 (Hamada et al., 2005). Likewise, the loss of avp3 integrins in mice results in an increase in V E G F receptor 2 / F l k l expression and phosphorylation/activation, amplifying the signals that promote proliferation and survival. I L K has been implicated in several of the processes described above and plays a role in the PI3 kinase cascade (Attwell et al., 2003; Delcommenne et al., 1998; Hannigan et al., 2005; Persad and Dedhar, 2003; Persad et al., 2001b). Therefore, it is possible that I L K is involved in the regulation of V E G F R 2 expression in endothelial cells and VEGF-mediated migration, a necessary mechanism in angiogenesis (Attwell et al., 2003; Ferrara, 2005; Ferrara et a l , 2003). Cel l migration is documented to be regulated by Rac/Cdc42 (Fryer and Field, 2005). Studies into the mechanism of ILK-mediated Rac activation suggest an important role for the ILK -P -pa rv in interaction and the activity of the Rac/Cdc42-specific guanine nucleotide exchange factor a - P I X downstream of I L K ; linking I L K with the Rac- and Cdc42-mediated actin cytoskeleton reorganization in epithelial cells (Filipenko et al., 2005) in a PI3 kinase dependent manner (Attwell et al., 2003). Together, these new findings imply that upon V E G F stimulation, the role of I L K in endothelial cells may partially involve the initiation of a positive autocrine/paracrine feedback loop by increasing the expression of V E G F R 2 and V E G F - A . In addition, I L K may be involved in the reorganization of the cytoskeleton in a -P IX, Rac and Cdc-42 dependent manner in endothelial cells. Moreover I L K may increase the levels of cyclin D , promoting 148 endothelial cells proliferation, as well as expression of anti-apoptotic molecules in an A k t / P K B dependent-manner upon V E G F stimulation. For the past 10 years, the identity of the kinase responsible for activation of A k t / P K B has been widely debated. In addition to I L K , several enzymes have been shown to phosphorylate A k t / P K B at serine 473 position, coined as P D K 2 or H M activity, including M A P K A P kinase-2, P K C isoforms, DNA-dependent protein kinase, A T M , PDK-1 and A k t / P K B itself (Bayascas and Alessi , 2005). Recently, Sarbassov and colleagues demonstrate that Rictor /GpL of the mTOR/Ric to r /GBL complex is also a possible P D K - 2 candidate (Sarbassov et al., 2004). In addition to phosphorylating A k t / P K B directly, data presented in this thesis demonstrate that I L K regulates m T O R through activation of A k t / P K B ; though it is not clear whether this is a rapamycin sensitive event. Still it is possible that I L K may directly or indirectly regulate the Rictor /GpL and subsequently A k t / P K B phosphorylation. In addition, I was able to show that the inhibition of I L K , with the I L K inhibitor, results in a decrease in angiogenesis and growth delay in xenograft prostate tumour size. Although the worked described in this thesis suggests that delayed tumor growth is mostly due to angiogenesis, the observed growth delay could be the result of concurrent decrease in angiogenesis and increased cancer cell death, as long term exposure to the I L K inhibitor results in cell death. Many heterogeneous tumors, such as prostate and colon cancer, display elevated C O X - 2 expression as a result of deregulated N F - K B function (Karin and Greten, 2005). Infiltrated within these tumors are special immune cells known as tissue activated macrophages ( T A M s ) (Lewis and Pollard, 2006), which have been observed to be the 149 main producers of prostaglandins and C O X - 2 enzyme (Karin and Greten, 2005). Furthermore, studies have shown that increased prostaglandin levels can stimulate malignant cells to produce V E G F , which initiates and promotes angiogenesis. This is the result of improper H I F - l a transcription factor activation under normoxic conditions (Huang et al., 2005). Thus, I L K may play a key role in at least two critical pathways and cell types within a heterogeneous tumor to promote and sustain the tumorgenic process. Specifically, I demonstrated that in vitro genetic and pharmacological inhibition of I L K suppresses LPS-stimulated, N F - K B mediated expression of i N O S and C O X - 2 , and the subsequent increase in N O and prostaglandin production. N F - K B is the primary mediator of proinflammatory molecules and evidence in vitro and in vivo suggests that the p65 subunit must interact with acetylated P A R P - 1 for full activation of N F - K B (Hassa et al., 2003). Recently, it has been shown that acetylation of P A R P - 1 is mediated by CBP/300 (Hassa et al., 2005), which is phosphorylated and activated by A k t / P K B activity (Du and Montminy, 1998). Since I L K can phosphorylate A k t / P K B (Delcommenne et al., 1998; Hassa et al., 2003; Persad et al., 2001a), it would be interesting to know whether I L K may further control N F - K B activity through the regulation of P A R P - 1 acetylation. In addition, the phosphorylation of p50, an N F - K B subunit, further activates its transcriptional activity, but the kinase which phosphosphorylates the subunit is not well defined (Hou et al., 2003; L i et a l , 1994a; L i et al., 1994b). Interestingly, the kinase responsible for this phopshorylation event has not been defined with I L K as a possible candidate kinase for this direct regulation of N F - K B transcriptional activity. In addition to proinflammatory molecules, N F - K B also upregulates prosurvival targets such as Bcl-2 , and drug resistance genes such as p-glycoprotein (Karin and Greten, 2005). Thus, over-150 activation of I L K may induce the expression of pro-survival targets simultaneously with the expression of C O X - 2 and i N O S to confer a survival and propagation advantage to diseased cells such as cancer cells. In summary, I have presented several lines of evidence supporting a key role for I L K in the positive regulation of: A k t / P K B and HIF-la-dependent expression of V E G F in cancer cells and NF-KB-dependent induction of i N O S and C O X - 2 . Further, my studies indicate that I L K is indispensable for the VEGF-mediated proliferation and migration of endothelial cells, and continue to highlight the importance of I L K as a kinase and adaptor in normal and malignant cell behaviour. Further studies w i l l be required to determine the precise mechanisms by which I L K regulates N F - K B and H I F - l a during processes such as cell survival, proliferation, and migration. In addition, further investigations would be required to understand the mechanisms involved in VEGF-stimulated, ILK-dependent migration and proliferation of endothelial cells. Elucidation of these mechanisms w i l l underscore the importance of I L K activity and I L K signaling pathway as targets for therapeutic intervention in treating cancer. 4.2 Future Directions I L K is pivotal in mediating and coordinating signal transduction events involving integrins and growth factor receptors during cell migration, survival and proliferation. Deregulation of these tightly controlled cellular processes can result in pathological conditions. The loss of I L K results in poor organogenesis, however, the overexpression or activation of I L K has directly been shown to transform cells to that which resembles a cancer cell (Hannigan et al., 2005; Hannigan et al., 1996; Novak et a l , 1998; Radeva et 151 al., 1997; Somasiri et al., 2001), exhibiting avoidance of apoptosis, increased proliferation, migration and invasion into the surrounding area. Indeed, these phenotypes are the result of specific interactions involving intracellular multiprotein complexes at the membrane, in turn, altering the various cascades of signals to further mediate nuclear complexes and gene transcription. Thus identifying proteins that interact with I L K during different cell activities may assist in further uncovering the role of I L K in development and disease cascades. I L K activity has been demonstrated to be important in blood vessel sprouting, tubulogenesis and morphogenesis both in vitro and in vivo (Cho et al., 2005; Friedrich et al., 2004; Hannigan et a l , 1996; Kaneko et al., 2004; K o u l et al., 2005; Vouret-Craviari et al., 2004; Watanabe et al., 2005; Y a u et al., 2005). However, little is known about the involvement of integrin and V E G F receptor cross-talk in mediating the negative role of the avp3 and av|35 (Stupack and Cheresh, 2004), and positive role of a 5 p i integrin during angiogenesis. Because I L K can associate with integrins, it is possible that it may be involved in coordinating these intracellular signals. Since the IPP complex can be a positive or negative regulator of downstream events, depending on the different P I N C H and Parvin isoforms bound to I L K , uncovering the identity of the proteins associated with the IPP complex would bring insight into the types of proteins and associations required for a specific phenotype. In order to address the dynamic nature of these protein complexes, tandem affinity purification (TAP) tag method (Puig et al., 2001; Rigaut et al., 1999) could be used to purify and identify proteins associating with avp3, avP5, a5p i integrins and I L K . This technique has been successfully used to identify over 200 unique multi-subunit complexes in yeast (Gavin et al., 2002). 152 Since T A P tag method is useful in identifying complexes of proteins, it may also be useful in unraveling the ILK-dependent complexes downstream from I L K activation. The subsequent evaluation of the post-translational modifications of the proteins in the complex wi l l also give insight into how they may be regulated. To further define the importance of each protein interaction, individual or combinations of proteins identified at the complex could be deleted with s i R N A , and the cells could be observed for changes in morphology, survival, proliferation and migration. This would also give insight into the downstream events that are dependent on specific upstream protein interactions. There are an incredible number of kinases and an amazingly complex network of protein phosphorylation pathways. Tools that can down regulate I L K activity, such as s i R N A , the Cre-Lox system, and a small molecule I L K inhibitor would help to deconvolute the complexity of I L K signaling and further our understanding into the underlying molecular mechanisms of I L K in cell behaviour, development and disease. Phosphoproteomics (Mumby and Brekken, 2005; Shu et al., 2004) could advance the study of I L K significantly by allowing the study of ILK-dependent protein phosphorylation on a proteome-wide scale. Small interfering R N A has proven to be an effective means of dramatically decreasing the levels of I L K protein expression (Dykxhoorn et al., 2003; Filipenko et al., 2005; Tan et al., 2004; Troussard et al., 2003). With this tool, future studies could look at how gene transcription and protein phosphorylation profile of cancer cells change upon a decrease of I L K protein expression. A s well , the ILK-dependent activation of A k t / P K B can be further evaluated. To answer these questions, cells displaying high I L K activity such as PC3 prostate cancer cells, M D A - M B - 2 3 1 breast cancer cells, and SW480 colon 153 cancer cells can be treated with either I L K s i R N A , A k t / P K B s i R N A or control s i R N A . Messenger R N A from treated and control cells at several time points would be used to prepare labeled c D N A to probe commercially available human microarray chips thus creating a temporal transcriptional profile. Transcripts which display a significant variance between control and s i R N A treated cells can be further validated by real-time P C R . To further evaluate the cellular phosphorylation profile in the absence of I L K , proteins from s i R N A treated and control cells could be differentially labeled using the stable isotope labeling of amino acid in cell culture ( S I L A C ) method (Amanchy et al., 2005; Peri et al., 2003; Peri et al., 2004). Quantification of phosphoprotein levels between the two conditions, and the unequivocal identification of phosphorylated proteins can be accomplished by L C - M S and tandem M S , respectively. Proteins displaying changes in phosphorylation status could be further confirmed by Western blot analysis. These techniques also offer the potential of identifying novel ILK-dependent pathways. In parallel, similar studies could also be done by treating cells with the I L K small molecule inhibitor. The tumorgenic process that leads a normal cell to a cancerous cell can be divided into three mechanistic phases: initiation, promotion and progression. To date, we have mounting evidence of the critical role of I L K in multiple stages o f tumorgenesis stemming from cell culture studies which have been further validated with histopathology studies (Hannigan et al., 2005). Several lines of evidence suggest that the role of I L K in carcinogenesis is at the level of tumor promotion and progression (Hannigan et al., 2005; Troussard et al., 2006). Troussard et al. demonstrated that breast cancer cells versus normal cells have a preferential dependence on I L K for protein kinase B / A k t activation 154 and cell survival (Troussard et al., 2006). Bravou et al. observed elevated I L K expression in metastatic lesions of colon cancer (Bravou et al., 2006), while Zhiyong et al. demonstrated that I L K regulates matrix metalloproteinase-2 and urokinase-type plasminogen activator expression to convey metastatic function in murine mammary epithelial cancer cells (Zhiyong et al., 2006). To dissect the role of I L K in various stages of tumorgenesis and angiogenesis, one could start by crossing I L K flox/flox mice with P T E N -/- mice, T R A M P or L A D Y mice, transgenic mice that spontaneously form prostate cancers (Gingrich et al., 1996; Gingrich and Greenberg, 1996; Greenberg et al., 1995; Greenberg et al., 1994). Progeny mice would be further crossed with tissue-specific promoter Cre-mice (Greenberg et al., 1994; Singh et al., 2002; Young and Dong, 2005), specifically for prostate (probasin promoter Cre) and endothelial cells (77e2Cre). A t various stages of spontaneous tumor promotion and progression, I L K expression can be silenced and the effect on tumorigenesis can be assessed. Hopefully, results from these I L K mouse model would give insight not only on the role of I L K in cancer, but also towards the development of preclinical animal models that would allow better prediction of the outcome and efficacy of anti-ILK therapy on human disease. A n accepted view of cancer is that they are stem cells that fail to differentiate (Harris, 2005). Thus the study of developmental pathways would give insight into the deregulated differentiation signals that may lead to cancer progression. Recently, M i l l s et al. demonstrated that I L K is associated with the regulation of Sonic Hedgehog (Shh) pathway (Mil ls et al., 2006), a pathway found to be deregulated in tumors of neurological origin (Wechsler-Reya and Scott, 2001). G S K - 3 is a constitutively active serine-threonine kinase thought to be involved in the regulation of several signaling pathways, 155 including the Wnt, Hedgehog and Notch pathway (Trowbridge et al., 2006). Furthermore, I L K has been demonstrated to regulate G S K - 3 through direct phosphorylation and indirectly through activation of A k t / P K B . To further define the role of I L K in these pathways, immunoprecipitation assays of I L K with G S K - 3 or A k t / P K B could be performed in the presence and absence of Wnt, Hedgehog and Notch exposure in HEK-293 cells and primitive hematopoietic stem cells to determine direct phosphorylation capabilities. Results from these experiments can provide immediate details of the role of I L K in the regulation of these pathways in a GSK-dependent manner. Fluorescence resonance energy transfer (FRET) technology could also be used to assess transient interactions. Hematopoeitic stem cells are a good model because all three pathways have been shown to be critical for cell proliferation, without affecting the mature cells (Trowbridge et al., 2006). These studies would serve to further define the role of I L K in development and provide insight into the function of I L K in cancer. In summary, I believe that I L K represents an attractive target for treatment of a variety of human diseases and cancers. Though we have evidence of I L K as a potential master regulator of critical biological processes that are relevant to cancer progression such cell-cell, c e l l - E C M signaling angiogenesis and development, there is still much work to be done. A long line of evidence shows that I L K is critical as a kinase as well as an adaptor in development and cancer progression, however, the proteins that interact with I L K to give signaling complexity and diversity to influence the specificity, strength and timing of intracellular signals are unclear. Another area to be explored would be to further determine the function of specific complex combinations and determine where and when I L K is expressed during tumor progression. 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