Glypican-3 Stimulates the Wnt Signaling Pathway by Facilitating/ Stabilizing the Interaction of Wnt Ligand and Frizzled Receptor

GLYPICAN-3 STIMULATES THE WNT SIGNALING

PATHWAY BY FACILITATING/ STABILIZING THE

INTERACTION OF WNT LIGAND AND FRIZZLED RECEPTOR

Tonya Leigh Martin

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Medical Biophysics University of Toronto

Tonya Leigh Martin

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Medical Biophysics, University of Toronto

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse.

This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd).

Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd.

The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

ii Acknowledgements

It is a pleasure to thank those who made this thesis possible. I would like to thank my supervisor, Dr. Jorge Filmus, for giving me the opportunity to be involved in a project that held my curiosity and interest from the beginning until the end. I am grateful also for his support, especially through the more challenging aspects of the project. I am very appreciative of his help with project direction, and indispensable guidance on how to communicate the story of GPC3 and Wnt signaling so that others may find it as interesting as I do.

I am indebted to many of my colleagues in the lab who provided guidance, expertise, technical know-how and entertainment on a daily basis. I would like to thank Mariana

Capurro for the countless protocols and tips she provided me in addition to many, many hugs; Wen Shi for being the queen of shortcuts and always knowing if something was too risky to try all while keeping me in line; Dr. Sandra Zittermann for much technical expertise, letting me borrow her solution recipe cards and many heart to heart talks;

Fuchuan Li for being ready and willing to do any favour you may ask of him, but especially for helping me tweak the washing solution that allowed me to get the binding assay working; and finally Joseph Antony who made a wonderful addition to the team toward the end, and is helping to get this work published. Together they have taught me virtually every lab skill that has made this research possible. They also gave me the great gift of believing in me when I didn’t believe in myself, which is what I needed to keep going.

Dr. Liliana Attisano and Dr. James Dennis were the brilliant and supportive members of my committee. I am thankful to them for providing suggestions that truly made me

iii look at my project, and my approach to research in a different way. I am extremely grateful to them for making committee meetings something that I looked forward to.

None of this experience would have been possible without the amazing support of my family and friends. Thanks so much to my parents, Karen and Darrel Martin for their encouragement through my entire education, and their eagerness to generously support whatever path I chose to take. Thanks to my brother, Jonathan Martin, and extended family for keeping tabs on me and sending encouraging emails throughout this time. A special thanks to the biocrew: Alex – for getting me in grad school, Alison – for keeping me in grad school and Colleen – for all the red, red wine. Thanks to Isuru for keeping

Sunnybrook fun with our regular lunch dates. Finally, thanks to my “permanent roommate” Tilak who always received an earful in response to the question, “how was your day”, but listened to every word and gave thoughtful responses. His ability to make me laugh, his stoic nature in the face of adversity and rock solid support made it possible for me to face new (and old) challenges every day.

Thank you to everyone who made this work possible.

iv Table of Contents

ABSTRACT II ACKNOWLEDGEMENTS III TABLE OF CONTENTS V ATTRIBUTIONS VI LIST OF ABBREVIATIONS AND SYMBOLS VII

CHAPTER 1 INTRODUCTION 1

1.1 GLYPICANS 2 1.2 GLYPICAN FUNCTION IN MODEL ORGANISMS 7 1.3 THE ROLE OF MAMMALIAN GLYPICAN-3 IN DEVELOPMENT 12 1.4 WNT SIGNALING PATHWAY 16 1.5 HEPATOCELLULAR CARCINOMA 21 1.6 HYPOTHESIS AND OBJECTIVES 23

CHAPTER 2 MATERIALS AND METHODS 24

2.1 CELL LINES, TRANSFECTIONS AND PLASMIDS 25 2.2 ANTIBODIES 25 2.3 LUCIFERASE ASSAY 26 2.4 SURFACE PLASMON RESONANCE 27 2.4 ALKALINE PHOSPHATASE BINDING ASSAY 28 2.5 CO-IMMUNOPRECIPITATION 29 2.6 IMMUNOCYTOCHEMISTRY 29

CHAPTER 3 RESULTS 31

3.1 GLYPICAN-3 STIMULATES WNT SIGNALING 32 3.2 GPC3 BINDS WNT 34 3.3 GPC3 INCREASES PHOSPHORYLATION OF LRP6 37 3.4 GLYPICAN-3 BINDS MULTIPLE FZD RECEPTORS VIA GAG CHAINS 39 3.5 GPC3 AND FZD INTERACT ON THE CELL MEMBRANE 43 3.6 HEPARIN INHIBITS BINDING OF GPC3 TO FZD 45 3.7 FZDCRD DOMAIN IS INVOLVED IN BINDING GPC3 47

CHAPTER 4 DISCUSSION 50

4.1 DISCUSSION 51

BIBLIOGRAPHY 62

v Attributions

All work contained in this thesis was performed by the author, with the exception of the data for GPC3 Wnt3a binding. Fuchuan Li performed the purifications and data analysis for the surface plasmon resonance assays.

vi List of Abbreviations and Symbols

ΔGAG Lacking glycosaminoglycan chains ΔGPI Lacking glycosylphosphatidylinositol anchor A/P Anterior/Posterior AFP α-fetoprotein AMP 2S-amino-2-methyl-1-propanol AP Alkaline phosphatase APC Adenomatous polyposis coli protein BMP Bone Morphogenic Protein BWS Beckwith-Wiedemann Syndrome CK1 Casein kinase 1 CRD Cysteine Rich Domain D/V Dorsal/Ventral Dl Dally Dlp Dally-like DMEM Dulbecco’s Modified Eagle Medium Dpp Decapentaplegic signaling pathway (Drosophila) FBS Fetal Bovine Serum FGF Fibroblast Growth Factor Fzd Frizzled GAG Glycosaminoglycan GalNAc N-acetylgalactosamine GlcA Glucuronic acid GlcNAc N-acetylglucosamine GlcNS Glucosamine-N-sulfate GPC3 Glypican-3 GPI Glycosylphosphatidylinositol GSK3 Glycogen synthase kinase 3

vii HCC Hepatocellular Carcinoma Hh Hedgehog signaling pathway HS Heparan Sulfate HSPG Heparan Sulfate Proteoglycan IGF Insulin Growth Factor IP Immunoprecipitation IRS Insulin Receptor Substrate LRP6 LDL-receptor-related protein 5/6 PCP Planar Cell Polarity pLRP5/6 Phosphorylated LDL-receptor-related protein 5/6 PNPP 4-nitrophenyl phosphate disodium salt hexahydrate SGBS Simpson-Golabi-Behmel Syndrome sGPC3 Soluble GPC3 Shh Sonic Hedgehog SPR Surface Plasmon Resonance TCF/LEF T-cell factor/ lymphoid enhancer factor WT Wild Type Xgly4 Xenopus glypican 4 YFP Yellow Fluorescent Protein

viii 1

Chapter 1

Introduction 2 1.1 Glypicans

Glypicans are a family of proteoglycans located on the cell membrane. There are 6 glypican family members in mammals and several homologues have been identified including dally and dally-like in Drosophila and Knypek in Zebrafish. All glypican proteins display glycosaminoglycan (GAG) chains and are linked to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor (Filmus and Selleck, 2001). Disruption of glypican function phenotypically resembles defects in regulatory signaling pathways.

Glypicans regulate several developmental processes such as body size determination and morphogenesis (Song and Filmus, 2002). Glypican expression occurs predominantly in the embryo, in a stage and tissue specific manner (Filmus, 2001).

Glypican core proteins are 60-70 kDa in size and share common structural features across the protein family. Each glypican can be divided into three structural domains. The linker domain is at the C-terminal end and connects the core protein to a GPI anchor in the cell membrane. Adjacent to the linker region, there are attachment sites for glycosaminoglycan (GAG) chains. The insertion sites are within 50 amino acid residues of the membrane anchor, positioning the GAG chains close to the cell membrane (Song and Filmus, 2002). The third glypican domain is a globular cysteine rich domain (CRD).

The tertiary structure of the CRD is thought to remain constant between glypican family members due to the presence of 14 highly conserved cysteine residues that are predicted to form stabilizing disulphide bonds. A schematic representation of a glypican protein is shown in Figure 1.1. 3

Figure 1.1 Schematic of Glypican-3 Glypican-3 is a member of the glypican family of proteins. Glypicans have an N-terminal domain containing 14 conserved cysteine residues that form stabilizing disulphide bonds. Glypican-3 has two insertion sites for heparan sulfate proteoglycan chains. Heparan sulfate chains carry a negative charge and can bind to proteins with positively charged domains. The C-terminal end of glypican-3 is a linker domain that connects the protein to a GPI anchor in the cell membrane. Glypican-3 has cleavage sites for phospholipase-D (PLD), Notum and members of the convertase family. Cleavage sites are marked in red. 4

Glypican GAG chains are linear sugar polymers consisting of a repeating disaccharide unit. The GAG chains of glypicans carry a negative charge, allowing promiscuous interaction with basic growth factors and morphogens in the extracellular space. The dominant GAG type is Heparan Sulfate (HS), but Chondroitin sulfate (CS) can also be found on Glypicans (Filmus et al., 2008). The disaccharide units of each

GAG chain are composed of an uronic acid and an amino sugar. Chondroitin sulfate contains glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), while HS is made up of GlcA and N-acetylglucosamine (GlcNAc) (Gandhi and Mancera, 2008). HS chains can undergo an array of modifications to create highly heterogeneous chains throughout different tissues (Figure 1.2) (Bülow and Hobert, 2006). The first modification of GAG chains is the removal of the N-acetate and replacement with N-sulfate by an N- deacetylase N-sulfotransferase enzyme to form glucosamine-N-sulfate (GlcNS).

Formation of GlcNS serves as a primer for other sugar chain modifications. Glucuronic acid can be epimerized to Iduronic acid and subsequently sulfated at the 2-O position.

GlcNS can likewise be sulfated at the 6-O position, and occasionally at the 3-O position

(Ai et al., 2003; Caterson et al., 1990). Sulfate groups are donated by 3’-phosphoadenyl-

5’phosphosulfate (PAPS), a high energy donor. The sulfate and carboxylic acid groups are deprotonated at physiological pHs, leaving the GAG chains with a strong negative charge (Capila and Linhardt, 2002). These negatively charged, heterogeneous sugar chains are known to promiscuously interact with extracellular proteins that display positively-charged domains (Powell et al., 2004). 5

Figure 1.2 Heparan sulfate biosynthesis and modifications. Heparan sulfate chains are linked to a serine residue on the glypican core protein. A linkage tetrasaccharide joins the sugar chain to the protein. Chain synthesis begins with copolymerization of N-acetylglucosamine and glucuronic acid residues. The first chain modification is the removal of N-acetate and replacement with N-sulfate by N-deacetylase N-sulfotransferase enzyme. Sulfotransferases add sulfate groups to iduronic acid (2-O) and N-acetylglycosamine (3-O, 6-O) residues. The sulfate groups are donated by 3-phosphoadenyl-5-phosphosulfate (PAPS). Varki, A. (2008). Essentials of Glycobiology second edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 6 Sugar modifications create domains within HS polysaccharides. Clusters of highly sulfated disaccharides have been found along the length of HS chains. Sulfate clusters have been shown to increase in number and length near the accessible non-reducing end of the sugar chain and mediate interaction with other proteins (Staples et al., 2010). HS sulfation patterning influences ligand binding and provides specificity for the regulation of signaling by HS (Esko and Selleck, 2002).

Glypican proteins are cleaved by two types of enzymes. Firstly, glypicans are cleaved at the GPI anchor by the lipases Notum and phospholipase D (Brunner et al.,

1994; Traister et al., 2007). Secondly, a family of convertases cleave the glypican core protein at an internal cleavage site. Cleavage by either category of enzyme can alter glypican function.

Notum and phospholipase D cleave glypican near the cell membrane. Phospholipase

D cleaves the GPI anchor before the inositol group, while Notum cleaves before the adjoining phosphate group. Glypican is cleaved by phospholipase D in the secretory pathway, before glypican reaches the cell membrane (Tsujioka et al., 1998). The functional consequences of cleavage by phospholipase D have not been well studied.

The ability of Notum to cleave glypicans has been implicated in morphogen gradient formation in Drosophila and inhibition of Wnt signaling in mammals (Ayers et al., 2010;

Giráldez et al., 2002; Traister et al., 2007). Notum is a transcriptional target of Wnt signaling, thus as Wnt signaling increases Notum expression increases (Giráldez et al.,

2002). Notum induces the release of glypicans, and any bound morphogen, from the cell surface (Torisu et al., 2008). Glypican cleavage by Notum is important for proper morphogen distribution during development. Notum has the ability to act in the extracellular environment, thus providing flexibility to release glypicans from the cell 7 surface in a regulated manner (Traister et al., 2007). Traister et al demonstrated that

Notum-induced cleavage of GPC3 inhibits Wnt signaling, and proposed that, upon cleavage, GPC3 acts as a competitive inhibitor for Wnt. Similarly, soluble GPC3

(sGPC3), a mutant form of GPC3 with the GPI linker domain deleted, is another competitive inhibitor of Wnt signaling because it is unable to attach to the cell membrane

(Capurro et al., 2005b). Although cleavage seems to be required for morphogen gradient formation in Drosophila, GPC3 requires attachment to the GPI membrane anchor to stimulate cell signaling. Notum is an important regulator of GPC3 membrane attachment, and consequently of GPC3 function.

Convertases are another family of enzymes that cleave glypicans. Convertase cleavage occurs within the globular CRD domain at a paired basic motif (De Cat et al.,

2003). Cleavage by convertases generates ~40kDa N-terminal and ~30 kDa C-terminal subunits that remain joined together by disulfide bonds, (De Cat et al., 2003). Glypican-3 induced modulation of Wnt signaling has been shown to require convertase cleavage in

Zebrafish development and in some cell lines. On the other hand, this cleavage is not required to stimulate Wnt signaling in hepatocellular carcinoma cell lines (Capurro et al.,

2005a; De Cat et al., 2003). Thus, selective cleavage provides an additional level of regulation of glypican activity.

1.2 Glypican Function in model organisms

Glypicans have homologues in several well studied model organisms including

Drosophila, Zebrafish and Xenopus laevis. Early evidence of glypican regulatory roles in several key signaling pathways was discovered in these model organisms. These pathways include, Wnt, Hedgehog (Hh), Bone Morphogenic Protein (BMP) and

Fibroblast Growth Factor (FGF) signaling (Desbordes and Sanson, 2003; Hartwig et al., 8 2005; Lin and Perrimon, 1999; Topczewski et al., 2001; Yan and Lin, 2007). An overview of glypican function in model organisms is provided here.

There are two glypican homologues found in Drosophila: Dally and Dally-like (Dlp).

Both transcripts are uniformly expressed in the early embryo (Baeg et al., 2001; Lin and

Perrimon, 1999). Drosophila wing imaginal disc development is a well used system to study morphogen regulation. Study of this system provided evidence that Dally and Dlp regulate gradient formation of Wingless (Wg), the Drosophila Wnt homologue, Hh and

Decapentaplegic (Dpp).

In wing discs, Wg is secreted from a narrow band of cells along the dorsal/ventral

(D/V) boundary and moves outward into each compartment forming a Wg gradient

(Cadigan, 2002). Transport of Wg to distant cells is necessary for transcription of long range target genes, such as Dpp. Genetic studies have demonstrated that Dally and Dlp have partially complementary roles in morphogen gradient formation. Flies deficient in

Dally are normal or have weak Wg-like phenotypes (Lin and Perrimon, 1999; Tsuda et al., 1999), while knocking out Dlp in Drosophila produces a phenotype similar to an incomplete Wg, Hh double mutant (Franch-Marro et al., 2005). Knocking out both Dally and Dlp increases the phenotypic severity, nearly replicating the double Wg/Hh knockout features (Franch-Marro et al., 2005). The evidence implies that each glypican has its own distinct role in signaling, but they also have partial functional redundancy.

Much effort has gone into deciphering the precise role of Dally and Dlp in Wg gradient formation and signaling. Evidence suggests that Dlp is crucial for transport of

Wg from the D/V border to cells further from the source (Baeg et al., 2001; Han et al.,

2005). If Dlp is absent, Wg migration is inhibited but, interestingly, Wg signaling will increase near the site of secretion (Han et al., 2005; Kirkpatrick et al., 2004). This finding 9 is counter intuitive, since Dlp mutants clearly show signs of deficient Wg signaling. It would appear the absence of Dlp increases Wg signaling near the site of secretion, but at the expense of long range signaling. Thus Dlp is important for transport of Wg signal from the site of secretion to long range target cells. The exact mechanism of transport has not been determined. There is evidence that Dlp may be released from the cell surface by Notum (Han et al., 2005; Kirkpatrick et al., 2004; Kreuger et al., 2004) or that it is able to present Wg to neighbouring cells via the GAG chains (Franch-Marro et al.,

2005). Dlp regulates transcytosis of Wg, another method of morphogen distribution. Dlp internalizes Wg from the apical membrane and then secretes it to the basolateral side

(Gallet et al., 2008). These are several proposed mechanisms through which Dlp may help form the Wg gradient.

Dally plays a smaller role than Dlp in Wg signaling and gradient formation. Dally has been shown to stimulate Wg signaling, although it is not strictly necessary for signal transmission (Franch-Marro et al., 2005). However, over-expression of Dally increases

Wg signaling without affecting the levels of available Wg (Tsuda et al., 1999). Based on this evidence, several groups have proposed that Dally acts as a non-essential co-receptor for Wg (Franch-Marro et al., 2005; Lin and Perrimon, 1999; Tsuda et al., 1999). Clearly more work is required to determine exactly how Dally contributes to Wg signal transmission and gradient formation.

The Hh morphogen gradient is maintained in a similar manner to Wg, but the roles of

Dally and Dlp are reversed. In the Hh signaling pathway Dally is crucial for gradient formation, while Dlp acts as a co-receptor. Notum has been implicated in regulating

Dally involvement in Hh gradient formation. Notum cleaves Dally from the apical cell surface which reduces Hh accumulation near the anterior/posterior (A/P) boundary and 10 promotes Hh movement to distant target cells (Ayers et al., 2010). As a co-receptor,

Dlp was recently shown to bind both Hh and Patched, preceding the internalization of the signaling complex (Gallet et al., 2008).

Dpp, a BMP homologue, is another important morphogen involved in wing disc patterning. Dpp is secreted along the A/P border and determines the fate of cells along that axis (Fujise et al., 2003). Dally and Dlp have redundant roles in stabilizing Dpp on the cell membrane and potentiating Dpp signal (Akiyama et al., 2008; Belenkaya et al.,

2004; Fujise et al., 2003; Jackson et al., 1997). Current evidence points to Dally as the most important HS Proteoglycan (HSPG) in wing disc Dpp signaling. Cells lacking

Dally are more deficient in Dpp signaling than cells without Dlp. However, the most severe Dpp signaling impairment is observed when both Dally and Dlp are absent

(Belenkaya et al., 2004). This indicates that Dally and Dlp have partially redundant roles, possibly as non-essential co-receptors in Dpp signaling (Belenkaya et al., 2004;

Fujise et al., 2003).

Gradient formation of Dpp is primarily dependant on the presence of Dally, with Dlp serving a smaller redundant role (Fujise et al., 2003). Dpp gradients formed by a truncated form of Dpp, unable to bind Dally or Dlp, are much more shallow than the wild type Dpp gradients (Akiyama et al., 2008). In addition, Double null mutants fail to transduce Dpp signal beyond cells adjacent to the line of Dpp secretion (Belenkaya et al.,

2004). Dally stabilizes Dpp on the cell surface by antagonizing the Dpp receptor thickveins and preventing rapid receptor mediated endocytosis (Akiyama et al., 2008).

This increases the in vivo half-life of Dpp and allows for glypican mediated dispersal of

Dpp across the wing (Akiyama et al., 2008; Belenkaya et al., 2004). 11 Recently Guo et al. showed that Dally plays a very important role in BMP gradient formation near germline stem cells (GSC) of the Drosophila ovary (Guo and Wang,

2009). Dally is expressed by cap cells at the anterior end of the ovary, which come into contact with two or three GSCs. BMP is stabilized and concentrated by Dally in close proximity to the GSCs, creating a steep BMP gradient that acts at a single cell resolution.

In this way, GSCs attain the high levels of BMP necessary to prevent differentiation.

Interestingly, Dally is required for BMP signaling in GSC cells, and not nearby somatic cells. BMP homologues, including Dpp, serve as another example of signaling pathways where Dally and Dlp are important as regulators of gradient formation and signal potentiation.

FGF signaling is another pathway regulated by Drosophila glypicans. Initially FGF signaling was shown to require HS GAG chains, but it was not known whether any

HSPG could fulfill this role, or if specific proteins were required (Lin et al., 1999). Dally has been shown to bind directly to vertebrate FGF proteins through the GAG chains although there are no reports on the functional consequence of this interaction

(Kirkpatrick et al., 2006). It was subsequently shown that Dlp mutant embryos exhibited severe defects in tracheal morphogenesis due to a reduction in FGF signaling (Yan and

Lin, 2007). The observed phenotype was similar to that observed when all HSPG synthesis is blocked, pointing to Dlp as the primary HSPG in tracheal morphogenesis.

Yan et al. further show that Dlp is only required in FGF receiving cells and not producing cells, suggesting that Dlp is important for FGF signaling but not gradient formation.

Taken together, this evidence is consistent with the model that Dally and Dlp act as FGF co-receptors, stabilizing FGF on the cell membrane and presenting it to the receptor. 12 Zebrafish and Xenopus systems have also been used to study glypican function, as each expresses a homologue of Glypican-4. In both systems, glypican homologues have been implicated in non-canonical Wnt signaling during gastrulation movements

(Ohkawara et al., 2003; Topczewski et al., 2001). Reducing levels of Xenopus glypican-

4 (Xgly4) reduces accumulation of Disheveled, a key Wnt signaling component, at the cell membrane (Ohkawara et al., 2003). This in turn disrupts well orchestrated cell movements during gastrulation. Zebrafish glypican-4 (Knypek) has been shown to regulate non-canonical Wnt signaling through Wnt11 (Topczewski et al., 2001).

Interestingly, disruption of Knypek membrane localization has also been shown to increase caveolin dependent Fzd7 endocytosis (Shao et al., 2009). Our understanding of the mechanism of action of Xgly4 and Knypek are incomplete, but the evidence in both cases suggests their roles are tied tightly to the stabilization of the interaction between

Wnt and its Fzd receptor.

The functions of glypicans in model organisms are important and diverse. Both the core protein and GAG chains of Glypicans interact with other cell signaling proteins.

The expression pattern of glypicans is crucial to their function as demonstrated by morphogen gradient formation and stem cell maintenance. The versatility of glypicans is increased through enzymatic modifications of their core protein or GAG chains. Many of these important glypican functions demonstrated in model organisms also hold true in the mammalian system.

1.3 The role of mammalian Glypican-3 in development

Glypican-3 (GPC3) is the most widely expressed glypican during mammalian development. GPC3 is found in most embryonic tissues, at varying developmental stages

(Song and Filmus, 2002). The embryonic expression pattern of GPC3 is in stark contrast 13 to expression in adults where it is found in only a few tissues including lung, kidney, ovary and breast (Kim et al., 2003). The myriad of conditions associated with a non- functioning GPC3 protein illustrates the extent to which GPC3 is necessary for proper development. Loss-of-function mutations of GPC3 cause Simpson-Golabi-Behmel overgrowth syndrome (SGBS) (Pilia et al., 1996). SGBS patients display a wide spectrum of clinical manifestations including pre and postnatal overgrowth, congenital heart defects, enlarged kidneys, skeletal irregularities and an increased risk for development of embryonal tumours (Neri et al., 1998; Pilia et al., 1996). At the time of this discovery, it was hypothesized that GPC3 was involved in regulating cell proliferation. This was shown to be true through investigation of cell proliferation in the developing kidney of GPC3-null mice (Grisaru et al., 2001). However, the mechanism by which GPC3 is able to regulate cellular processes remains a topic of intense investigation.

Mammalian GPC3 regulates several different signaling pathways in a stage and tissue specific manner. GPC3 null mice have been used to determine the various mechanisms of GPC3 action during mammalian development (Cano-Gauci et al., 1999;

Paine-Saunders et al., 2000). GPC3 null mice share several features with SGBS patients, most strikingly the developmental overgrowth (Cano-Gauci et al., 1999). GPC3 -/ mice also exhibit many other SGBS traits including perinatal death, cystic and dysplastic kidneys, abnormal lung development and skeletal abnormalities (Cano-Gauci et al., 1999;

Paine-Saunders et al., 2000).

Several laboratories have investigated the question of which signaling pathway mediates the regulation of body size by GPC3. Initial reports suggested that insulin-like growth factor 2 (IGF2) pathway might be involved due to similarities in presentation 14 between SGBS and Beckwith-Wiedemann syndrome (BWS), an overgrowth disease caused by IGF2 over-expression (Pilia et al., 1996). However, this hypothesis has been thoroughly refuted. GPC3 null embryos show no alterations in IGF signaling (Cano-

Gauci et al., 1999). Furthermore, our laboratory demonstrated that GPC3 cannot interact with IGF2 (Song et al., 1997). The most convincing evidence came from GPC3 null mice mated with insulin receptor substrate 1 (IRS-1) null mice (which are deficient in IGF signaling). If overgrowth is indeed caused by an increase in IGF signaling in the absence of GPC3, then removing the ability of IGF to signal should rescue the overgrowth phenotype. However GPC3/IRS-1 double knock-out mice display levels of overgrowth that are similar to those of GPC3-null mice, indicating that GPC3 is in fact modulating body size through a different pathway (Song et al., 2005).

Recent work from our laboratory has shown that GPC3 regulates body size, at least partially, through the Hh pathway (Capurro et al., 2008). GPC3 acts as an Hh signaling inhibitor, in contrast to Drosophila glypican Dlp, which acts as a co-receptor for Hh to stimulate signaling. GPC3 is able to bind Sonic Hedgehog (Shh), one of three mammalian Hhs, and sequester it from its receptor Patched. GPC3 and Shh are internalized and targeted for degradation (Capurro et al., 2008). Therefore, in the absence of GPC3, more Shh is available for signaling and the Hh pathway is stimulated.

Consequently, GPC3 null mice display higher Shh protein levels, and are 30% larger, than their wild type littermates (Capurro et al., 2009). Cell sizes in GPC3 null mice are similar to that of wild type mice indicating that overgrowth is caused by an increase cell proliferation (Chiao et al., 2002). Confirmation that GPC3 modulation of Hh signaling affects body size was obtained by crossing GPC3 null mice with mice that were lacking another of the three Hh ligands, Indian Hedgehog (Ihh). Mice lacking functional GPC3, 15 with wild type Ihh expression, were 30% larger than wild type mice (Capurro et al.,

2009). However, when GPC3 null mice were in an Ihh null background, the overgrowth phenotype was partially rescued, as the mice were only 20% larger than GPC3 +/+ Ihh-/- mice. Since there are two other Hh ligands, it is reasonable to assume that a complete knockdown of all Hh ligands would further rescue the overgrowth phenotype.

Unfortunately since an Hh null phenotype is lethal in very early embryonic stages it is impossible to determine the exact contribution of Hh signaling to overgrowth in GPC3 null mice (Capurro et al., 2008). Taken together results indicate that the most relevant signaling pathway for GPC3-mediated modulation of body size is the Hh pathway.

GPC3 also regulates Hh signaling indirectly through the FGF signaling pathway.

GPC3 null mice have coronary artery fistulas, formed through uneven Hh expression, which leads to the overactive development of the coronary artery (Ng et al., 2009). In wild type mice, FGF9 stimulates Hh signaling which promotes heart development. Ng et al. propose that GPC3 acts as a co-receptor for FGF9. In the absence of GPC3, FGF9 signaling is reduced, followed by a reduction in Shh expression. Interestingly, even though overall Shh levels are reduced compared to wild type, certain populations of cardiac cells maintain normal levels of Shh signaling, leading to formation of coronary artery fistulas (Ng et al., 2009). Uneven Shh expression in the absence of GPC3 demonstrates that the regulatory function of GPC3 acts in a cell-type specific manner.

GPC3 has been reported to regulate Wnt signaling in mouse embryos and several cell lines (Capurro et al., 2005b; Song et al., 2005). Wnt is a known regulator of Cyclin D1, which is up regulated in GPC3 null mice compared to wild type. In addition, GPC3 knockout mice have decreased non-canonical Wnt signaling and increased canonical Wnt signaling (Wnt pathways described below) (Song et al., 2005). This led to the hypothesis 16 that in wild type mouse embryos, GPC3 stimulates the non-canonical signaling pathway, which in turn inhibits the canonical Wnt signaling pathway. This hypothesis was supported by the finding that transfection of GPC3 into a mesothelioma cell line potentiates non-canonical Wnt5a signaling at the expense of canonical signaling, and slowed proliferation of cells in culture (Song et al., 2005). Interestingly, GPC3 regulates

Wnt signaling differently in hepatocellular carcinomas where it stimulates canonical Wnt signaling and cell proliferation.

Wnt and Hh are both crucial signaling pathways in normal development (Ingham and

McMahon, 2001; Logan and Nusse, 2004). GPC3 is able to regulate both pathways and is therefore is an important regulator of developmental processes. The mechanism by which GPC3 regulates Hh signaling is largely solved, while the mechanism by which

GPC3 regulates Wnt signaling is not clear.

1.4 Wnt Signaling pathway

The Wnt signaling pathway is essential for proper control of embryonic development and tissue homeostasis (Logan and Nusse, 2004). Wnt signaling has been identified as an important regulator of stem cell differentiation and self-renewal (Nusse,

2008). These properties, as well as dysfunctional Wnt activity in many cancers, have made Wnt signaling an area of intensive research (Reya and Clevers, 2005).

Wnt signaling is very complex, owing to large families of both ligands and receptors. In mammals, there are 19 Wnt ligands and 10 Fzd receptors in addition to several other pathway activators (van Amerongen and Nusse, 2009). Ryk and Ror are two receptor tyrosine kinases that function as non-Fzd receptors for some Wnt ligands (van

Amerongen et al., 2008). Norrin is a Wnt ligand that stimulates canonical Wnt signaling 17 through Fzd4 in the retina and inner ear (Xu et al., 2004). The exact function of each receptor-ligand pair is still being investigated.

The β-catenin Wnt signaling pathway, also known as canonical Wnt signaling, leads to the stabilization of β-catenin in the cytoplasm, and is a major area of research due to its role in cancer growth (Giles et al., 2003). Non-canonical Wnt signaling pathways do not rely on β-catenin stabilization for signal transmission. Stimulation of non-canonical signaling pathways activates a plethora of signaling molecules including

Rho, Rac, Jnk and Src that carry out functions such as establishment of cell polarity, axon guidance and convergence and extension movements; the mechanisms in these pathways are not as well defined as those for canonical Wnt signaling (McNeill and Woodgett,

2010; van Amerongen et al., 2008).

Canonical Wnt signaling regulates the stability of β-catenin via a large cytoplasmic protein aggregate termed the destruction complex. The destruction complex consists of the structural protein Axin, adenomatous polyposis coli protein (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). β-catenin is bound and phosphorylated by the destruction complex, marking it for proteosomal degradation. In the absence of Wnt ligand, cytoplasmic levels of β-catenin kept are low through constant degradation (Figure 1.3A).

In the presence of Wnt ligand, Wnt binds Fzd and co-receptor LDL-receptor- related protein 5/6 (LRP5/6) on the cell membrane. The Wnt/Fzd/LRP complex recruits intracellular proteins Disheveled, Axin, and GSK3 to the cell membrane (Bilic et al.,

2007; Mao et al., 2001). This triggers multimerization of LRP5/6 and, presumably, Fzd receptors into a large signaling aggregate termed the signalosome (Bilic et al., 2007;

Junge et al., 2009). Aggregation of LRP5/6 leads to phosphorylation of multiple sites 18 along the intracellular domain by several kinases including GSK3 and CK1 (Niehrs and Shen, 2010). The destruction complex is rendered inactive after the recruitment of

Axin and GSK3 kinase to the cell membrane. β-catenin accumulates in the cytoplasm and eventually translocates to the nucleus where it binds transcription factors of the T-cell factor/ lymphoid enhancer factor (TCF/LEF) family. Genes controlling cell proliferation, apoptosis and other crucial cellular functions are expressed (Figure 1.3B) (Cadigan and

Liu, 2006; Zerlin et al., 2008).

Increased levels of β-catenin have been found to be a stimulant for proliferation of several types of cancer. Mutations causing stimulation of the Wnt signaling pathway have been found a majority of Hepatocellular Carcinomas (HCC), making Wnt signaling a critical topic in HCC research (Kim et al., 2008). 19 A

B 20

Figure 1.3 Wnt Signaling A) Inactive Wnt signaling. When Wnt is absent, a destruction complex is formed in the cytoplasm consisting of adenomatous polyposis coli, Axin, glycogen synthase kinase 3 and casein kinase 1. The destruction complex binds and phosphorylates β-catenin, targeting it for destruction. B) Active Wnt signaling. Wnt binds to Fzd and co-receptor lipoprotein receptor related protein5/6 (LRP5/6). Clustering and phosphorylation of LRP5/6 creates a binding site for Axin, which is subsequently drawn away from the destruction complex. In the absence of Axin, the destruction complex is no longer functional, and is unable to tag β- catenin for degradation. β-catenin is able to accumulate and translocate to the nucleus. Here, it associates with TCF/LEF transcription factors to allow transcription of Wnt responsive genes. 21 1.5 Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the 5th most common cancer in males and 8th in females in the developed world (Parkin et al., 2005). Rates of disease incidence in

Canada are expected to rise by 73% in males and 28% in females from 1996 to 2015

(Pocobelli et al., 2008). Even more alarming is that HCC ranks third in rates of cancer mortality (Parkin et al., 2005). The poor prognosis of HCC is due to several factors linked to disease progression and treatment options. Symptoms of HCC do not arise in patients until later stages of the disease when the cancer is more difficult to treat.

Secondly, HCC often arises in a background of liver cirrhosis due to previous hepatitis infections or alcohol consumption. The underlying liver inflammation means that cytotoxic treatments are not well tolerated; treatments such as radiation, chemotherapy and cytotoxic drugs can only be used sparingly (Thomas et al., 2008). Radioablation, a non-cytotoxic treatment, is initially effective but ultimately has high rates of recurrence in late stage cancers (Choi et al., 2007). Liver transplantation has high rates of success, but there are clearly challenges with access to donors and the complexity of the procedure

(Mazzaferro et al., 2009). Therefore it is recognized that targeted molecular therapeutics are the preferred treatment option for HCC.

Investigation of HCC on a molecular level revealed that GPC3 expression is increased in HCC lesions, compared with normal or cirrhotic liver (Capurro et al., 2003).

GPC3 is expressed in 70-80% of HCC lesions, while being virtually absent in normal liver (Kandil and Cooper, 2009). These characteristics have made GPC3 an excellent candidate as an HCC marker. There is some evidence that GPC3 is especially valuable in detecting poorly differentiated HCC lesions (Shafizadeh et al., 2008). GPC3 is more 22 sensitive and specific than the leading HCC marker, α-fetoprotein (AFP) as a serum test for early diagnosis of HCC (Capurro et al., 2003).

As the diagnostic potential for GPC3 was being evaluated, the question was posed as to whether GPC3 affected the progression of HCC malignancies. Given that glypicans are required for optimal signaling of various growth factors, GPC3 was thought to play a role in driving the proliferation of HCC through one of these pathways. The Wnt pathway was a good candidate, as its hyperactivation is an early event in HCC progression, and is found to be up regulated in 50-70% of all HCC cases (Wong et al., 2001). It was found that indeed, GPC3 acts through the Wnt pathway to stimulate HCC proliferation (Capurro et al., 2005b). Capurro et al. found that HCC cell lines expressing GPC3 proliferated faster than cell lines without GPC3. The stimulatory effect of GPC3 was also observed in vivo, where HCC xenografts expressing GPC3 grew faster than controls (Zittermann et al., 2009). HCC cell lines expressing GPC3 had increased levels of cytoplasmic β- catenin, a key component in the transmission of Wnt signaling. GPC3 was shown to bind

Wnt, and GPC3-expressing cells had higher expression of a Wnt induced reporter gene.

Taken together, the evidence clearly shows that GPC3 stimulates growth and proliferation of HCC through the Wnt signaling pathway (Capurro et al., 2005b). This discovery presented a novel target for potential HCC therapy.

Targeting GPC3 stimulation of HCC has already been shown to have some anti- tumourigenic effects on human HCC cell lines. A recent study from our lab indicated that sGPC3 was able to inhibit the growth of HCC cell lines in nude mice. (Zittermann et al., 2009). In some of these cell lines sGPC3 inhibited Wnt signaling. However, in others sGPC3 seemed to act by inhibiting different signaling pathways. This was expected, since GPC3 binds to various growth factors through its HS chains. Therefore, using 23 sGPC3 a therapeutic treatment for HCC has potential to inhibit HCC growth by simultaneously targeting multiple signaling pathways.

1.6 Hypothesis and Objectives

The hypothesis of this study is that GPC3 stimulates Wnt signaling in HCC by facilitating/stabilizing the interaction between Wnt and Fzd.

The objectives of this study are to demonstrate GPC3 stimulation of Wnt signaling as well as binding of GPC3 to both Wnt and Fzd. In addition, I will establish the role of

GPC3 GAG chains and the Fzd CRD domain in the interaction of GPC3 and Fzd.

Understanding the mechanism of GPC3 induced stimulation of Wnt signaling will help provide molecular targets for development of novel therapeutic treatments for HCC. 24

Chapter 2

Materials and Methods 25 2.1 Cell lines, Transfections and Plasmids

HEK293T cells were cultured in DMEM + 10% fetal bovine serum (FBS) in a 37˚C,

5% CO2 incubator. Transfections were done using Lipofectamine 2000 (Invitrogen).

HEK293T cells were plated at the following cell densities for transfection: 1.8x106 cells per 60mm plate, 1x106 cells per 35mm plate, 4.3x105 cells per well of 24 well plate.

Flag-tagged Fzd constructs were gifts from the lab of Dr. L. Attisano (University of

Toronto, Biochemistry). Fzd8-YFP plasmid was a gift from the lab of Dr. C. Niehrs

(German Cancer Research Centre) (Bilic et al., 2007). The lab of Dr. Jeremy Nathans

(Johns Hopkins University, Molecular Biology and Genetics) generously provided the

FzdCRD constructs. Expression vectors for wild type GPC3, GPC3ΔGAG and sGPC3

(also known as GPC3ΔGPI) were previously described (Gonzalez et al., 1998). The sGPC3-AP and sGPC3ΔGAG-AP vectors were prepared by inserting the human sGPC3 and sGPC3ΔGAG cDNAs into the BspE1 site of the pAP-Tag2 vector (GeneHunter®

Corporation).

2.2 Antibodies

The antibodies used were as follows: monoclonal mouse α-GPC3 antibody

(1G12)(Capurro et al., 2003); rabbit α-pLRP6 Ser1490 (Cell Signaling); monoclonal mouse α-LRP6 (Abcam); monoclonal mouse α-Flag M2 antibody (Sigma); rabbit α-Flag

F7425 antibody (Sigma), monoclonal mouse α-Myc (Santa Cruz); Goat α-mouse IgG

FITC (Jackson ImmunoResearch); rabbit α-mouse texas red (Jackson ImmunoResearch); goat α-rabbit IgG HRP (Stressgen); goat α-mouse IgG HRP (Stressgen). 26 2.3 Luciferase Assay

HEK293T cells were plated in a 6 well plate and co-transfected with a β- galactosidase vector (125ng), and a luciferase reporter driven by TOPFLASH, a β- catenin responsive promoter (500ng). Cells were also transfected with either GPC3 or EF empty vector control. Cells were trypsinized and re-plated into a 96-well plate at a density of 4.0x104 cells per well 16 hours post transfection. After a further 24 hour incubation, growth media was removed and purified mouse Wnt3a (R&D) was added to each well, in varying concentrations. Wnt3a was removed from the cells after 2.5 hours of incubation at 37˚C. Cells were washed with PBS and fresh growth media was applied to the cells for an additional 4 hours to allow for the expression of the luciferase reporter gene. After incubation, media was removed, cells were washed with PBS and the plates were sealed and stored at –80˚C overnight.

Luciferase assay reagents and buffers were from the Promega Luciferase Assay system. Cells were thawed in 15µl 1x cell culture lysis reagent and lysed in the plate for an additional 20 minutes. Lysates were moved to eppendorf tubes and cell debris pelleted for 10m at 17,000 rcf. For the Luciferase assay, 5 µl of lysate was added to 25 µl of luciferase assay reagent, vortexed, and light output was read for 10 seconds on a

Berthold Lumat LB9501 luminometer.

β-galactosidase activity was determined using 5µl of cell lysate. Lysate was mixed with 100 µl β-galactosidase assay reagent (60mM Na2HPO4, 40mM NaH2PO4, 10mM

KCL, 1mM MgCl2, 50mM β-mercaptoethanol, 330ng/ml O-Nitrophenyl-β-D- galactopyranoside), incubated at 37˚C until yellow colour developed (approximately 15 minutes) and samples were measured for light absorbance at 405 nm. Luciferase assay 27 readings were normalized by transfection efficiency (β-galactosidase activity). The assay was done in triplicate.

2.4 Surface Plasmon Resonance

Purification of His-tagged GPC3

Secreted his-tagged GPC3 in conditioned media was purified by a combination of anion exchange chromatography on DEAE-sephacel and affinity chromatography on Ni-

NTA-Agarose beads (QIAGEN). DEAE-sephacel gel (0.5 ml) (Pharmacia Biotech) was added into the conditioned medium (300 ml) and incubated at 4 °C overnight on a nutator mixer, centrifuged and loaded into an empty column. After washing with at least 10 volumes of 0.2 M NaCl in 50 mM phosphate buffer (pH 6.5), the bound material was eluted with 3-5 volumes of 2 M NaCl in 50 mM phosphate buffer (pH 6.5). The eluted fraction was diluted 10 fold with water and loaded on a column containing Ni-NTA- agarose beads (0.5 ml) (repeated 3 times). After washing the column with 5 volumes of wash buffer (50 mM Phosphate Buffer containing 300 mM NaCl and 10 mM Imidizole, pH 7.0), his-tagged GPC3 was eluted with 3-5 volumes of elution buffer (50 mM

Phosphate Buffer containing 300 mM NaCl and 200 mM Imidizole, pH 7.0). Finally, the glypican preparation was desalted by washing with phosphate buffered saline (PBS) using a Microcon YM-10 centrifugal filter (Millipore).

Biotinylation of GPC3

2 µg of GPC3 in 20 µl PBS was added to 2 µl of 1 mg/ml fresh EZ-LinkSulfo-NHS-

LC-Biotin (Pierce) in water (prepared just before use) and mixed well, then incubated at

RT for 30 minutes. The excess biotin was removed with 5 washes of 200 µl phosphate buffered saline (PBS) using a Microcon YM-10 centrifugal filter (Millipore). 28 Surface Plasmon Resonance

The kinetic constants of the interaction of Wnt3a with GPC3 or GPC3ΔGAG were evaluated using a BIAcore 3000 biosensor system. Biotinylated GPC3 (sGPC3/His) or nonglycanated GPC3 (sGPC3ΔGAG/His) were individually immobilized (1000 RU) in flow cells 2 and 3 on a SA sensorchip (BIAcore AB). Flow cell 1 without ligand was used as a correction reference for nonspecific binding. The indicated concentrations of

Wnt3a (R&D) in running buffer HBS-EP (BIAcore AB) were injected over these flow cells at 50 µl/minute for 90 seconds at 25 °C. After a 3 minute wash with HBS-EP, the flow cells were regenerated with 1 minute pulses of HBS-EP containing 1 M NaCl and

10 mM NaOH. Data were analyzed with BIAevaluation 3.0 software.

2.4 Alkaline Phosphatase Binding Assay

HEK293T cells were plated in 60 mm tissue culture plates and transfected with 1µg

Flag-tagged Fzd constructs, 1µg Myc-tagged FzdCRD constructs or vector control for 36 hours. Cells were lysed in RIPA buffer containing phenylmethanesulfonylfluoride

(PMSF) and aprotinin as protease inhibitors. Equal amounts of cell lysate were incubated with α-Flag or α-Myc antibody overnight at 4˚C. Tagged Fzds or FzdCRDs were immunoprecipitated with protein G beads for 1 hour at 4˚C. Beads were washed 3 times with RIPA buffer. The samples were blocked for 2 hours at room temperature (RT) in 1 ml 5% BSA in PBS. Each 1 ml fraction was split into 9 aliquots (3 per condition) and 300

µl of the appropriate conditioned media (alkaline phosphatase (AP), sGPC3-AP or sGPC3ΔGAG-AP) was added. Samples were rotated for 2 hours at RT. Beads were washed 4 times with wash buffer (75 mM BSA, 20 mM Hepes pH 7, 0.5% Triton, 150 mM NaCl). One 5 mg tablet of p-nitrophenyl phosphate disodium salt hexahydrate

(PNPP) (Sigma) was added to 2.5 ml alkaline phosphatase (AP) assay solution (0.5 M 29 2S-amino-2-methyl-1-propanol (AMP), pH 10.5 and 5 mM MgCL2) to create the AP substrate. Samples were incubated with 300 µl of substrate, and left at RT until colour developed (approximately 45 minutes). Absorbance at 405 nm was read for each sample.

Heparin competition: Indicated concentrations of purified porcine heparin (Sigma) were added to each AP conditioned media at the time of incubation with sample.

Conditioned media: HEK293T cells in 60 mm plates were transfected with 12 µg sGPC3-AP plasmid, 12 µg sGPC3ΔGAG-AP plasmid or .5 µg AP control plasmid.

Media was changed to 2.5 ml fresh DMEM+10% FBS 16 hours post transfection.

Conditioned media was harvested 48 hours later. To test activity of the conditioned media, 5 µl was incubated with 150 µl AP substrate solution for 10 minutes at 37˚C and assayed for light absorbance at 405 nm. Conditioned media was diluted with

DMEM+10% FBS as necessary so that all conditioned media had similar activities.

2.5 Co-immunoprecipitation

HEK293T cells were plated in 60 mm plates and transfected with 1µg GPC3 plasmid and 100ng Fzd7-Flag or Myc-tagged FzdCRDs. Cells were lysed with RIPA buffer containing aprotinin and PMSF and pre-cleared with protein G sepharose beads (Sigma) for 1 hour at 4˚C. Samples were incubated with antibodies against GPC3 (1G12) or

FzdCRD (α-Myc) overnight at 4˚C. Target proteins were immunoprecipitated with protein G sepharose beads for 1 hour at 4˚C. Beads were washed 3 times in RIPA buffer.

Immunoblot analysis was performed on each sample.

2.6 Immunocytochemistry

HEK293T cells were plated on coverslips in 4-well plates. Coverslips were treated in the following manner: 2 hour RT incubation in 2 N NaOH, 3 washes with water, 20 min RT incubation in 1% poly-L-lysine (Sigma), air dried and sterilized in 70% ethanol. 30 Cells were transfected with the indicated plasmid (200 ng GPC3, 200 ng Wnt3a, 20 ng

Fzd8-YFP or 20 ng Fzd4). One day after transfection cells were washed with 1xPBS and fixed in 4% paraformaldehyde for 20 minutes at RT, washed with PBS and permeabilized with 0.1% triton in PBS for 15 minutes at RT. For staining, all samples were blocked with 5% milk in PBS prior to 1 hour RT incubations with primary and secondary antibodies. Samples were washed 3 times with 1xPBS between antibody incubations.

Cover slips were mounted using Dako fluorescent mounting medium. 31

Chapter 3

Results 32 3.1 Glypican-3 Stimulates Wnt signaling

A range of Wnt concentrations in which GPC3 stimulates canonical Wnt signaling was determined. To this end, we performed a TOPFLASH-luciferase reporter assay in

HEK293T cells in the presence of varying concentrations of Wnt3a. HEK293T cells were transfected with a β-catenin-responsive promoter-driven luciferase reporter gene, β- galactosidase for transfection efficiency normalization and either GPC3 or EF empty vector control. One day after transfection, cells were incubated with the indicated concentrations of purified Wnt3a for 2.5 hours at 37˚C. Wnt3a conditioned media was removed, and cells were incubated in DMEM+FBS for a further 4 hours to allow for gene expression. Cells were then lysed and assayed for luciferase and β-galactosidase activity.

GPC3 is able to increase the cellular response to exogenous Wnt3a across a range of concentrations (Figure 3.1). We observe that GPC3 transfected cells show an increase in

Wnt signaling over EF transfected cells even when no purified Wnt3a has been added.

This can be explained by the presence of Wnts in the growth media with serum, in addition to the Wnts being expressed by HEK293T cells and acting in an autocrine/paracrine manner. The data also shows that cells expressing GPC3 reach a signaling plateau that is higher than that of the control cells. This suggests that GPC3 increases the signaling capacity of these cells. 33

Figure 3.1 Effect of GPC3 on Wnt responsive luciferase activity. Luciferase activity of transfected HEK293T cells (EF or GPC3) in response to different concentrations of purified Wnt3a in DMEM+10%FBS. Luciferase activity was normalized for transfection efficiency using β-galactosidase activity. The relative luciferase activity represents the ratio of activities between cells in the presence and absence of Wnt3a. The experiment was done twice in quadruplicate. One representative experiment is shown. Error bars represent ±SE. 34 3.2 GPC3 binds Wnt

GPC3 must be able to bind both Wnt and Fzd in order to facilitate/ stabilize their interaction. GPC3 has previously been shown to bind Wnt in HCC cell lines, and analogous findings have been shown in model organisms with other glypicans (Franch-

Marro et al., 2005). In order to verify that GPC3 is able to bind Wnt, we performed surface plasmon resonance (SPR) analysis. We tested the ability of Wnt to bind to

GPC3ΔGAG, a mutant variation of GPC3 with two point mutations that disrupt the attachement of HS chains to the protein core. Biotinylated Wild type GPC3 and

GPC3ΔGAG were each immobilized on a Streptavidin coated sensor chip. Several concentrations of Wnt3a were injected onto the sensor chips, allowed to bind to the immobilized GPC3, and then washed off with buffer.

The analysis showed that affinity for Wnt3a is very similar for GPC3 and

GPC3ΔGAG (Kd values: 3.0± 2.1 nM, 2.1±1.0 nM respectively). However the association and dissociation constants differed by an order of magnitude between the two protein forms (Table 3.1). Binding curves are shown in Figure 3.2 for Wnt3a with wild type GPC3 (Figure 3.2 A) and GPC3ΔGAG (Figure 3.2B). The first section of the curve, up to 120 seconds, indicates the association phase and the dissociation curve falls after the 120 second mark. During the association phase, Wnt3a binds faster to GPC3ΔGAG than to wild type GPC3. During dissociation, Wnt3a remains bound to wild type GPC3 longer than GPC3ΔGAG. This confirms earlier studies (Capurro et al., 2005b) indicating that GPC3 core protein has a major role in binding Wnt while GAG chains are required for optimal Wnt3a binding. 35

Table 3.1 Kinetic parameters for the interaction of Wnt3a with immobilized GPC3

*ka, kd and Kd values were determined using a 1:1 Languimuir binding model. Each value is expressed as the mean ± SE of five different concentrations. 36

Figure 3.2 SPR analysis of Wnt3a interacting with GPC3 and GPC3ΔGAG. Biotinylated wild type GPC3 (A) and GPC3ΔGAG (B) were each immobilized in flow cells on a streptavidin sensor chip. A blank flow cell without GPC3 was used as a reference to correct for nonspecific binding. Arrows mark the termination of ligand injection (120 seconds). Increasing concentrations of Wnt3a (bottom to top: 6.7, 13.3, 26.7, 53.3 and 106.7nM) were injected on the surfaces of flow cells. The nonspecific binding was subtracted from the sensorgram. RU, relative units. 37

3.3 GPC3 increases phosphorylation of LRP6

It has been previously reported that GPC3 increases β-catenin levels in the cytoplasm

(Capurro et al., 2005b). An increase in β-catenin levels is one of the final steps in the

Wnt signaling cascade. If GPC3 regulates Wnt signaling at the level of Wnt binding to

Fzd, then we should observe changes in the signaling cascade prior to β-catenin stabilization.

Phosphorylation of LRP6 is an early event in Wnt signaling. In response to clustering of Wnt receptors Fzd and LRP5/6 on the cell membrane, the intracellular domain of LRP5/6 is phosphorylated at multiple sites. LRP5/6 has 5 conserved Pro-Pro-

Ser-Pro (PPSP) motifs in its intracellular domain (Niehrs and Shen, 2010). Each of these sites is constitutively phosphorylated at low levels and is phosphorylated further in response to Wnt. Serine 1490 (S1490) is within PPSP motif A, and is phosphorylated by

GSK3 and GRK5/6 in response to Wnt (Niehrs and Shen, 2010). Because the action of

GPC3 is predicted to be upstream of this phosphorylation, we expect to observe increased levels of pLRP6 at S1490 when cells express GPC3.

To investigate the effect of GPC3 expression on phosphorylation of LRP6, we transfected HEK293T cells with GPC3 or vector control plasmids. Cells were lysed 24 hours post transfection and lysate samples were run on a gel for immunoblotting. Levels of endogenous pLRP6-S1490, total endogenous levels of LRP6, transfected GPC3 were determined by immunoblotting. β-actin levels were used as a loading control. Cells expressing GPC3 have higher levels of pLRP6 than cells transfected with empty vector control (Figure 3.3). These results indicate that GPC3 acts upstream of LRP6 phosphorylation. 38

Figure 3.3 GPC3 increases phosphorylation of LRP6 HEK293T cells were transfected with GPC3 or vector control. Cells were harvested 24 hours post transfection, lysed and assayed for levels of the indicated proteins. Immunoblots show endogenous pLRP6, LRP6 and transfected GPC3. β-actin was used to compare protein loading between samples. This figure is representative of two experiments. 39 3.4 Glypican-3 binds multiple Fzd receptors via GAG chains

Since GPC3 is able to bind to Wnt3a, and GPC3 has been shown to stimulate canonical Wnt signaling in this context, we hypothesized that GPC3 would also be able to bind to the Wnt receptor Fzd. To demonstrate that GPC3 and Fzd interact, we first performed a co-immunoprecipitation experiment in HEK293T cells (Figure 3.4-A).

GPC3 and Flag tagged Fzd7 (Fzd7-Flag) were co-transfected into HEK293T cells. Cells were harvested 48 hours post transfection and lysed with RIPA buffer plus protease inhibitors. GPC3 was immunoprecipitated from lysates using an α-GPC3 mAb (1G12), and the precipitate was immunoblotted for Fzd7-Flag. Fzd7 bound to GPC3 in the immunoprecipitation assay, as shown in Figure 3.4 A.

To confirm these results we performed a cell binding assay in which sGPC3 binds to

Fzd4 on the surface of HEK293T cells. HEK293T cells were transfected with sGPC3 and either Fzd4-Flag or vector control. Cells were fixed 24 hours post transfection and blocked with 5% milk in preparation for immunofluorescent staining. Mouse α-GPC3 mAb was used as a primary antibody followed by goat α-mouse IgG FITC. In the absence of Fzd4 transfection, there is no sGPC3 bound to the cells. However, upon Fzd4 transfection, sGPC3 is visibly bound to cells in the monolayer (Figure 3.4 B). These observations further support the model that GPC3 is able to bind Fzd at the cell membrane.

The final binding assay was designed to avoid co-expression of GPC3 and Fzd. This assay confirms that binding between GPC3 and Fzd is real and not an artifact of co- overexpression. Flag tagged Fzds 1,7 and 8 were transfected into HEK293T cells. Cells were lysed 36 hours post transfection and Fzd proteins were immunoprecipitated with protein G sepharose beads. Protein G beads, with bound Fzd proteins, were blocked with 40 5% BSA and incubated with alkaline phosphatase (AP)-tagged sGPC3, sGPC3ΔGAG-

AP or control AP conditioned media. Fzd and AP tagged constructs were allowed to bind for 2 hours before being washed and evaluated for AP activity. AP activity for each Fzd sample represents the amount of GPC3 bound to Fzd. Each Fzd receptor tested showed significant binding to sGPC3-AP over AP control, and interestingly sGPC3ΔGAG-AP was not able to bind Fzd (Figure 3.4 C).

Using three different methods we deomstrated that GPC3 binds to the Wnt receptor,

Fzd. In addition, we conclude that the GAG chains of GPC3 are required for interaction with Fzd. 41 42

Figure 3.4 Glypican-3 binds to Frizzled (A) HEK293T cells were transfected with Flag-tagged Fzd7 alone or in combination with GPC3. GPC3 was immunoprecipitated from cell lysate with αGPC3 (1G12) 48h post-transfection. Top Fzd7 was probed with αFlag (M2). Presence of Fzd7 (Middle) and GPC3 (bottom) in whole lysate, was assessed by Immunoblot. (B) HEK293T cells were transfected with Fzd4 and sGPC3 (left) or vector control and sGPC3 (right). Differential Interference Contrast (DIC) image shows bright field of cells. Cells were stained for GPC3. Secreted GPC3 is only visible bound to cells transfected with Fzd4. (c) HEK293T cells were transfected with Fzd1, Fzd7, Fzd8 or vector control and harvested 48h post transfection. Fzds were immunoprecipitated and incubated with sGPC3-AP, sGPC3ΔGAG-AP or AP alone. After washing, AP substrate was added to samples, and binding of sGPC3/sGPC3ΔGAG was quantified by measuring absorbance at 405nm. Final values were obtained by subtracting the binding to vector control. Bars represent ±SE. This is a representative figure of 4 experiments. 43 3.5 GPC3 and Fzd interact on the cell membrane

The model of GPC3 facilitation/stabilization of Wnt and Fzd predicts that GPC3 and

Fzd interact on the cell membrane. Therefore, we next investigated whether Fzd and

GPC3 co-localize on the membrane of HEK293T cells. This will also confirm that previously described Fzd-GPC3 pull down assay (Figure 4.3 A) was representative of the proteins interacting on the cell membrane, and not as over-expression artifacts within the cell.

To this end we co-transfected GPC3 and Fzd8-YFP in HEK293T cells. Cells were fixed 24 hours post transfection and GPC3 was fluorescently labeled with rabbit α-mouse texas red. The cellular localization of both proteins was assessed by immunofluorescence. As expected, both GPC3 and Fzd8-YFP have a strong membrane presence (Figure 3.5). Co-localization of Fzd8 and GPC3 (yellow) on the cell membrane is shown in the third panel of Figure 3.5. This is evidence that Fzd and GPC3 interact on the cell membrane in our over-expression system. 44

Figure 3.5 GPC3 and Fzd co-localize on the cell membrane HEK293T cells were transfected with GPC3, Fzd8-YFP or both plasmids and fixed 24h post transfection. Cells were stained with α GPC3 (1G12) and α mouse IgG texas red. This is a representative figure of 3 experiments. 45 3.6 Heparin inhibits binding of GPC3 to Fzd

Previous data (Figure 3.4 C) indicates that GPC3-Fzd interaction is mediated by the

HS GAG chains of GPC3. If this finding is correct, binding of GPC3 to Fzd would be prevented by competitive binding of free HS. Heparin is a short, highly sulfated GAG chain commonly used in binding experiments as an HS competitor. Thus, we performed a heparin competition binding assay to confirm the necessity of GAG chains in GPC3-Fzd binding. For this assay, HEK293T cells were transfected with Fzd7-Flag or empty vector control. Cell lysates were incubated with α-FLAG antibody and Fzd7-Flag was immunoprecipitated from the lysate with protein G sepharose beads. Next, the protein G beads were washed and blocked with 5% BSA before being incubated with sGPC3-AP and the indicated concentrations of heparin. Samples were then assayed for AP activity.

Activity of vector control samples was subtracted from the activity of samples containing

Fzd7-Flag. As the model predicts, increasing amounts of heparin are able to reduce binding of GPC3 to Fzd in a dose dependant manner (Figure 3.6). In fact, heparin competition was able to eliminate binding completely, demonstrating the critical role of

GAG chains in GPC3-Fzd binding. Using Graph Pad Prism software, we performed non- linear regression using a variable slope, estimating an IC50 of 2.1x10-3 mg/ml. 46

1.5

1.0

0.5

0.0

-0.5 -5 -4 -3 -2 Log Heparin (mg/ml)

Figure 3.6 Heparin inhibits binding of Glypican-3 to Frizzled HEK293T cells were transfected with Fzd7 or vector control and harvested 48h post transfection. Fzd7 was immunoprecipitated and incubated with sGPC3-AP along with increasing concentrations of heparin. After washing, AP substrate was added to samples. Binding of sGPC3-AP was quantified by measuring absorbance at 405nm. Final values were obtained by subtracting the binding of sGPC3-AP to immunoprecipitated from vector control cells. Samples were processed in triplicate. Bars represent ±SE. This is a representative figure of 3 experiments. 47 3.7 FzdCRD domain is involved in binding GPC3

We have shown that GPC3 binds Fzd, and that binding is mediated by the GAG chains of GPC3. Next, we sought to identify a domain of Fzd involved in binding to

GPC3. Fzd is a large, 7 span transmembrane protein and thus much of the protein is hidden from interaction with GPC3 because it is located within the cell membrane, or in the intracellular compartment. The N-terminal, extracellular portion of Fzd contains a cysteine rich domain that is known to bind Wnt proteins. We decided to test whether the

FzdCRD domain is able to bind GPC3. To this end, we performed a co- immunoprecipitation of Myc tagged, GPI-anchored FzdCRD constructs with wild type

GPC3. The FzdCRDs and GPC3 were co-transfected in HEK293T cells and 48 hours post-transfection, cells were harvested and lysed with RIPA buffer plus protease inhibitors. FzdCRD was immunoprecipitated from cell lysates using an α-Myc antibody.

Immunoblots probed for GPC3 show that the CRD domains from multiple Fzd receptors are able to co-immunoprecipitate with GPC3 (Figure 3.7). The immunoblot also shows strong bands corresponding to GPC3 core protein. This is contradictory to results showing that GPC3 GAG chains are required for binding to Fzd.

GPC3-FzdCRD interaction was also assessed using the binding assay described in section 3.4. AP-tagged sGPC3 was incubated with FzdCRDs bound to sepharose protein

G beads. The amount of sGPC3 bound to each FzdCRD was assessed by a colourmetric assay for AP activity. For each FzdCRD tested, AP and sGPC3ΔGAG showed negligible binding, while sGPC3 bound to each one (Figure 3.8). Therefore we conclude from these two experiments that the CRD domain of Fzd, at least partially, mediates binding to

GPC3. 48

Figure 3.7 Frizzled cysteine rich domain co-immunoprecipitates with GPC3. HEK293T cells were transfected with myc tagged GPI linked FzdCRDs 3-8 or vector control in addition to GPC3. Myc tagged GPI-FzdCRDs were immunoprecipitated from cell lysate 48h post transfection. (Panel 1) Bound GPC3 was probed with αGPC3 antibody (1G12). (Panel 2) Presence of FzdCRDs in the precipitate and (Panel 3) GPC3 in whole lysate was assessed by immunoblotting. This is a representative figure of 2 experiments. 49

0.6 AP 0.5 ΔGAG-AP 0.4 GPC3-AP

0.3

0.2

0.1

0.0

Figure 3.8 GPC3 binds the cysteine rich domain of Frizzled HEK293T cells were transfected with membrane bound FzdCRD 4-8 or vector control and harvested 48h post transfection. FzdCRDs were immunoprecipitated and incubated with sGPC3-AP, sGPC3ΔGAG-AP or AP alone. After washing, AP substrate were added to samples, and binding of sGPC3/sGPC3ΔGAG/AP was quantified by measuring AP activity. Final values were obtained by subtracting the binding to vector control. Samples were processed in triplicate. Bars represent ±SE. This is a representative figure of 3 experiments. 50

Chapter 4

Discussion 51 4.1 Discussion

In this study I sought to uncover the mechanism by which GPC3 stimulates canonical

Wnt signaling. I hypothesized that GPC3 promotes Wnt activity by stabilizing/facilitating the interaction between Wnt and Fzd. This hypothesis is based on the fact that GPC3 is able to bind both Wnt and Fzd, and GPC3 does not act as a competitive inhibitor. My results showing that GPC3 interacts with Fzd on the cell membrane, together with the previous finding that GPC3 interacts with Wnts, provides strong support to this hypothesis.

This study shows a novel interaction between GPC3 and the Wnt receptor, Fzd.

Furthermore, it has demonstrated that binding is mediated by the GAG chains of GPC3 and, at least partially, by the FzdCRD domain.

GPC3 stimulates Wnt signaling at the cell membrane

If our hypothesis is correct, it is expected that GPC3, acting on autocrine Wnts, would induce the phosphorylation of LRP5/6, an early event in the canonical Wnt signaling pathway. Here we show that GPC3 up regulates the phosphorylation of LRP6 at

S1490, a site phosphorylated in response to Wnt signaling (Niehrs and Shen, 2010). The increase of pLRP6 levels in response to GPC3 expression indicates that GPC3 is acting upstream of LRP6 phosphorylation. This is consistent with the hypothesis that GPC3 facilitates/stabilizes the interaction between Wnt and Fzd by forming a tripartite complex.

GPC3 binds Fzd

It has previously been speculated that glypicans interact with Fzd, but convincing experimental support for this hypothesis was lacking. One previous study by Ohkawara et al. found that Xgly4 was able to pull-down Fzd7. However, in the same experiment they 52 show that Xgly4 also co-immunoprecipitates with two other receptors that were used as negative controls (Ohkawara et al., 2003).

In the present study, using mammalian GPC3 and mammalian Fzd constructs, it is conclusively shown for the first time that GPC3 binds Fzd. In addition to observing specific co-immunoprecipitation of GPC3 and Fzd7, we performed three other assays that support this finding. First, sGPC3 bound to HEK293T cells transfected with Fzd4. A binding assay was also done using immunoprecipitated Fzds and sGPC3-AP conditioned media. Finally to confirm that GPC3 and Fzd were interacting on the cell membrane and not as over-expression artifacts inside the cell, we observed GPC3 and Fzd co-localize on the cell surface.

Of the 10 mammalian Fzd receptors, we chose several to work with. The rational behind this selection was based on expression profile of Fzds in HCC. It has been reported that Fzds 3,6 and 7 are routinely over-expressed in HCC cell lines (Bengochea et al., 2008). Therefore we chose Fzd 7 to represent Fzds over-expressed in HCC and Fzds

1, 4 or 8 to represent Fzds that were not routinely found in HCC. Our results suggest that

GPC3 interacts similarly with both groups of Fzds.

Model for GPC3 binding receptors and ligands

Our results showing that GPC3 binds Fzd receptors are new and interesting findings.

Classically, the role of GPC3, and homologues in lower organisms, has been described solely by the interaction with secreted morphogens and ligands. This study provides the first example of a glypican binding to a signaling receptor. Another example of this type of interaction has been found recently in our lab. It was observed that Glypican-5 is able to bind the Hh receptor Patched, also through its GAG chains (Li, unpublished data). Our findings provide evidence for a new model of glypican action where the ligand binds to 53 glypican with low affinity and is presented to a signaling receptor that is bound to the glypican GAG chains (Figure 4.1).

Figure 4.1 Glypican-3 facilitates/stabilizes Wnt and Frizzled GPC3 core protein binds to Wnt while the GPC3 GAG chains bind Fzd. In this manner GPC3 facilitates/stabilizes the interaction between Wnt and Fzd resulting in an increase in Wnt signaling. 54 Role of GAG chains

The role of GPC3 HS chains remains an important area of study. In this study I determined that binding of GPC3 to Fzd is mediated by the HS chains of GPC3.

However, Wnt signaling is still activated by GPC3ΔGAG, although not to the same extent as by wild type GPC3 (Capurro et al., 2005b). We observed that the HS GAG chains initially interfere with direct binding of Wnt3a to the GPC3 core protein (Figure

3.2A). However, during dissociation, the GAG chains delay the release of Wnt3a by

GPC3 (Figure 3.2B). In wild type GPC3 the GAG chains are able to assist the core protein in keeping Wnt at the cell surface.

The HS GAG chains of GPC3 bind to both Wnt and Fzd, keeping Wnt in close proximity with the receptor. Consequently, there is clear stimulation of Wnt signaling by wild type GPC3. This draws into question how the GPC3 core protein can activate Wnt signaling in the absence of GAG chains.

Previous work has shown that in the absence of GAG chains Wnt is still able to bind the GPC3 core protein on the cell surface (Capurro et al., 2005b). Even without direct interaction of GPC3ΔGAG and Fzd, the local concentration of Wnt would be increased at the cell surface and increased Wnt signaling would occur. Therefore, GPC3ΔGAG still increases the availability of Wnt to Fzd, although it is not as efficient as the wild type

GPC3.

There are two ways GPC3ΔGAG may continue to interact with Fzd despite the lack of GAG chains. First, both Fzd and GPC3 are able to bind Wnt and thus each could bind a common Wnt protein. This would essentially create a Wnt bridge between GPC3 and

Fzd when the two proteins came in close contact. GAG chains may act as a net, capturing

Fzd proteins for Wnt presentation. In the absence of GAG chains, GPC3ΔGAG would 55 collide with Fzd less frequently, but binding would still occur. This would result in less stimulation of Wnt signaling.

The second possibility is that both the GPC3 core protein and Fzd bind another component of the Wnt signaling complex. A strong candidate would be LRP5/6 as it is known to associate with Fzd through mutual binding of Wnt. We have preliminary data showing that GPC3 is able to bind LRP6, but the role of the GAG chains in this coupling remains unknown (data not shown). Both of these are plausible scenarios to explain the somewhat contradicting evidence regarding GAG chain function. Although we conclude that the GAG chains are essential for direct receptor binding, we propose that through an indirect association with Fzd, GPC3ΔGAG is still able to increase Wnt signaling.

It is interesting to note that GPC3 binds to all the Fzd family members tested in this study. There is some indication that various GAG chain modifications could render different binding consensus sequences allowing for targeted binding (Gandhi and

Mancera, 2008). This study provides no evidence that GPC3 binds preferentially to any given Fzd. However GAG modifications are cell type specific and thus it would be interesting to investigate the ability of GPC3 to bind to various Fzds in a different cell type – perhaps one where GPC3 does not increase canonical Wnt signaling.

Fate of membrane complex

There is currently little evidence as to the fate of the putative GPC3/WNT/Fzd complex. There are two scenarios currently being investigated. First, it is possible that the GPC3/Wnt/Fzd complex is part of a much larger aggregate termed the signalosome.

Secondly, the complex may undergo endocytosis. These fates are not mutually exclusive, and both may play a role in GPC3 activation of Wnt signaling. 56 Several years ago Bilic et al. proposed the existence of a signalosome, a large canonical Wnt signaling aggregate defined by a clustering of phosphorylated LRP6 that forms quickly in response to Wnt treatment (Bilic et al., 2007). The signalosome also contains other Wnt signaling proteins including Fzd, Dishevelled, Axin and GSK3β. The signalosome model predicts that clustering of pLRP6 leads to further LRP6 phosphorylation and downstream signaling. Given the interaction of GPC3 with Wnt and

Fzd, both found in the signalosome, it would not be surprising if GPC3 was also found to be one of the proteins in this aggregate.

Recently it has been shown that a soluble proteoglycan, Tspan12 stimulated Wnt signaling by assisting Fzd receptors to form large aggregated complexes (Junge et al.,

2009). This raises the possibility that GPC3 may assist the formation of a protein aggregate on the cell membrane. The key differences between Tspan12 and GPC3 are that Tspan12 is not membrane bound and it doesn’t bind Wnt, limiting the possible ways it could influence Wnt signaling. Although GPC3 has the ability to facilitate/stabilize the interaction between Wnt and Fzd, it is possible that it could facilitate the aggregation of

Wnt signaling components.

The second possible fate is that the complex is endocytosed. There is a plethora of literature regarding the importance of endocytosis in Wnt signaling. Debate exists over whether it is required for the transmission of Wnt signaling or for the down regulation and degradation of signaling components (see review (Gagliardi et al., 2008)). Given the complexity and flexibility of Wnt signaling, it seems likely that endocytosis is required in some scenarios and not in others.

Whether endocytosis is required for GPC3 mediated stimulation of Wnt signaling is unknown, although we have observed low levels of GPC3 internalization in HEK293T 57 cells (data not shown). Yamamoto et al. looked at Wnt signaling in HEK293 cells (the same cell system used in this study) and found that caveolin dependent internalization of

Wnt signaling components was necessary for Wnt dependent accumulation of β-catenin

(Yamamoto et al., 2006). Bilic et al. have reported some limited co-localization of the signalosome with Caveolin (Bilic et al., 2007) and our group obtained similar results in a preliminary study of GPC3 and caveolin interaction (unpublished data). However, caveolin internalization is not the only endocytic pathway relevant to Wnt signaling.

Canonical Wnt signaling in L-cells is dependent on clathrin mediated endocytosis

(Blitzer and Nusse, 2006). Yamamoto et al. also showed that in the absence of LRP6,

Fzd5 and Wnt3a were internalized by clathrin coated vesicles (Yamamoto et al., 2006).

Therefore, the first step in characterizing internalization of GPC3 and Wnt will be to determine the relevant endocytic pathway. Furthermore, it will be interesting to investigate whether GPC3 affects the route of internalization or the rate of endocytosis in

Wnt signaling.

Role of FzdCRD in GPC3 binding

Fzds are a family of ten 7 span transmembrane proteins that act as high affinity receptors for Wnt proteins (Huang and Klein, 2004). The extracellular domain of each

Fzd contains a highly conserved cysteine rich domain containing 10 cysteine residues

(Xu and Nusse, 1998). Previous biochemical and crystallography studies have shown binding of Wnt to the FzdCRD (Dann et al., 2001; Hsieh et al., 1999). The FzdCRD domain is extracellular, easily accessible to GPC3 and has a high affinity for Wnt. We hypothesized that GPC3 presents Wnt to Fzd at the CRD domain. In this scenario, we would expect the GAG chains of GPC3 to bind to the FzdCRD domain. 58 Using GPI anchored FzdCRD regions from six different Fzd family members, we co-transfected these constructs with GPC3 into HEK293T cells and performed co- immunoprecipitation assays. The results show that GPC3 binds each FzdCRD tested.

These results indicate that GPC3 is interacting with the CRD domain of Fzd.

The second assay tested the ability of sGPC3-AP or sGPC3ΔGAG-AP to bind to 5 different immunoprecipitated FzdCRD domains. sGPC3-AP showed significant binding to each FzdCRD tested. However, echoing the binding results of GPC3 and Fzd, sGPC3ΔGAG showed no binding to any of the FzdCRD domains. This indicates, as expected, that the GAG chains of GPC3 are required for binding to the FzdCRD domains.

Of note is the fact that GPC3 core protein is very prominently pulled down with several FzdCRD domains in the co-immunoprecipitation assay. A possible explanation for this observation is GPC3 aggregation on the cell membrane. In an over expression system, it is unlikely that all GPC3 proteins are properly glycanated, due to lack of sufficient glycanation enzymes. In addition, we have observed that GPC3 is able to aggregate on the cell surface (data not shown). We propose that the strong band of GPC3 core protein observed in the co-immunoprecipitation assay is due to the co- immunoprecipitation of aggregated non-glycanated GPC3 with wild type GPC3. In addition, we have observed in the lab that our GPC3 antibody, 1G12, preferentially binds to non-glycanated protein which would further emphasize the imbalance between core protein and glycanated GPC3 levels.

Therapeutic Application

Understanding the mechanism of GPC3 stimulated Wnt signaling in HCC may help in the development of therapeutic treatment for HCC. Recently, it has been shown that sGPC3 acts as an inhibitor of tumorigenic signals including Wnt (Zittermann et al., 59 2009). In our current understanding of Wnt/Fzd/GPC3 interactions, GPC3 captures

Wnt at the cell membrane and presents it to Fzd to initiate Wnt signaling. In contrast, sGPC3, which is not membrane bound, binds Wnt and removes it from the cell surface causing a reduction in Wnt signaling. HCC cell lines expressing sGPC3 injected into the flanks of SCID mice proliferated slower than the controls (Zittermann et al., 2009). In some cases tumour penetrance, the number of mice that developed tumours, was also decreased. Interestingly, the Huh6 cell line showed the highest activation of Wnt signaling and also showed the greatest response to sGPC3 treatment with almost complete inhibition of tumour growth. Zittermann et al. also demonstrated that sGPC3 affects more than just Wnt signaling. Treatment with sGPC3 inhibited phosphorylation of ERK1/2, AKT and reduced levels of phosphotyrosine in various HCC cell lines. The study by Zittermann et al. serves as proof-of-principle that GPC3 acts on several pathways that stimulate HCC to reduce tumour growth. The sequestration of growth factors by sGPC3 fits with our hypothesis of membrane bound GPC3 binding Wnt and presenting it to Fzd when they come in contact on the cell membrane. If GPC3 is no longer present on the cell membrane, it is no longer able to present ligand to receptor and the result is signaling inhibition.

Future Study

There are still many questions to be answered concerning the role of GPC3 in Wnt signaling. As noted in the introduction, GPC3 stimulation of canonical Wnt signaling has been reported in Hepatocellular Carcinoma (HCC). GPC3 stimulates non-canonical signaling at the expense of canonical Wnt signaling in most tissues during normal development (Song et al., 2005). The mechanism of GPC3 tissue specificity is unknown, but may be related to the binding profile of GPC3 with Wnt signaling components. The 60 present study showed that GPC3 can bind several Fzds and previous work has shown binding to Wnt7b and Wnt3a (Capurro et al., 2005b). An important future study will be to systematically determine which of the 10 mammalian Fzds and 19 Wnts are able to bind to GPC3.

We speculate that GPC3 is able to bind the majority of Wnts and Fzds. If this is the case, than binding specificity of GPC3 to various signaling components is not crucial to determining its function. Instead, GPC3 may bind to all Fzds and Wnts that are available.

In this case, the function of GPC3 would be dependant upon the expression profile of

Wnt receptors and ligands in each cell. GPC3 would undergo the same mechanism of action in each context, gathering Wnt from the extracellular environment and presenting it to Fzd. The Wnts and Fzds available to bind GPC3 would be dependant upon the expression profile of the cell. Variation in the expression of Wnts and Fzds would alter which Wnt signaling pathways were activated.

However, preferential binding of GPC3 to various Wnts and Fzds has not been ruled out. Binding of GPC3 to Wnt and Fzd is at least partially mediated by GAG chains in both cases. Modification of GAG chains, such as sulfation states and epimerization, influences interaction with various signaling molecules (Ai et al., 2003). It could be that

GPC3 GAG chains are modified to preferentially bind certain Wnt signaling components in various tissues. In addition, GAG chains could be differentially modified between cell types, causing binding to Wnt or Fzd to vary across cell types. Ultimately, a balance of biochemical specificity and cellular context will likely determine the specificity of GPC3 for various Wnts and Fzds.

The role of GPC3 HS chains remains an important area of study. GAG chains are required for optimal stimulation of Wnt signaling, but GPC3ΔGAG is also able to 61 stimulate signaling through one of the scenarios described in previous sections. Further work is needed to determine the exact mechanism of action of GPC3ΔGAG. It is important to note that the concentration of signaling components may also be important when determining the function of GAG chains. When Wnt signaling components are present in high concentrations, HS chains may not be needed, as GPC3, Wnt and Fzd would readily encounter each other without the extra reach provided by the long sugar chains. In very low concentrations of Wnt, signaling GPC3 may require GAG chains to facilitate interaction with Wnt. Conversely, with extremely high concentrations of Wnt,

GAG chains may prove unnecessary as binding of Wnt to Fzd would be saturated. Thus, future study into the role of GAG chains should pay close attention to ratios of GPC3,

Fzd and Wnt in experimental systems.

Summary

In this study we demonstrate that GPC3 stimulates the canonical Wnt signaling pathway and that GPC3 is able to bind both Wnt and its receptor Fzd. The binding of

GPC3 to Fzd is mediated by the GAG chains of GPC3, which interact with the cysteine rich domain of Fzd. Since Fzd and GPC3 are both able to bind to Wnt, and no competitive inhibition takes place, this indicates they are part of the same signaling complex. These observations are consistent with the model of GPC3, Fzd and Wnt forming a tripartite complex on the cell surface that facilitates/stabilizes the interaction of

Wnt with Fzd, resulting in increased signaling.

This mechanism will be valuable for future study of targets for therapeutic treatments of hepatocellular carcinomas. 62

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