GLYPICAN-3 STIMULATES THE WNT SIGNALING

PATHWAY BY FACILITATING/ STABILIZING THE

INTERACTION OF WNT LIGAND AND FRIZZLED RECEPTOR

by

Tonya Leigh Martin

A thesis submitted in conformity with the requirements

for the degree of Master of ScienceGraduate Department of Medical Biophysics

University of Toronto

© Copyright by Tonya Leigh Martin (2010)

ii

Glypican-3 stimulates the Wnt signaling pathway by facilitating/ stabilizing theinteraction of Wnt ligand and Frizzled receptor

Tonya Leigh Martin

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

Graduate Department of Medical Biophysics, University of Toronto

© Copyright by Tonya Leigh Martin (2010)

Abstract

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.

iii

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

iv

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.

v

Table of Contents

ABSTRACT IIACKNOWLEDGEMENTS IIITABLE OF CONTENTS VATTRIBUTIONS VILIST OF ABBREVIATIONS AND SYMBOLS VII

CHAPTER 1INTRODUCTION 1

1.1 GLYPICANS 21.2 GLYPICAN FUNCTION IN MODEL ORGANISMS 71.3 THE ROLE OF MAMMALIAN GLYPICAN-3 IN DEVELOPMENT 121.4 WNT SIGNALING PATHWAY 161.5 HEPATOCELLULAR CARCINOMA 211.6 HYPOTHESIS AND OBJECTIVES 23

CHAPTER 2MATERIALS AND METHODS 24

2.1 CELL LINES, TRANSFECTIONS AND PLASMIDS 252.2 ANTIBODIES 252.3 LUCIFERASE ASSAY 262.4 SURFACE PLASMON RESONANCE 272.4 ALKALINE PHOSPHATASE BINDING ASSAY 282.5 CO-IMMUNOPRECIPITATION 292.6 IMMUNOCYTOCHEMISTRY 29

CHAPTER 3RESULTS 31

3.1 GLYPICAN-3 STIMULATES WNT SIGNALING 323.2 GPC3 BINDS WNT 343.3 GPC3 INCREASES PHOSPHORYLATION OF LRP6 373.4 GLYPICAN-3 BINDS MULTIPLE FZD RECEPTORS VIA GAG CHAINS 393.5 GPC3 AND FZD INTERACT ON THE CELL MEMBRANE 433.6 HEPARIN INHIBITS BINDING OF GPC3 TO FZD 453.7 FZDCRD DOMAIN IS INVOLVED IN BINDING GPC3 47

CHAPTER 4DISCUSSION 50

4.1 DISCUSSION 51

BIBLIOGRAPHY 62

vi

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.

vii

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

viii

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

1

Chapter 1

Introduction

21.1 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 glypicanfamily of proteins. Glypicans have an N-terminal domain containing 14 conservedcysteine residues that form stabilizing disulphide bonds. Glypican-3 has twoinsertion sites for heparan sulfate proteoglycan chains. Heparan sulfate chainscarry a negative charge and can bind to proteins with positively chargeddomains. The C-terminal end of glypican-3 is a linker domain that connects theprotein to a GPI anchor in the cell membrane. Glypican-3 has cleavage sites forphospholipase-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 arelinked to a serine residue on the glypican core protein. A linkage tetrasaccharide joins the sugarchain to the protein. Chain synthesis begins with copolymerization of N-acetylglucosamine andglucuronic acid residues. The first chain modification is the removal of N-acetate andreplacement with N-sulfate by N-deacetylase N-sulfotransferase enzyme. Sulfotransferases addsulfate groups to iduronic acid (2-O) and N-acetylglycosamine (3-O, 6-O) residues. The sulfategroups are donated by 3-phosphoadenyl-5-phosphosulfate (PAPS).Varki, A. (2008). Essentials of Glycobiology second edition. Cold Spring Harbor, NY: Cold Spring HarborLaboratory Press.

6Sugar 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

7surface 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.,

82005; 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

9is 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

10promotes 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).

11Recently 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.

12Zebrafish 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

13to 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

14between 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,

15with 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

16that 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

17through 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

18along 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).

19A

B

20

Figure 1.3 Wnt Signaling A) Inactive Wnt signaling. When Wnt is absent, adestruction complex is formed in the cytoplasm consisting of adenomatouspolyposis coli, Axin, glycogen synthase kinase 3 and casein kinase 1. Thedestruction complex binds and phosphorylates β-catenin, targeting it fordestruction. B) Active Wnt signaling. Wnt binds to Fzd and co-receptorlipoprotein receptor related protein5/6 (LRP5/6). Clustering andphosphorylation of LRP5/6 creates a binding site for Axin, which issubsequently drawn away from the destruction complex. In the absence ofAxin, 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 thenucleus. Here, it associates with TCF/LEF transcription factors to allowtranscription of Wnt responsive genes.

211.5 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

22sensitive 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

23sGPC3 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

252.1 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).

262.3 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

27readings 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).

28Surface 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

292S-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.

30Cells 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

323.1 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. Luciferaseactivity of transfected HEK293T cells (EF or GPC3) in response to differentconcentrations of purified Wnt3a in DMEM+10%FBS. Luciferase activity wasnormalized for transfection efficiency using β-galactosidase activity. The relativeluciferase activity represents the ratio of activities between cells in the presence andabsence of Wnt3a. The experiment was done twice in quadruplicate. Onerepresentative experiment is shown. Error bars represent ±SE.

343.2 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. Eachvalue is expressed as the mean ± SE of five different concentrations.

36

Figure 3.2 SPR analysis of Wnt3a interacting with GPC3 and GPC3ΔGAG. Biotinylatedwild type GPC3 (A) and GPC3ΔGAG (B) were each immobilized in flow cells on astreptavidin sensor chip. A blank flow cell without GPC3 was used as a reference tocorrect for nonspecific binding. Arrows mark the termination of ligand injection (120seconds). Increasing concentrations of Wnt3a (bottom to top: 6.7, 13.3, 26.7, 53.3 and106.7nM) were injected on the surfaces of flow cells. The nonspecific binding wassubtracted 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 weretransfected with GPC3 or vector control. Cells were harvested 24 hours posttransfection, lysed and assayed for levels of the indicated proteins. Immunoblotsshow endogenous pLRP6, LRP6 and transfected GPC3. β-actin was used tocompare protein loading between samples. This figure is representative of twoexperiments.

393.4 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

405% 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-taggedFzd7 alone or in combination with GPC3. GPC3 was immunoprecipitated from cell lysate withαGPC3 (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 cellswere transfected with Fzd4 and sGPC3 (left) or vector control and sGPC3 (right). DifferentialInterference 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 weretransfected with Fzd1, Fzd7, Fzd8 or vector control and harvested 48h post transfection. Fzds wereimmunoprecipitated 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 bymeasuring absorbance at 405nm. Final values were obtained by subtracting the binding to vectorcontrol. Bars represent ±SE. This is a representative figure of 4 experiments.

433.5 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 weretransfected 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 arepresentative figure of 3 experiments.

453.6 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

-5 -4 -3 -2-0.5

0.0

0.5

1.0

1.5

Log Heparin (mg/ml)

Figure 3.6 Heparin inhibits binding of Glypican-3 to Frizzled HEK293T cellswere transfected with Fzd7 or vector control and harvested 48h posttransfection. Fzd7 was immunoprecipitated and incubated with sGPC3-APalong with increasing concentrations of heparin. After washing, AP substratewas added to samples. Binding of sGPC3-AP was quantified by measuringabsorbance at 405nm. Final values were obtained by subtracting the binding ofsGPC3-AP to immunoprecipitated from vector control cells. Samples wereprocessed in triplicate. Bars represent ±SE. This is a representative figure of 3experiments.

473.7 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 vectorcontrol in addition to GPC3. Myc tagged GPI-FzdCRDs were immunoprecipitatedfrom cell lysate 48h post transfection. (Panel 1) Bound GPC3 was probed withαGPC3 antibody (1G12). (Panel 2) Presence of FzdCRDs in the precipitate and(Panel 3) GPC3 in whole lysate was assessed by immunoblotting. This is arepresentative figure of 2 experiments.

49

0.0

0.1

0.2

0.3

0.4

0.5

0.6AP

ΔGAG-APGPC3-AP

Figure 3.8 GPC3 binds the cysteine rich domain of Frizzled HEK293T cells weretransfected with membrane bound FzdCRD 4-8 or vector control and harvested 48hpost 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 wereprocessed in triplicate. Bars represent ±SE. This is a representative figure of 3experiments.

50

Chapter 4

Discussion

514.1 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

52show 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

53glypican 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 coreprotein binds to Wnt while the GPC3 GAG chains bind Fzd. In this manner GPC3facilitates/stabilizes the interaction between Wnt and Fzd resulting in an increase inWnt signaling.

54Role 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

55collide 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.

56Several 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

57cells (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.

58Using 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.,

592009). 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

60present 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

61stimulate 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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