High-Throughput Screen to Identify Small Molecule Inhibitors of the Canonical Wnt Signaling Pathway

by

Stephen J Perusini

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

Department of Biochemistry University of Toronto

© Copyright by Stephen J. Perusini 2008

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High-Throughput Screen to Identify Small Molecule Inhibitors of

the Canonical Wnt Signaling Pathway

Stephen J, Perusini

Masters of Science

Department of Biochemistry University of Toronto

2008

Abstract

Wnt signaling is important in human development and disease, thus dysregulated β-catenin

constitutes an attractive target for drug intervention. The few functional inhibitors currently

available target transcriptional activation, therefore, identifying novel upstream modulators

would be of tremendous importance to elucidating the mechanisms involved in regulating β-

catenin activity.

To achieve this, I developed a high-throughput screen to assess β-catenin stability in mammalian

cells using a luciferase tagged β-catenin molecule. This assay was used to screen three chemical

libraries to identify small molecule modulators of the pathway. Identified inhibitors/activators of

the pathway were investigated via secondary assays. The most promising inhibitor, 21H7,

significantly attenuated activated β-catenin signaling in colon cancer cells, decreasing β-catenin

stability. The inhibitory effects of 21H7 and a structurally similar compound were shown to not

only inhibit Wnt target gene expression in colon cancer cells, but also prostate cancer lines.

Thus, 21H7 represents an attractive lead compound for further study.

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Acknowledgments

I wish to thank my supervisor, Dr. Liliana Attisano for allowing me to join her team, for her guidance throughout my graduate studies and the various revisions of this thesis. My thanks and appreciation also goes to my thesis committee members, Dr. Craig Smibert and Dr. Aaron Schimmer, and collaborators Alessandro Datti and Jeffrey Wrana. I am greatly indebted to my peers in the Attisano lab, for their expertise, kindness, and most of all, for creating an enjoyable atmosphere with worthwhile memories.

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Table of Contents Acknowledgments........................................................................................................................... ii

Table of Contents........................................................................................................................... iii

List of Tables ................................................................................................................................ vii

List of Figures .............................................................................................................................. viii

1 Introduction.................................................................................................................................. 1

1.1 Wnt signaling ...................................................................................................................... 1

1.1.1 Wnt genes and proteins........................................................................................... 1

1.1.2 The Wnt extracellular environment ........................................................................ 1

1.1.3 Wnt signal transduction .......................................................................................... 2

1.1.4 The canonical branch of the Wnt pathway ............................................................. 3

1.1.5 Additional factors involved in mediating a Wnt response...................................... 6

1.2 Wnt signaling in development ............................................................................................ 7

1.2.1 Invertebrates............................................................................................................ 7

1.2.2 Xenopus development............................................................................................. 8

1.2.3 Mammalian development........................................................................................ 8

1.3 Wnt signaling in human disease ....................................................................................... 10

1.3.1 Wnt signaling in Cancer........................................................................................ 11

1.3.2 Other human diseases and Wnt signaling ............................................................. 13

1.4 Small molecule screening ................................................................................................. 13

1.4.1 Drug discovery...................................................................................................... 14

1.4.2 Small molecule libraries ....................................................................................... 16

1.4.3 Chemical genomics............................................................................................... 17

1.4.4 Known small molecule modulators of the Wnt pathway...................................... 17

1.5 Rationale for project……………………………………………………………………...22

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2 Materials and methods ............................................................................................................. 23

2.1 Reagents............................................................................................................................ 23

2.2 Cell lines and maintenance ............................................................................................... 23

2.3 Production of Wnt3A ligand in conditioned media .......................................................... 24

2.4 Constructs, transcriptional reporter assays and immunoblotting...................................... 24

2.5 High-throughput assays .................................................................................................... 25

2.6 Real time quantitative reverse transcription-PCR analysis............................................... 27

3 Results ...................................................................................................................................... 28

3.1 High throughput assay development................................................................................. 28

3.1.1 Beta-catenin nuclear translocation assay .............................................................. 28

3.1.2 Firefly luciferase β-catenin stabilization assay (Ff-luc) ....................................... 32

3.1.3 Ff-luc assay optimization...................................................................................... 32

3.2 Pilot high-throughput Ff-luc assay run ............................................................................. 39

3.3 Final screening results and data analysis .......................................................................... 42

3.3.1 Comparison of various statistical hit-selection methods ...................................... 42

3.3.2 Summary of results from the Lopac library.......................................................... 43

3.3.3 Summary of results from the Prestwick library .................................................... 48

3.3.4 Summary of results from the Maybridge library .................................................. 48

3.3.5 Total assay hits from the entire HTP screen ......................................................... 53

3.4 Investigating selected hits via secondary analyses ........................................................... 53

3.4.1 Assessing the reproducibility of effects ................................................................ 53

3.4.2 Effects of hits on Wnt transcriptional activity and assessing pathway specificity.............................................................................................................. 57

3.5 Analysis of 21H7, a selective Wnt pathway inhibitor ...................................................... 59

3.5.1 Compound 21H7 preferentially inhibits Wnt responsive Topflash activity over the non-responsive Fopflash ................................................................................. 59

3.5.2 21H7 blocks Wnt-induced β-catenin stabilization................................................ 61

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3.5.3 21H7 inhibits Wnt dependent gene expression in colon cancer cell lines............ 61

3.5.4 Expression of endogenous Wnt target genes is inhibited by 21H7 in CRC cell lines ....................................................................................................................... 63

3.5.5 21H7 inhibits expression of endogenous Wnt target genes in prostate cancer cell lines. ............................................................................................................... 69

3.5.6 Wnt signaling is inhibited by 21H7 at the level of β-catenin ............................... 69

3.5.7 Structurally related compounds mimic the effect of 21H7………………………72

4 Discussion ................................................................................................................................. 75

5 References................................................................................................................................. 82

6 Appendices…………………………………………………………………………………….92

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List of Tables

Table 1: Known Small molecules modulators of the Wnt pathway…………………………21

Table 2: Pilot screen hits indicate a reproducible Ff-luc hit rate…………………………….40

Table 3: Summary of results from all compounds screened…………………………………54

Table 4: Summary of secondary assay results for twenty-two selected inhibitors and random control ..………………………………………………………………………………55

.

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List of Figures Figure 1: Overview of the canonical Wnt signaling pathway…………………………………4

Figure 2: Known Chemical inhibitors of the Wnt signaling pathway………………………..19

Figure 3: The β-catenin Nuclear translocation assay displays high variability…………........30

Figure 4: Development of the β-catenin-firefly-luciferase (Ff-luc) assay……………………33

Figure 5: The Ff-Luc assay …………..………………………..…………………………….36

Figure 6: Validation of the Ff-luc screening protocol…………………………………………37

Figure 7: Validating the pipetting accuracy and chemiluminescent readout of the robotics platform ………………………………………………………………………………………..38

Figure 8: Comparison of different statistical hit-selection methods……………………….....44

Figure 9: Lopac library screening results ……………………………...………………….....46

Figure 10: Prestwick library screening results ……………………………………………....49

Figure 11: Maybridge library screening results………………………………………………51

Figure 12: Re-testing of select compounds using the Ff-luc assay .………...………………56

Figure 13: Compound 21H7 preferentially inhibits the Wnt pathway ..…………………….58

Figure 14: 21H7 preferentially inhibits the transcriptional activity of Wnt3A treated cells ...60

Figure 15: Compound 21H7 inhibits the Wnt3A induced stabilization of endogenous β-catenin ……………………………………………………………………………………...62

Figure 16: Differential suppression of Wnt-dependent gene expression in colon cancer cell lines harboring activating mutations in the Wnt pathway by 21H7 ……………………...64

Figure 17: Wnt target gene expression is inhibited by 21H7 in colorectal cancer (CRC) cell lines irrespective of the differing Wnt pathway…………………………………………..66

Figure 18: 21H7 inhibits Wnt target gene expression in prostate cancer cells …………...…70

Figure 19: Epistatic analysis indicates 21H7 effects Wnt-dependent signaling at the level of β-catenin………………………………………………………………………………71

Figure 20: Screened Maybridge compounds with significant structural similarity to 21H7…73

Figure 21: Compound 21A8, the molecule of greatest structural similarity to 21H7 inhibits the expression of Wnt target genes in colon cancer cell lines…………………………………74

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1 Introduction

1.1 Wnt signaling

1.1.1 Wnt genes and proteins

Wnt genes, of which there are 19 in humans and mouse comprise a family of highly conserved

secreted glycoproteins that regulate processes such as cell motility and differentiation during

embryonic development by inducing diverse signaling cascades to effect cell growth and survival

(Clevers, 2006; Logan and Nusse, 2004). The Wnt proteins are found in species ranging in

complexity from sea anemone to humans, and effect embryo development by acting locally and

at distances in a morphogenic manner.

Due to the diverse roles of Wnts, they have typically been identified via sequence similarity and

not functional properties. All possess a highly conserved spacing of 22 cysteine residues, several

highly charged amino-acid with conserved positioning, a signal sequence for secretion and

multiple potential sites of glycosylation (Miller, 2002). Post-translational processing such as

glycosylation and lipid modification plays a significant role in Wnt functionality either during

secretion from the ligand producing cell or signal transduction at the receiving cell. Loss of

particular glycosylation sites has been shown to hinder the ability of Wnt3A and Wnt5A

producing cells to efficiently secrete the ligand, however, the ability of Wnts to bind their

receptors and transduce signals are unaffected (Coudreuse and Korswagen, 2007). Conversely,

palmitoylation of Wnt ligands is crucial to ligand receptor binding and bears no effect on

secretion (Kikuchi et al., 2007). Therefore, many Wnt modifications are essential for proper

secretion profiles and initiating a response in signal receiving cells in development and beyond.

1.1.2 The Wnt extracellular environment

Once secreted, the extracellular milieu allows Wnts to interact with a multitude of additional

Wnt signaling co-factors that modulate the effect of Wnts in a variety of ways (Kikuchi et al.,

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2007). Genetically identified factors such as secreted frizzled related protein (SFRP) and Wnt-

inhibitory factor (WIF-1) were shown to bind Wnts, sequestering them from the Wnt receptor

Frizzled (Fz) and thus antagonizing Wnt signaling (Kim et al., 2005). However, not all effectors

which bind Wnts are antagonistic. R-spondin binds Wnts and was recently demonstrated to bind

both Fz and its co-receptor LRP6 to synergistically activate the Wnt pathway (Kim et al., 2005;

Nam et al., 2006). Additional factors such as the Dikkopf (Dkk) family of proteins antagonize

the Wnt pathway not by binding Wnts, but through binding to the LRP co-receptor and blocking

receptor availability (Mao et al., 2001). Norrin is not a Wnt ligand as those previously described,

but was shown to activate the Wnt signaling pathway by independently binding select Fz

isoforms and thus effects Wnt signaling without modulating ligand activity (Planutis et al.,

2007). Consequently, the Wnt pathway extracellular environment is undoubtedly complex and

influences Wnt signaling through a combination of agonists and antagonists vying for the ability

to bind both ligands and their receptors

1.1.3 Wnt signal transduction

The various Wnt ligands induce one of three distinct but interconnected signaling pathways;

canonical, planar cell polarity, and the Ca2+ pathway. The canonical pathway is by far the best

understood of the three and it transduces signals via a distinct signaling cascade. The other two

Wnt pathways are far less understood. Both the Wnt/Ca2+ and non-canonical pathways transduce

signals through the Frizzled (Fz) receptors, but diverge afterwards. The non-canonical or planar

cell polarity pathway involves the activation of RhoA and Jun Kinase to regulate spatial and

temporal cytoskeletal rearrangements, cell migration and tissue polarity during embryogenesis

and afterwards (Adler, 2002). It is believed that the group of non-canonical Wnts initiate

signaling via binding the Frizzled receptors without co-receptor involvement and transduce

signal through Disheveled (Dvl) proteins, activating the two independent and parallel small

GTPase pathways, Rac and Rho.

The Wnt/Ca2+ pathway is best understood via its effect on intracellular Ca2+ levels, subsequent

activation of Protein Kinase C and Calmodulin kinase and principally affects cell movement

(Kikuchi et al., 2007). Wnt associated Frizzled activation is thought to affect Ca2+ levels through

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the activation of heterotrimeric G-proteins, resulting in the production of DAG and inositol

triphosphate second messengers (Slusarski et al., 1997).

1.1.4 The canonical branch of the Wnt pathway

By far the best understood of the Wnt pathways is canonical Wnt signaling which influences cell

fate through the activation of the LEF/TCF family of transcription factors. The central

component of the canonical Wnt signaling pathway is β-catenin. In the absence of Wnt ligand,

its cytoplasmic levels are kept low via the sequential phosphorylation by Casein Kinase 1 (CK1)

and GSK3β (Glycogen synthase kinase-3) on β-catenin. This phosphorylation permits it to be

recognized by the E3 ubiquitin ligase, βTrCP, and undergo continuous proteosome-mediated

degradation (Fig. 1) (Lustig and Behrens, 2003). This constitutive destruction of β-catenin is

interrupted as the Wnt pathway is activated once canonical Wnt ligands bind the Frizzled (Fz)

and LRP5/6 (low density lipoprotein related) co-receptors (Tamai et al., 2004). Current

knowledge indicates LRP functions solely in the canonical Wnt pathway and is dispensable for

the others (Wehrli 2000). These receptors signal to both Axin and the Disheveled (Dvl)

proteins, both key intracellular components of the Wnt pathway. By a yet unknown mechanism,

receptor activation allows Dvl to promote the dissolution of a multi-component cytoplasmic

complex (Destruction complex) that phosphorylates β-catenin (Wharton, 2003). Furthermore,

receptor activation recruits Axin, a member of the destruction complex, to LRP receptors

phosphorylated on both conserved Ser/Thr residues and the PPPSP motif (Tamai et al., 2004). In

a Wnt induced manner, CK1γ phosphorylates the Ser/Thr cluster regions of LRP5/6 while

GSK3β subsequently phosphorylates the PPPSP motif, allowing for Axin recruitment to the

plasma membrane and away from a multi-component destruction complex (Liu et al., 2002;

Tamai et al., 2004). This event, in combination with Dvl’s association with this multi-

component scaffolding/destruction complex containing Axin, GSK3β, the APC (Adenomatous

Polyposis Coli) tumor suppressor further promote complex dissociation, thereby preventing β-

catenin from being phosphorylated by both CK1 and GSK3β.

In a non-Wnt stimulated state, the destruction complex is believed to contain Axin as the primary

scaffolding molecule from which, CK1 (Liu et al., 2002), GSK3β (Dajani et al., 2003), and APC

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Figure 1. Overview of the canonical Wnt signaling pathway. In the basal state, defined by

an absence of Wnt signal, β-catenin is held within a multicomponent complex. Complex

formation facilitates the sequential phosphorylation of β-catenin by CK1α and GSK3β on

conserved serine and threonine residues. Phosphorylation on its amino terminus allows β-TrCP,

an E3 ligase, to bind β-catenin and promote ubiquitin-mediated proteosomal degradation

resulting in low levels of β-catenin. In the presence of Wnt ligand, the cell surface interaction

between Wnt and its two co-receptors Frizzled and LRP5/6 promotes the formation of a Frizzled-

Dishevelled complex and the phosporylation of LRP by CK1γ,ε and GSK3β. These

modifications allow for Axin to bind the LRP intracellular domain. Axin is sequestered away

from the destruction complex, to the cell surface, resulting in the dissociation of the

multicomponent complex. β-catenin is freed, is not efficiently modified by CK1α and GSK3β

and therefore goes unrecognized by the E3 ligase allowing for β-catenin levels to accumulate

and β-catenin enters the nucleus. Once in the nucleus, β-catenin interacts with members of the

TCF/LEF transcription factor family displacing transcriptional repressors and recruiting a host of

co-activators to promote the expression of a variety of proliferative and cell-fate determining

genes

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Figure 1

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(Kimelman and Xu, 2006; Spink et al., 2000) bind. β-catenin enters the complex by binding

Axin through the conserved armadillo repeats and the 15 amino acid repeats on APC. These

associations are thought to position the N-terminus β-catenin adjacent to the two kinases in the

complex, enabling CK1 to phosphorylate β-catenin at Ser45 (Hagen and Vidal-Puig, 2002) .

This primary modification serves as a primer, permitting GSK3β to sequentially phosphorylate

β-catenin at adjacent Thr 41, Ser 37 and Ser 33 (Amit et al., 2002). Upon Wnt stimulation, the

complex dissociates in the manner described above and β-catenin is freed from these interactors

and therefore is not efficiently modified by CK1 and GSK3β.

Upon release from the scaffolding complex, the non-phosphorylated form of β-catenin goes

unrecognized by the proteosome destruction complex. Thus, free β-catenin is stabilized and

accumulates in the cytosol, allowing its translocation to the nucleus where it binds to

transcription factors of the TCF/LEF (T cell-specific transcription factor/lymphoid enhancer-

binding factor) family. This interaction transiently converts TCF factor repression into

transcriptional activation thereby inducing the expression of a variety of downstream genes,

many of which have been implicated in cancer (Logan and Nusse, 2004). Canonical Wnt target

genes include cell cycle and anti-apoptotic factors such as survivin (Zhang et al., 2001a), Cyclin

D1 (Tetsu and McCormick, 1999), C-myc (He et al., 1998), in addition to a variety of secreted

growth factors including VEGF (Zhang et al., 2001b) and WISP (Xu et al., 2000). Furthermore,

Wnt signaling promotes the expression of Wnt pathway component Axin2 (Jho et al., 2002),

resulting in negative feedback.

1.1.5 Additional factors involved in mediating a Wnt response

Like the Wnt extracellular environment, there are many co-factors outside of the core Wnt-

pathway components which modulate intracellular Wnt signal transduction. In fact, there are

currently 32 purported intracellular modulators outside of the core canonical pathway

components (The Wnt homepage, http://www.stanford.edu/~rnusse/wntwindow.html). For

instance, members of the protein phosphatase family PP2A and C, are key components of

canonical Wnt signaling and are often found within the multi-component destruction complex

(Lustig and Behrens, 2003; Seeling et al., 1999). Their roles, however remain unclear,

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obfuscated by the ability of PP2A to bind both Axin and APC while acting as both a positive and

negative regulator of canonical Wnt signaling, in a yet unknown manner (Seeling et al., 1999;

Willert et al., 1999). PP2C has been shown to interact with Dvl2 though its PDZ domain and as

a result destabilizes Axin (Strovel et al., 2000). Other major cytoplasmic modulators such as

Frodo and Dapper possess 90% sequence identity, yet have been shown to inversely effect

canonical signaling. Required for proper development, both bind Dvl, but on different domains,

Dapper is a negative regulator, while Frodo has been shown to positively regulate Wnt signaling

by mechanisms still unknown (Wharton, 2003).

Once in the nucleus, a variety of factors directly influence β-catenin/TCF/LEF activity. ICAT,

for example, binds β-catenin and is thought to displace it from its transcriptional partners

TCF/LEF and CREB binding protein (Reifenberger et al., 2002). Furthermore, commonly seen

splice variants of TCF/LEF are known to act as dominant-negatives lacking either β-catenin or

DNA binding domains (Hoppler and Kavanagh, 2007). Furthermore, factors such as the

TCF/LEF co-repressor Groucho, competes with β-catenin for TCF/Lef binding. All of these

effectors contribute to the complexity of Wnt signal transduction and transcriptional activation.

1.2 Wnt signaling in development

1.2.1 Invertebrates

Wnt signaling is a crucial factor during embryonic development in organisms of varied

complexity. Wnt-β-catenin signaling is a key regulator of germ layer formation in invertebrate

species such as nematodes, sea urchina and cnidarians, in which factors stored in oocytes directly

activate canonical Wnt signaling in a spatially restricted manner specifying germ layers and

defining body axis patterns (Marikawa, 2006). This role of germ layer specification is most

evident in C. elegans, where the Wnt3 homologue mom-2 plays a critical role in establishing

embryonic polarity and inducing germ layer formation. After the second division of the zygote,

in a four-cell-staged embryo, one of the cells (P2) becomes polarized and adjacent daughter cells

which from there-on divide separately give rise to the endoderm or mesodermal lineages. In the

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absence of Wnt signaling, both daughter cells adopt the mesodermal cell fate and genetic screens

have linked mom2 as the essential P2 cell factor responsible for the polarization (Rocheleau et

al., 1997). Furthermore, activity of a Lef-1 homologue in the polarized daughter cells is critical

for the cells to adopt differing fates (Lin et al., 1995).

1.2.2 Xenopus development

Like the invertebrates, frog body axes are defined in the first few cellular divisions, establishing

the three germ layers, endoderm, ectoderm and mesoderm in the polarizing oocyte. Major body

axis organization is largely coordinated via a region of the developing embryo called the

Spemann organizer. This organizer consists of a small group of cells in the vertebrate embryo

which possess inductive morphogenic properties that establish the frog body plan (Garcia-

Fernandez et al., 2007). Wnt signaling plays a crucial role in the development of the organizer,

as activated β-catenin is present on the dorsal side of the blastula at the same time and location

the organizer develops. Injection of murine Wnt1 mRNA into early Xenopus blastomeres

resulted in the induction of dorsal mesoderm formation and a duplicate body axis (McMahon and

Moon, 1989). This quintessential Wnt experiment demonstrated a significant role for β-catenin

signaling in vertebrate body plan formation. Moreover, it demonstrated the highly conserved

nature of the canonical Wnt signaling pathway whereby murine Wnt1 was able to effect frog

development. Equally important, Wnt signaling was shown to play a critical role in specifying

germ layer and body plan in invertebrates and vertebrates alike. Subsequent studies revealed that

identical axis duplications could be induced by injecting any of the canonical Wnt ligands and a

wide variety of downstream Wnt pathway activating components including β-catenin, Dvl, and

dominant-negative forms of GSK3β (Clevers, 2006; Wodarz and Nusse, 1998).

1.2.3 Mammalian development

Although Wnt signaling is responsible for establishing the dorso-ventral body axis in lower

vertebrates, regulates germ layer formation and specifies body axes amongst invertebrate

species, data on higher vertebrates is not as well established. The Wnt influence in mammalian

development is significantly more complex and less delineated, however, significant insights

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have been garnered from mouse studies. Wnts have been shown to play critical roles during

various stages of development. Compared to other species, mammalian development is

significantly different; the initial events of germ layer and body axis formation in non-

mammalian species are secondary events in mammalian development. The first event in

mammals is placenta development allowing for interacting with the mothers uterus, body plan

and axis formation occur post implantation (Marikawa, 2006). A variety of studies have

investigated the role of canonical Wnts during pre-implantation development using loss-of-

function, gain-of-function, and overexpression analysis and none have reported any

abnormalities until post implantation (Haegel et al., 1995; Kemler et al., 2004). Therefore,

canonical Wnt signaling solely plays its role post implantation, despite a variety of ligands and

receptors being expresses pre-implantation (Hamatani et al., 2004; Wang et al., 2004).

The initial event of germ-layer formation occurs around E4.5, with the formation of the primitive

endoderm in the developing blastocyst, defining the dorso-ventral (D-V) axis of the embryo.

The role for Wnt in this event is unclear. β-catenin-null embryos develop normal primitive

endoderm (Haegel et al., 1995), however, in vitro models suggest up-regulation of β-catenin

signaling induces primitive endoderm formation (Liu et al., 1999). The first clear role for

Wnt/β-catenin signaling appears to be at E5.5 in patterning the visceral endoderm and promoting

its migration by regulating the expression of secreted molecules controlling the anterior

movement of the visceral endoderm (Huelsken et al., 2000).

After the formation of the endoderm, epiblast cells move opposite the anterior visceral ectoderm

and undergo epithelial-mesenchymal transformation (gastrulation) to generate the primitive

streak. It is from this structure that cells give rise to endoderm and mesoderm. Similar to

Xenopus development, non-phosphoryated β-catenin accumulation is present in the region of

epiblast cells where the primitive streak emerges during the onset of gastrulation (Marikawa,

2006; Mohamed et al., 2004). Furthermore, reporter constructs indicate TCF-mediated

transcription is spatially and temporally activated during gastrulation (Mohamed et al., 2004).

Also, several Wnt pathway components have demonstrated indispensability for both primitive

streak and eventual axis formation (Liu et al., 1999), while excessive Wnt signal causes

expansion and disorganization of the streak in mouse embryos (Kemler et al., 2004). The data

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indicates a critical role for canonical Wnt signaling in the events leading up to and encompassing

mouse gastrulation.

After gastrulation, the anterior visceral endoderm (AVE) and the anterior visceral mesoderm

(AME) which derive from the anterior primitive streak play critical roles in the formation of

anterior structures. Interestingly, both of these regions express high levels of Wnt antagonists

such as DKK, SFRP and CER1 (Biben et al., 1998; Glinka et al., 1998). Consequently, ectopic

expression of canonical Wnts has been shown to suppress the formation of anterior structures,

along with inducing partial axis duplication in mice. These experiments indicate critical roles for

AME and AVE factors in suppressing local Wnt signaling for normal anterior structure

development (Marikawa, 2006). Further, anterior involvement implicates canonical Wnts as

inducers of the neural crest, promoting its formation, closure and dispersal throughout the

embryo that eventually gives rise to most of the nervous system and the cranial skeleton

(LaBonne, 2002).

In addition to the early events in embryonic development, canonical Wnt signaling is required

for early stages of mammalian osteoblastogenesis (Day et al., 2005), late stages of brain (Thomas

and Capecchi, 1990), kidney (Herzlinger et al., 1994), tooth, and mammary gland development

by exerting its influence upon proliferation, cell migration and differentiation similar to events

surrounding gastrulation. It is through combinations of ligand expression patterns, agonists and

antagonists vying for the ability to bind both ligands and their receptors and in combination with

a variety of other embryonic factors, that Wnts contribute to the precise temporal and spatial

patterning of the developing mouse embryo

1.3 Wnt signaling in human disease Developmentally important signaling pathways like that of Wnts, which coordinate diverse

processes such as cell migration, proliferation, adhesion and death, play significant roles not only

in development but also in tissue homeostasis (Clevers, 2006). When a critically important

pathway such as Wnt becomes dysregulated, it predictably leads to a variety of pathological

conditions. These conditions range from various types of cancers (Polakis, 2000), to multiple

diseases related to unbalanced tissue homeostasis (Krishnan et al., 2006). The actions of these

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diseases are mediated mostly through the canonical branch of the pathway and the number of

Wnt-induced proliferative and or anti-apoptotic genes.

1.3.1 Wnt signaling in Cancer

The best studied of diseases involving Wnt signaling relate to its role in a variety of cancers

(Barker and Clevers, 2006; Giles RH, 2003; Polakis, 2000). Most evident are the implications

within the intestinal epithelium in colorectal cancer (CRC) where mutations of key pathway

components appear very early in adenoma development in approximately 90% of human CRC’s.

Mutational hot-spots located in APC account for 80% of inherited and random cases of colon

cancer, while mutations in β-catenin are seen in 9% of such cases. Additional Wnt pathway

components are mutated at a lesser frequency, yet all result in the stabilization of β-catenin,

promoting constitutive activation of canonical Wnt signaling (Barker and Clevers, 2006; Giles

RH, 2003; Polakis, 2000). Familial adenomatous polyposis (FAP) is an autosomal, inheritable

disease predisposing patients to develop thousands of colon polyps typically progressing to

carcinoma by early to mid adulthood. The majority of FAP associated mutations reflect

heterozygous truncations of the APC gene and are associated with a subsequent loss of

heterozygosity (Galiatsatos and Foulkes, 2006). This phenotype is mimicked in the Apcmin

mutant mouse heterozygous for a truncated form of APC, in which mice readily develop both

polyps and colon carcinomas (Moser et al., 1995).

Canonical Wnt signaling is required for maintaining epithelial homeostasis within the intestinal

tract. The colon is comprised of both villi (protrusions) and invaginations (crypts). The basal

crypt cells are highly proliferative cells that give rise to multiple cell types lining the intestinal

tract. Canonical Wnts exert a proliferative effect on these crypt progenitor cells through the

regulation of Wnt target genes c-Myc and CyclinD1 (Gregorieff and Clevers, 2005). Both

factors are highly overexpressed in cases of both FAP and colon cancer. Studies aimed at

blocking their expression in CRC cell lines have indicated their cell proliferative effect can be

mitigated and cellular differentiation programs induced via overexpression dnTCF4 (van de

Wetering et al., 2002). The precise mechanism by which Wnt signaling regulates the

proliferative homeostasis of these cells remains a mystery. In the colon, however, it is believed

that adenoma formation is promoted through Wnt induced unabated expansion of progenitor

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cells which somehow retain their crypt phenotype, yet continue to expand outside the intestinal

invaginations (Gregorieff and Clevers, 2005).

In addition to the multiple examples of activating mutations of Wnt signal transduction

components, recently, a variety of epigenetic regulatory mechanisms have been noted in cases of

colon cancer. A number of studies have reported hypermethylated promoters of secreted Wnt

ligand genes such as WIF1 (Taniguchi et al., 2005), DKK1 (Aguilera et al., 2006) and SFRP’s

(Suzuki et al., 2004) in multiple colon cancer cell lines and patients. These factors all act as

inhibitors of Wnt signaling in the extracellular matrix where they either sequester Wnt ligands

via direct binding or prevent Wnts from binding their target receptor. In effect,

hypermethylation lessens the ability of these factors to quell an already hyperactive Wnt signal in

many instances of colon cancer.

A common theme within the Wnt field surrounds the related effects of Wnt in cancer and cellular

or tissue self-renewal. While this theme is best characterized in the intestine, it is also evident in

hair follicle tumours, and in cases of leukemia. Hair follicle tumours are the first reported case

of a Wnt inactivating mutation seen to be highly correlative with a tumor (Takeda et al., 2006).

A significant percentage of follicular sebaceous tumours harbour a Lef1 mutation rendering it

unable to efficiently bind β-catenin. The end result is a set of early progenitor cells being

directed towards the wrong cellular fate in the absence of sufficient Wnt signal. Obfuscating the

picture in follicular tumours are the cases in which activated Wnt signaling is seen to play a

significant role in promoting tumor development (Lo Celso et al., 2004). This disparity implies a

more complex role for β-catenin/Wnt signaling in the determination or balance of follicular cell

fate.

This balance between cell-fate and cancer seen in the colon and hair follicles is also seen in

leukemias, whereby hyperactive Wnt signaling is believed to enhance the self-renewal activity of

CML granulocyte-macrophage progenitors leading to increased leukemic potential (Jamieson et

al., 2004). Moreover, similar instances of activated canonical Wnt pathways are seen and

implicated as effectors in cases of breast, lung, prostate, ovarian, thyroid and a variety of

additional cancers (Lustig and Behrens, 2003; Mazieres et al., 2005; Nusse and Varmus, 1982;

Tekmal and Keshava, 1997; Yardy and Brewster, 2005).

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1.3.2 Other human diseases and Wnt signaling

In addition to cancer, canonical Wnt signaling has been implicated in diseases as far ranging as

Alzheimer’s, bipolar disorders, skin diseases, cardiovascular diseases, type two diabetes and a

limb development disorder (Holmen et al., 2004; Johnson and Rajamannan, 2006; Luo et al.,

2007). However, the best documented case outside of cancer is that relating to skeletal disorders.

This crucial role for canonical Wnt signaling has only been documented in the last decade, as

Wnts play an essential role in post-natal bone tissue homeostasis.

In adult life, bone homeostasis is maintained by the balanced activities of osteoblasts, which

produce bone matrix and their cellular counterpart osteoclasts, which resorb the same matrix.

Therefore, bone mass is determined by the tightly coupled relative activities of these two cell

types (Bennett et al., 2005). Loss-of-function mutations of LRP5, the canonical Wnt co-receptor,

are associated with osteoporosis-pseudogloma syndrome, which is characterized by a loss of

bone density, hence skeletal fragility (Krishnan et al., 2006). Conversely, gain-of function

mutations in the LRP5 N-terminal domain which reduces its affinity for the Wnt antagonist

DKK1 confers a high bone mass phenotype (Boyden LM, 2002). The bone density phenotype in

LRP5 null mice is exacerbated by the heterozygous loss of LRP6, adding credence to the notion

that the entire LRP family of receptors critically influence bone mass through canonical Wnt

signaling (Holmen et al., 2004). While the molecular mechanisms by which Wnts influence

bone mass are not fully understood, GSK3β inhibition was seen to stimulate precursor cells to

differentiate into osteoblasts (Bennett et al., 2005). Additional studies have demonstrated

stabilized β-catenin and Wnts 3, 1 and 10b, all canonical Wnts, induce osteoblastogenesis

strongly indicating Wnts stimulate bone formation via directing cells towards an osteoblastic cell

fate (Krishnan et al., 2006).

1.4 Small molecule screening Sequencing of the human genome has provided researchers with a multitude of new biological

targets to study based on the approximately 25,000 genes likely to encode millions of protein

products. Of these proteins, fewer than 1000 are currently targeted by small molecules (Drews,

14

2000), and are predominantly pharmaceutical targets. The popularity of small molecule

screening is likely linked to the abundance of information provided by the genomics revolution.

New screening technologies and improved small molecule libraries have the potential to

accelerate the transition of genomic data into biological or therapeutic significance (Austin,

2003).

Used as a complement to traditional research, small molecules have a number of advantages;

they target a gene product rather than mRNA or a genetic locus, can be agonistic or antagonistic,

and they can selectively affect their target for a defined period in vivo and in vitro. With

virtually limitless structural diversity, there are conceivably specific agonists and antagonists for

every protein. Therefore, by screening chemical libraries against a variety of assays, it is

possible to identify specific effectors and develop small molecules as pharmaceuticals and as

research tools. Conversely, it is also possible to screen chemicals with known molecular

properties to dissect the function of novel genes and cellular pathways.

1.4.1 Drug discovery

The process of drug discovery involves biological target identification, assay development, and

high-throughput screening followed by identifying the mechanism of action and hit-to-lead

validation. Most molecules in the drug discovery process fail due to lack of potency, toxicity or

unsound biological targeting (Caldwell, 2007). The benefits, however, of being able to screen a

quality drug target against thousands of potential chemical compounds are invaluable and have

increased with the availability of drug-like compound libraries and robotics capabilities

(Goodnow, 2001). High-throughput screening, therefore, has become commonplace in industry

and increasingly so in academia as a means of drug discovery and as a tool to address key

biological questions.

Much like biological research itself, these screens take one of three distinct approaches; cell-

based, in vitro, and in silico. All possess distinct benefits and limitations. For example, in vitro

screens are predominantly used when a specific molecule of interest is directly targeted. In vitro

assays provide researchers with direct readouts of activity or function of the drug target, and can

be easier than cell-based screens. With an in vitro approach, however, it is not a certainty

15

whether similar effects will be duplicated in vivo, nor whether the compound is cytotoxic, or has

multiple off target effects. Cell-based screens bypass two of these issues. With the proper

controls, cytotoxic small molecules can be easily identified or screened for (Zaman, 2004) and in

vivo effect are not in question. However, cell-based screening is not without problems. False

positive rates are significantly higher, due to off target effects even in the best designed screen

(Zaman, 2004). However, the biggest advantage of cell based screens is the ability to perform

pathway and phenotypic screens. These screens allow researchers to address greater biological

questions through a broader range of applications. The third approach, in silico screening, also

has broad applications. In contrast to both in vitro and cell-based screening, in silico screens

draws information from previous screens and literature to make computer generated predictions

about compound effects on a particular protein. Structural modeling is used to generate

inhibitors/activators of enzymatic activity or of protein-protein interactions. In silico screens,

however, are limited to proteins with solved structures or with known small molecule effectors

as a starting point (Shan et al., 2005).

Regardless of screen type, of principal screening importance is identifying a proper target from

the onset. Particularly more so when screening for anti-cancer drugs, as their properties are often

toxic to normal tissues as well as the cancerous target cell population. Coupled with cancer

being a diverse disease due to environmental factors as well as patient genetic variability, a

screening target molecule or pathway must be very sound from the onset. This does not rule out

the possibility of targeting a component or pathway essential to general cell survival, because the

host of secondary issues these targets bring with them that can be satiated through appropriate

dosing and directed drug distribution (Kamb et al., 2007). Furthermore, using a cell-based assay

for drug screening can be advantageous in weeding out compounds with more general cytotoxic

effects.

An equally important part of the screening process is the development of a high-throughput

readout to measure the activity of the target molecule or pathway. The recent advances made in

cell-based screening technology including automated systems for cell manipulation, small

molecule delivery tools, signal detection reagents and apparatus, along with better chemical

libraries have all made cell-based small molecule implementation increasingly more reliable and

viable.

16

The most difficult and time consuming aspect of the drug discovery process is not the screen

itself, but the subsequent work validating the identified hits. The first goal is turning screened

hits into lead chemical compounds (Goodnow, 2001). Small molecule leads need not only have

a verified effect, but their cellular targets, and mechanism of action need to be identified in order

to proceed to optimization. Target identification needs to be determined in phenotypic and or

pathway screens, as opposed to targeted enzymatic screens in which the target is already known.

While genomic and proteomic tools have been in place for a long time in yeast to more easily

identify target molecules, these tools are not fully adaptable to mammalian cells (Luesch, 2006).

Therefore other than coupling a small molecule screen with a genomic screen (siRNA), a large

amount of secondary work is necessary for target and/or mechanistic identification. After target

identification, lead compounds typically go through an optimization stage in which their

structure is refined through medicinal chemistry to produce a clinically viable candidate lead

(Goodnow, 2001). From there, the long process of enabling their entry into animal and then

human studies is started, with the vast majority of leads failing due to lack of potency, toxicity or

specificity (Goodnow, 2001). It is precisely due to this high failure rate that a screens need to be

designed for a sound druggable target, and hit validation and optimization be thorough and

comprehensive.

1.4.2 Small molecule libraries

As important as it is to design the proper screen against a well researched target, hits will only

generate quality lead compounds if the chemical library contains compounds with biological

activity. Medicinal chemistry has traditionally favored particular scaffolds from which drugs are

derived. Therefore, molecules in a screening collection typically adhere to conventional rules for

drug-like properties, taking into consideration solubility, H-bond donating and accepting groups,

and size restrictions (Lipinski, 1997). The advantage of these chemical properties is

considerable, as they not only predict drug activity, but also demonstrate adequate physiological

absorption, distribution, metabolism, and excretion profiles (Yu and Adedoyin, 2003).

Therefore, they are ideal compounds for post-screening purposes.

The measure of chemical space is equally as important as the aforementioned properties. A

chemical library designed to randomly populate chemical space is not of significant biological

17

use if chemical and biological space does not overlap (McMillan and Kahn, 2005). Therefore,

compound libraries such as Maybridge (Thermo Fisher scientific), which are designed using core

structures common to medicinal chemistry as scaffolds, from which diversity and variability of

chemical space is explored, are ideal for drug screens. Moreover, a library of this type is best

suited to identify novel modulators of a cellular target while also adhering to typical drug-like

properties. Libraries such as the LOPAC (Sigma) and Prestwick (Prestwick chemicals), which

consist of biologically active compounds and off-patent drugs, are not likely to produce selective

inhibitors of a particular pathway of interest owing to their activity towards other cellular targets.

However, these libraries are of chemical genomic interest, and can identify interactions or

commonalities between different signaling pathways.

1.4.3 Chemical genomics

In parallel with the drug discovery process is the emerging field of chemical genomics. Forward

chemical genetic screens are identical to drug based screens, but differ in their end goals. While

drug discovery aims to generate lead medicinal compounds, chemical genomics aims to identify

a small molecule which can produce a particular phenotype (Lokey, 2003). In a manner very

similar to forward genetics, chemical genomics seeks compounds possessing properties able to

mimic a particular genetic mutation by altering the effects of one or more gene products

(Bellows and Tyers, 2004). In many situations, the molecular targets or phenotypes studied

through chemical genomics screens are the ones also of interest for drug discovery. At the core

of excitement surrounding chemical genomics, are its wide-ranging application. Once a screen

has been used to identify a chemical modulator, and its specificity of action upon a molecular

target is verified, the small molecule can then be used as a tool to dissect the biology of the

molecular target, its interactions, and its in vivo role. Moreover, the simplicity of use gives it an

advantage over the myriad of genetic tools that can be used in parallel.

1.4.4 Known small molecule modulators of the Wnt pathway

Dysregulated Wnt signaling was first linked to colon cancer in the late 1990’s (Korinek et al.,

1997), and since then, a significant amount of research from both private and public sectors has

18

investigated that link. Therefore, as expected, studies of purported small molecule antagonists of

canonical Wnt signaling have emerged (Fig. 2 and Table 1). The first inhibitor reported was

nitric oxide donating aspirin compounds already in drug testing as Cox2 inhibitors (Nath et al.,

2003). Subsequently, additional compounds such as hydrogen peroxide (Shin et al., 2004),

Green tea compounds (Kim et al., 2006) and Quercetin (Park et al., 2005) were also reported as

inhibitors. However, a mechanism for the activity of these and most other inhibitory compounds

remains largely undefined.

The largest class of compounds demonstrating an antagonistic effect on canonical Wnt signaling

is the NSAIDS. Included in this category are Aspirin, the NO-donating forms of Aspirin, and

sulindac sulfide. All have long been used for the treatment of pain and inflammation. The

mechanisms by which they affect Wnt signaling are varied. Aspirin is believed to inhibit PP2A

activity (Bos et al., 2006), while NO-donating aspirin have been suggested to disrupt the β-

catenin/TCF complex in the nucleus (Nath et al., 2003), and Sulindac was shown to inhibit

nuclear β-catenin accumulation in FAP patient colonic cells through an undetermined

mechanism (Boon et al., 2004; Koornstra et al., 2005). While these compounds have

demonstrated an inhibitory effect upon the canonical Wnt pathway, the effects were seen at high

concentrations; moreover, they are not specific to Wnt signaling.

In addition to reports of already studied compounds, three groups have used small molecule

screens in an attempt to identify novel compounds inhibiting canonical Wnt signaling. The first

of these reports characterized the compound ICG-001, identified in a cell-based screen using a

Wnt responsive transcriptional reporter, Topflash, as assay readout. This synthetic compound

demonstrated an inhibitory effect on Wnt transcriptional activity through its binding to the β-

catenin transcriptional partner CREB Binding Protein (Emami KH, 2004). A more directed

approach was taken by Leproucelet et al., in which they screened a chemical library in an

ELISA-based high throughput screen for inhibitors of β-catenin-TCF interaction (Lepourcelet et

al., 2004). The screen identified six inhibitors with IC-50 values in the low μM range, all of

which demonstrated in vivo effects on canonical Wnt signaling. The third screen was

accomplished in silico, using a structure-based approach. Utilizing the crystal structure of Dvl

binding protein Dapper, Shan et al. ran a virtual screen of 250,000 compounds to identify small

19

Figure 2. Known Chemical inhibitors of the Wnt signaling pathway. Small molecules with

antagonistic Wnt pathway activity are displayed at their molecular targets. Four of the six

known inhibitors are downstream effectors of the pathway acting at the transcription level.

Additional modulators, including agonists are listed in Table 1.

20

molecules which would bind Dvl and interrupt its association with the Frizzled receptor (Shan et

al., 2005). One such molecule was identified and demonstrated an in vivo effect upon Wnt

signaling, however, its effects were only seen at a very high concentration.

21

Table 1. Known small molecules modulators of the Wnt pathway. A number of small-

molecules have demonstrated the ability to effect β-catenin mediated Wnt signaling. While

some molecules were identified through high-throughput screening as indicated above, most

have been identified by alternative methods. All known Wnt pathway modulators are reported

along with the associated IC50 value (mM) and the molecular target, if identified. ND indicates

no data is available.

22

1.5 Rationale for project

Due to the importance of Wnt signaling in human disease, dysregulated β-catenin constitutes an

attractive target for drug intervention. However, in addition to anti-cancer therapy, the Wnt

pathway plays a critical role in animal development. The few functional inhibitors in existence

are downstream effectors of transcriptional activation. Therefore, identification of novel

upstream small molecule inhibitors/activators would be of tremendous importance as a research

tool to elucidate the mechanisms involved in regulating β-catenin stability, and thus Wnt

signaling.

My research endeavor therefore centered on the development and implementation of a high-

throughput screen to assess the status of β-catenin activity in mammalian cells. Using a

luciferase tagged β-catenin molecule to detect its stabilization, we screened the Maybridge,

LOPAC and Prestwick chemical libraries to identify small molecule modulators of canonical

Wnt signaling. Compounds identified as inhibitors or activators of the pathway were

subsequently investigated via β-catenin stabilization assay, and pathways specific reporter assay,

to assess repeatability and pathway specificity. The effects of the most promising inhibitory

compound was tested in colon cancer cell lines and was shown to significantly attenuate

activated β-catenin signaling via quantitative PCR analysis. Furthermore, through dissection of

the Wnt signaling pathway, we were bale to pinpoint the effect of the compound upon β-catenin

stability, through a currently undefined mechanism. Lastly, the inhibitory effects of this

compound and ones of significant structural similarity were shown to not only inhibit Wnt target

gene expression in colon cancer cell lines, but also effect prostate cancer models.

23

2 Materials and methods

2.1 Reagents The Lopac1280, Prestwick and Maybridge libraries housed at the S.M.A.R.T Robotics Facility at

the Samuel Lunenfeld Research Institutes (http://38.112.98.3/), were purchased from Lopac

Chemicals, Prestwick Chemicals and Maybridge Chemicals respectively. Aliquots were stored

at -900C in 96 and 384 well storage plates (Costar, Corning). Individual compounds used in

secondary analyses were obtained from Ryan Scientific, dissolved in DMSO (Sigma) and stored

in small 50 mM aliquots at -200C.

2.2 Cell lines and maintenance All cell lines were maintained in media according to American Type Culture Collection

guidelines (www.ATCC.org), supplemented with 10% FBS (Hyclone), incubated at 37 0C with

5% CO2 and went without additional additives unless otherwise indicated. Briefly, SW480,

SW620, LS1034, LS174T and HepG2 cells were cultured in alpha-MEM. HepG2 cells also

received non-essential amino acid supplementation (Gibco). The prostate derived cell lines

Du145 and PC3 were maintained in regular MEM. Colo205 cells were sustained in RPMI

media, while L-cells, HEK293 and HEK293T cells were cultured in DMEM. Wnt3A expressing

L-cells (Labbe E, 2000) were maintained in DMEM containing 0.4 mg/ml G418 (Gibco). The

Flag-β-catenin-Luciferase stable cell lines were generated by transfecting HEK293 cells with

pCAGIP-Flag-β-catenin-Ff-luciferase (see below) using calcium phosphate transfection (see

below) in DMEM. Cells stably expressing the fusion protein were selected with 1.5 μg/ml

puromycin (Sigma) by ring cloning after one week. Early passages were frozen down and

expanded when needed.

24

2.3 Production of Wnt3A ligand in conditioned media Mouse fibroblast L cells stably expressing the murineWnt3A gene were generated by Dr. Etienne

Labbe using the pPGK-neo-Wnt3A construct (Shibamoto et al., 1998). Cells were plated in 10

cm dishes and allowed to grow to 70% confluence, after which, the media was changed for

ligand collection. Both control and Wnt3A conditioned media was collected from cells cultured

for five days in DMEM supplemented with 0.2% fetal bovine serum. After collection, the media

was filtered through a 0.22 μm bottle top filter (Corning) and stored at 4o C for up to four weeks

without significant loss of activity. Confirmation of ligand activity was performed on each new

batch using Topflash transcriptional reporter assays to assess Wnt transcriptional activity and

immunoblot analysis to detect stabilization of endogenous β-catenin in L-cells (see below).

2.4 Constructs, transcriptional reporter assays and immunoblotting

Topflash and Fopflash reporter constructs were originally obtained from the Vogelstein Lab and

are the same as those previously used in our lab (Labbe E, 2000). IBRE-lux (Benchabane and

Wrana, 2003) and 3TP-luc (Wrana et al., 1992) reporters were obtained from the Wrana lab, and

the NFκB reporter is from Upstate Biotechnology. Wnt pathway components, Dvl2, β-catenin,

Lef1 and LRP6 all possess a C-terminal triple-Flag-epitope-tag and were cloned into the pCMV5

vector by Dr. Bryan Miller. The Flag(N-terminal)-β-catenin-firefly-luciferase(C terminal) fusion

construct was excised from a pCMV5B construct generated by Dr. Letamendia and cloned into

the ClaI and BglII sites of the pCAGIP vector, which contains a Puromycin resistance cassette

used to generate stable cell lines.

For transcriptional reporter assays, cells were transiently transfected with a reporter plasmid

specific to the Wnt, TGF-β, BMP or NFκB pathways, pCMVβ-gal, and any indicated constructs.

To induce the luciferase reporters, cells were treated overnight with the combinations of ligand

and compound as indicated. Luciferase activity in cell lysates was measured using the luciferase

assay system (Promega) in the EG&G Berthold microplate luminometer and normalized to β-gal

levels. HEK293T and HepG2 cells were transiently transfected using calcium-phosphate DNA

precipitation. Briefly, cells plated in 24-well dishes were given a fresh complete medium change

25

2 hr prior to transformation. DNA (3.0 μg/6 wells) was added to 15 μl of 2.5 M CaCl2 mixed

with 150μl of 2× HBS (280 mM NaCl, 50 mM HEPES, and 1.5 mM sodium phosphate [pH

7.05]) and incubated at room temperature for 20 min. The precipitate was added to cells, which

were incubated overnight (Labbe et al., 1998). Colo205, SW620 and SW480 cells were also

transfected with a total of 0.5μg cDNA per individual well of a 24 well plate, using 1 μl

Lipofectmine (Invitrogen) according to standard protocols. After six hours, the medium was

changed for overnight incubation. The following day, cells were treated with compounds and the

assay was performed as described above.

To determine endogenous β-catenin protein levels, lysates were prepared from cells plated in 12

well plates, treated with combination of Wnt and/or compound. Proteins from cell lysates were

separated by SDS–PAGE and transferred to nitrocellulose membranes. β-catenin was detected

using the anti-β-catenin antibody (1:10,000, BD Transduction laboratories) and HRP conjugated

secondary antibody via chemiluminesence as recommended by the manufacturer (ECL kit,

Amersham). Actin levels were detected using an anti-actin (1:2000, Sigma) antibody.

In the manual Ff-luc assay, stable 7 cells generated to express the flag-β-catenin-firefly-

luciferase fusion construct (see above for details) were manually plated into 48-well plates

(Greiner-Bio-One) at a density of 2500 cells/well and incubated for 24 hours. To the cells,

media containing the compounds was added. After a one hour pre-incubation with compound,

Ligand or control media was added and incubated for an additional 15 hours. Media was

removed, cells were lysed and luciferase activity measured using the luciferase assay system

(Promega).

2.5 High-throughput assays

For the β-catenin nuclear translocation assay, NIH3T3 cells (5000/well) plated in 96-well flat-

bottom plates (Costar, Corning) were treated with control or Wnt (50/50) conditioned media.

After a sixteen hour incubation, cells were washed twice in PBS prior to being fixed in 4%

paraformaldehyde (in PBS) for 10 minutes, washed twice, permeabilized with 0.5% triton X-100

(90 seconds), and washed an additional two times (PBS). Cells were prepared for

immunofluorescence by incubating with the mouse monoclonal anti-β-catenin antibody (1:1000,

26

BD Transduction Laboratories) for one hour, followed by a FITC conjugated secondary antibody

(Jackson ImmunoResearch) and Hoesct nuclear staining for one hour. A Cellomics Arrayscan II

HCS imager (Cellomics) was used to image 50-100 cells per well from each 96-well plate. The

Arrayscan V3.5 software captured images using a 10x lens and an XF100 filter. The molecular

translocation application on the Arrayscan was configured to define the nuclear (Hoechst

stained) and cytoplasmic cellular regions and to quantify the FITC fluorescent intensity

difference of β-catenin in these compartments defined as (mean nuclear intensity-cytoplasmic

intensity). These values were calculated for each cell with averages generated for individual

wells.

For the automated Ff-luc assay, stable 7 cells previously generated to express the flag-β-catenin-

firefly-luciferase fusion construct (see above for details) were manually plated into 96-well

plates (Greiner-Bio-One) at a density of 1250 cells/well and incubated for 24 hours at 37 0C plus

5% CO2. One hour prior to initiating the run, the medium was replaced with 50 μL of a

starvation media containing 0.2% FBS via manual pipettor. All plate handling thereafter and

reagent additions were performed by an integrated arm robotics platform (CRS robotic arm

controlled by the Polara software, Thermo Electron). Chemical aliquots from the libraries were

added to cells for a final assay concentration of approximately 1.2 μM and a final DMSO

concentration of 0.2% (compound solvent) via the Multimek automated pipettor (Beckman

Coulter) pinning apparatus. Control wells, treated with solvent alone were in the first and last

columns of each plate for control purposes. Following a one hour pre-incubation with the

compound at 2x final assay concentrations, 50μl of Wnt or control ligand was added to each well

by the multidrop dispensor (Thermo Electron). After a 16 hour compound treatment, 50μl of a

luciferase substrate/lysis reagent (Promega Bright-Glo) was directly added to each well via the

multidrop dispensor and shaken for 10 minutes. Luciferase activity in each well was measured

by the CLIPR (Molecular Devices), a high-throughput luminometer system set to quantify the

luciferase activity for all wells on a plate in a 30 second read. The run was performed in

duplicate, once with compound treatment and Wnt3A ligand, followed by compound and control

media treatment. For consistency, Wnt-stimulated and unstimulated runs were executed together

with the same pool of reagents.

27

2.6 Real time quantitative reverse transcription-PCR analysis Total cellular RNA was isolated from cells cultured in 35mm dishes (BD-Falcon) using RNeasy

Mini Kits (Qiagen) according to standard procedures. Briefly, media was aspirated and cells

washed with PBS, cells were then lysed and harvested with a cell scraper. RNA was then

isolated according to kit instructions. RNA samples were quantified on a spectrophotometer

(Eppendorf) and 6μg of each was treated with 3 units of DNase1 (Fermentas), and reverse

transcribed using random hexamers (2 μL of 100 μM stock) and 400 units of RevertAid H Minus

M-MuLV Reverse Transcriptase (Fermentas). Real-time PCR was performed using the SYBR

Green PCR master mix (Applied Biosystems) on the ABI Prism 7000 or 7900 sequence detection

system (Applied Biosystems). Primer pairs used at 100 nmol/L were designed and previously

reported by Dr. Etienne Labbe (Labbe et al., 2007), and validated against glygeraldehyde-3-

phosphate dehydrogenase (GAPDH) by comparative standard curve analysis. Relative

quantitation was calculated by the ΔΔCt method normalized to GAPDH levels

(docs.appliedbiosystems.com/pebiodocs/ 04303859.pdf).

28

3 Results

3.1 High throughput assay development Development of a high-throughput robotics-based screen requires an assay with high

reproducibility, efficiency and the appropriate sensitivity to chemical compounds. To identify

small molecule inhibitors of the canonical Wnt pathway we developed and tested two assays to

evaluate Wnt signaling. These included an immunoflourescence-based microscopy assay

directed at quantifying β-catenin nuclear translocation and a luciferase-tagged β-catenin

molecule to measure β-catenin stabilization

3.1.1 Beta-catenin nuclear translocation assay

Activation of the Wnt pathway induces the nuclear translocation of β-catenin (Polakis, 2000),

thus the first assay tested was an immunofluorescent-based assay to measure the nuclear

translocation of β-catenin in Wnt stimulated and untreated samples. For this assay, Wnt-

responsive NIH3T3 cells seeded at a density of 12,000 cells per well were plated in a 96-well

dish and treated with Wnt3A containing cell culture media. For this and all other assays, Wnt3A

containing media was generated by collecting media from mouse L cells stably expressing and

secreting the Wnt3A ligand, with the appropriate control media from non-Wnt3A transfected

cells collected in parallel for the assay. In brief, the Articulated arm robotics system at the

Samuel Lunenfeld Research Institute was used for ligand treatment, fixing, washing and staining

of endogenous β-catenin using an anti-β-catenin primary and FITC-conjugated secondary

antibody. A Cellomics Arrayscan II apparatus was used to visualize and quantify multiple wells

in 96 well plates. The Arrayscan V3.5 molecular translocation software is configured to define

the nuclear (Hoechst stained) and cytoplasmic cellular regions and to quantify the fluorescent

intensity difference of β-catenin in these compartments. By counting 50-100 cells per well, β-

catenin nuclear accumulation was quantified in response to Wnt stimulation in the presence or

absence of small molecule inhibitors.

29

The trial runs showed a clear difference in nuclear localization between the treated and untreated

samples (Fig. 3), however, the variability was consistently too high for high-throughput

screening (HTS) purposes. The Z-values, which are a statistical function indicative of the

feasibility of an assay for HTS ranged from 0.01-0.25, which is below the threshold of 0.4

considered to be a feasible assay. Use of higher ligand concentrations or other cell lines did not

improve the Z-values (data not shown). In summary, variability in the treated samples from

multiple tests was consistently too high for a viable screen, even when run in duplicate, and was

not further pursued.

30

Figure 3: The β-catenin nuclear translocation assay displays high variability. NIH3T3 cells

were treated with Wnt3A or control media, fixed, washed and stained with a b-catenin antibody

on the robotics platform. Nuclear and cytoplasmic subcellular regions were defined through

staining patterns and the difference in β-catenin fluorescent intensity between these

compartments was quantified using the Cellomics Arrayscan II apparatus. A) Raw data

collected during a run from all six plates is plotted collectively, and indicates considerable

variability amongst the Wnt treated samples. B) Data from the best (plate 1) and worst (plate 4)

plates in the run are plotted with their associated Z-factor values. Z-factor values, which are a

measure of assay viability are substantially under the threshold of 0.4 considered to be a viable

assay C) Z-factor equation, which accounts for the dynamic range between control and

experimental values, and the variability within the data set. Four parameters are needed to

calculate Z-factor; the mean (m) and standard deviation (s) of both the control (c) and sample

values (s).

31

Figure 3

32

3.1.2 Firefly luciferase β-catenin stabilization assay (Ff-luc)

As the stabilization of β-catenin is the key event in dysregulated Wnt signaling, I next developed

a method to measure β-catenin protein levels by tagging β-catenin with a firefly luciferase

protein (Fig. 4A). The luciferase tag was fused C-terminally to human β-catenin in the pCAGIP

plasmid under the control of a β-actin promoter. Multiple clones in two mammalian cell lines

(HEK 293 and L’s) stably expressing the fusion protein were isolated via puromycin selection

and were tested for Wnt induced increases of luciferase signal, indicative of β-catenin

stabilization. Most of the HEK 293 cell clones displayed roughly a two-fold increase in

luciferase activity in response to Wnt treatment (Fig. 4B), while those obtained from L cells were

less responsive (data not shown). Subsequent testing showed that HEK293 clone number 7 gave

the most reproducible results, and a signal sufficiently high to be amenable for HTP detection

and was selected for high-throughput screening. The cell density that yielded maximal β-catenin

stabilization was next determined by seeding a varying number of cells (from 500-6000) in each

well of a 96-well dish. The following day, media was aspirated, cells lysed, and the lysates were

then measured for luciferase activity. A density dependent decrease in fold activation was

observed (Fig. 4C). Thus, seeding of 1250 cells per well, roughly corresponding to 25%

confluence, achieved a reproducible two-fold activation and was easily detectable with the

CLIPR, a CCD camera used to measure the luciferase signal on the robotics platform. Further

testing indicated that DMSO did not affect the viability of the cells, nor did it affect the readout

(Fig. 4D).

3.1.3 Ff-luc assay optimization

Due to the limited (two-fold) dynamic range of the Ff-Luc assay, it was necessary to conduct a

series of manual and then robotic runs to optimize and validate the HTP protocol. Thus a number

of seemingly modest details of the assays were optimized to achieve best results. First, I tested a

variety of different plate types and brands settling on Greiner Bio-One plates which allowed for

easy robotics handling, permitted high cell adherence and the deep wells minimized glow

contamination from adjacent wells. We varied the reading time on the CLIPR from 10-120

seconds and established that 30 second reads were in the linear range of detection and gave a

33

Figure 4: Development of the β-catenin-firefly-luciferase (Ff-luc) stabilization assay. A)

Schematic of the Ff-luc assay, which allows for high-throughput detection of b-catenin stability

in human cell lines. B) In the presence and absence of Wnt3A, β-catenin levels were

determined by measuring the luciferase signal of whole cell lysates in HEK293 clones stably

expressing Ff-Luc-β-catenin. Most clones displayed a Wnt3A induced stabilization β-catenin.

Clone 7 was selected for screening because it reproducibly displayed a robust Wnt3A

stabilization and possessed a high signal intensity. C) The Ff-luc assay is influence by seeding

density. Clone 7 cells were plated in 96-well dishes at the indicated cell densities and assayed

for luciferase signal. A density of 1250 cells/well yielded a repeatable two-fold Wnt induction

and a signal intensity high enough for detection on the robotics platform. D) Using the Ff-luc

assay as readout, DMSO has no effect on assay signal or cell viability at concentration

comparable to proposed screening conditions.

34

Figure 4

35

sufficiently high signal. In addition the ideal volumes of media, ligand and luciferase/lysis

reagent were determined taking into consideration, cost, ease of use and reproducibility.

The final established assay protocol involves manual plating of 1250 cells/well in a 96 well

format, followed by a 24 hour incubation. One hour prior to initiating the run, the medium is

replaced with 50 μL of media containing 0.2% FBS. Compounds are added by pinning DMSO

soluble compounds to a final concentration of approximately 1.2 μM and then incubated for one

hour prior to the addition of 50 μl of Wnt or control ligand overnight. On day three, 50μl of a

luciferase substrate/lysis reagent (Promega Bright-Glo) is directly added to cells, the plate is

incubated in a shaker for 10 minutes at room temperature and luciferase activity quantified using

a CCD camera. The readout indicates Wnt induced stabilization of the β-catenin fusion protein

(see Fig. 5). On each plate, the two outer columns are used for control purposes, with cells in

one column treated with Wnt ligand and the other with control media, while the inner 10

columns are all treated with individual compounds.

Prior to undertaking a HTS, it is essential to validate assay performance. However, in the case of

Wnt signaling, upstream inhibitors were not available. As an alternative, we used an inhibitor of

GSK3β, Indirubin-3’-monoxime (I3M, Sigma), which activates Wnt signaling, for proof of

principle experiments (Polychronopoulos et al., 2004). I first confirmed compound activity in

manual assays. I3M stabilized the β-catenin-luciferase fusion construct in a dose dependent

manner in both Wnt treated and untreated samples (Fig. 6A). The stabilizing effect on β-catenin

observed using the Ff-Luc assay was mimicked when endogenous β-catenin was examined by

immunoblotting in a mouse fibroblast cell line (L cells) (Fig. 6B). To test for reliability and

reproducibility of the HTP platform, we added I3M to a final concentration of 5 μM, which

elicits signal stabilization roughly equivalent to Wnt3A treatment. Robotic runs showed minimal

variability within I3M treated wells across plates, indicating accurate and reliable compound

addition by the robotic platform (Fig. 7). Therefore, not only was it possible to identify chemical

modulation of the pathway in the assay, the robotics protocol readout and compound addition

steps were functioning as expected.

36

Figure 5: The Ff-luc assay. A) HEK293 cells (Clone 7 cells) stably expressing Firefly

Luciferase tagged β-catenin were used to indentify compounds affecting b-catenin stability in the

Ff-luc assay. B) Step-by-step flowchart of the Ff-luc assay. C) Raw data from plate 21 is

shown for both the Wnt-treated and unstimulated runs. H7 denotes an inhibitory hit identified

from the screen.

37

Figure 6: Validation of the Ff-luc screening protocol. A) Using the Ff-luc assay as readout,

Wnt pathway agonist Indirubin-3'-monoxime (I3M, Sigma), a GSK3β inhibitor, was used to

validate the screening protocol. Here, the small molecule I3M stabilizes the Ff-luc-β-catenin

reporter in a dose-dependent manner. B) I3M is also shown to stabilize endogenous levels of β-

catenin in mouse L-cells as determined by immunoblotting in a similar dose-dependent manner.

38

Figure 7: Validating the pipetting accuracy and chemiluminescent readout of the robotics

platform. The robotics platform was programmed to add a 120 nL aliquot of concentrated

Indirubin-3'-monoxime (I3M) using the Multimek pinning tool into alternating wells of four 96-

well plates in the Ff-luc assay. The desired final concentration of (5 µM) is known to stabilize b-

catenin similar to Wnt3A treatment. The Ff-luc assay was performed entirely on the robotics

platform, and averages from the four different treatments across all four plates is displayed. The

data from the robotic compound addition indicates low variability and consistency across the

assay.

39

3.2 Pilot high-throughput Ff-luc assay run After having established a screening protocol, our next goal was to identify the ideal compound

concentration critical for screening purposes. Identifying the proper screening concentration

allows us to balance the search for compounds with high specificity at a low concentration while

minimizing false negatives. Typical concentrations used in cell based screens range from 1-10

μM, therefore to determine optimal concentrations for our assay we first screened through the

first 800 compounds (10 plates) of the Maybridge library at roughly 1.2 and 7 μM. Analysis of

the 10-plate screen revealed that higher chemical concentration significantly increased variability

in the assay. Average Z’-values for the first 10 plates screened at the lower concentration was

0.379 compared to -0.135 for the same 800 compounds screened at the high dose. Therefore, all

future screens were conducted using the lower (1.2 μM) concentrations, we expect this would

enhance identification of more potent compounds.

We next conducted a pilot screen using the first 4000 compounds of the Maybridge chemical

library to assess the hit rate and reproducibility of the assay. For this, runs with and without Wnt

ligand were performed together, while a replicate Wnt treated run was executed on a separate

day. Results from the initial run (Wnt3A #1) of the first 4000 compounds (Table 2) showed 37

individual activating hits of the pathway, 25 of which were duplicated in the replicate Wnt

treated run (Wnt3A #2), and 24 of these activators were also detected in the control run (Wo).

The high degree of correlation amongst the three runs suggests a high rate of reproducibility for

the screen. A similarly pattern of reproducibility was seem among inhibitory compounds. We

identified fewer inhibitors with a total of 9 from the first Wnt3A screen; 5 duplicated in the

second stimulated run with 4 being detected in all three runs. Furthermore, the 4 inhibitors

common to all runs can be further broken down into those displaying an increased inhibitory

effect in the Wnt treated samples, and those with similar levels of inhibition in both treated and

untreated runs. One compound, 21H07, fit the profile of preferential Wnt inhibition.

Using B-score analysis, the hit rate for activators from the initial run (Wnt3A #1) was 0.93%.

Inhibitors, which were the more desired lead compound, had a lower rate of 0.23%. Based on

these hit rates we predicted a total of 465 activating and 115 inhibiting lead compounds from the

entire 50,000 molecule Maybridge library if we were simply to screen the library with one

stimulated and one unstimulated run. Because the majority of the hits were duplicated in the

40

Table 2: Pilot screen hits indicate a reproducible Ff-luc hit rate. The pilot Ff-luc screen was

performed on the first 4000 Maybridge compounds to assess the hit rate and reproducibility of

the assay. For this, two duplicate Wnt treated runs were performed on separate days, while a

single unstimulated run was run in conjunction with the first stimulated one. Hits (both

activators and inhibitors) were defined by those points surpassing the 3 standard deviation

threshold above or below the mean. The associated B-score for each compound is listed.

41

42

second Wnt treated run and considering screening costs, we concluded that a single stimulated

run combined with an unstimulated run would be sufficient for the final screening procedure.

Moreover, the 46 chemical modulators identified in the initial stimulated run (37 activators, 9

inhibitors), 30 were duplicated in the second run and 28 of these were identified in the

unstimulated run. Consequently, a large increase in false positives is not expected to be very

pronounced from using single runs, therefore, all subsequent runs were performed without a

duplicate Wnt treated run.

3.3 Final screening results and data analysis Three diverse sets of compound libraries were available for our screen, the Maybridge, Lopac

and Prestwick chemical sets. The Maybridge chemical library is a 50,000 molecule diverse

library set, whose compounds possess typical drug-like properties. The Prestwick set (Prestwick

chemicals) includes an 1120 molecule set of largely off-patent drugs and the 1280 molecule

Lopac set contains drug compounds with some known molecular targets and properties.

3.3.1 Comparison of various statistical hit-selection methods

As there is only a two-fold dynamic range in the Ff-Luc assay, the data analysis method would

be critical to our hit selection process. To establish the most appropriate data analysis method,

we focused on the Lopac library results due to its small size and the known properties of many of

its compounds. The data from stimulated runs was analyzed using three distinct statistical

methods; percent activity, fold over median and B-score. The percent activity measure is the

simplest method, in which experimental data points (compound treated wells) are compared to

control wells within the same plate. The data is converted into a percentage value, whereby the

value of 100 indicates no effect and activators or inhibitors will show an increase or decrease

relative to 100. The fold over median method is a measure of the compound treated wells with

respect to each other on an individual plate, and does not take the control wells of the plate into

consideration. The B-score is a more complex function which takes into consideration the

experimental points (compound treated), yet also factors in plate location by relating the data

43

points to adjacent wells within a plate, thereby correcting for possible plate trends in a run

(Brideau et al., 2003).

The three data analysis methods for the Lopac library returned roughly the same number of hits

for the Wnt3A treated run (Fig. 8). Fold over median (using a three standard deviation cutoff)

identified 4 activators and 26 inhibitors, percent activity (also using three SD) showed 4

activators and 23 inhibitors, while B-score revealed 2 activators and 21 inhibitors. Notably, all

the effectors, both β-catenin stabilizers and destabilizers indicated by the B-score method were

hits in the other methods as well. Both fold over median and percent activity measures identified

some hits that did not appear in the other methods. This analysis revealed two important

conclusions. First, B-score appeared to be the most reliable of the methods as miscellaneous hits

were apparently weeded out and secondly that irrespective of which statistical method is used to

determine hits, most of the same hits are identified amongst all methods. This analysis

confirmed that even though the dynamic range of the Ff-Luc assay is not very large, it is in fact a

viable screen and variability within and between plates is best analyzed using the B-score

method.

3.3.2 Summary of results from the Lopac library

As reported above, B-score analysis of the Wnt stimulated run identified 4 compounds which

stabilized the β-catenin-Ff-Luc construct and hence were possible Wnt pathway agonists, and 23

molecules that destabilized β-catenin and might work as Wnt antagonists (all listed in Table 3).

Additionally, the unstimulated run displayed 15 inhibitors and 6 activators. Twelve of these

inhibitors are common to both stimulated and unstimulated runs; therefore, 11 inhibitors were

detected uniquely in the Wnt treated run (Fig. 9). These are the compounds that appear from

screening results to possess a preferential inhibitory effect in the presence of Wnt treatment and

are more likely to be Wnt pathway antagonists as opposed to general effectors of β-catenin

stability. Interestingly, the inhibitory hit rate on average was about 4 fold higher that the

activators.

44

Figure 8: Comparison of different statistical hit-selection methods. Fold over median, B-

score and percent activity measurements were used to analyze raw data from Lopac library

screen. A) Data from the Lopac library is depicted for each method of analysis, Fold over

median, B-score and percent activity. The upper and lower lines mark the three standard

deviation threshold we defined as a hit in the high-throughput screen. B) Summary of Lopac hits

using the different methods of analysis, and those common amongst all three statistical methods

of analysis.

45

Figure 8

46

Figure 9: Lopac library screening results. A) Summary of total hits and hit rate identified

through B-score analysis of the 1280 molecules screened from the Lopac chemical library. Hits

were defined by those points surpassing the 3 standard deviation threshold above or below the

mean. B) Raw data plotted from B-score analysis of the entire screened Lopac run is depicted.

Lines mark the 3 standard deviation threshold above and below the mean (middle line).

47

Figure 9

48

3.3.3 Summary of results from the Prestwick library

The largely off-patent drug containing Prestwick library displayed a pattern similar to that of the

Lopac compounds, where the vast majority of effecter molecules had a destabilizing effect on β-

catenin. The inhibitory hit rate for the combined (stimulated and unstimulated) runs was 2.37%.

Twenty seven destabilizers were identified in the Wnt treated run and 26 in the untreated run,

with the vast majority (21) common to both (Fig. 10A and B). With only five activators

identified in the Wnt3A treated run and three from the unstimulated run, it is interesting to note

that only two are common to both screens, three are unique to stimulated while one is unique to

the non-Wnt treated run.

3.3.4 Summary of results from the Maybridge library

Hits identified from the Maybridge chemical library would be particularly interesting as any

inhibitors or activators identified would be novel and undocumented, unlike the Lopac and

Prestwick sets. Of the entire 50,000 Maybridge set, 130 plates corresponding to 10400

compounds were screened.

Based on the hit rates of activators (0.93%) and inhibitors (0.23%) from the initial 4000

Maybridge compound pilot screen, we predicted a total of 97 activating and 24 inhibiting lead

compounds from the 10400 screened of the Maybridge set. The activator hit rate for the entire

10,400 compounds screened adhered roughly to the expected rates culminating in an overall

activator hit rate of 1.52% or 158 compounds for the stimulated run. The unstimulated run

displayed a higher activator hit rate of 188 compounds, 77 of which were unique to the

unstimulated run. In contrast, the inhibitory hit rate increased significantly from the predicted

0.23% to 0.6%, consisting of 63 inhibitory compounds in the stimulated run and 0.44% or 46

compounds in the unstimulated run, with 40 hits unique to the stimulated run (Fig. 11 A and B).

Of note, the Maybridge library displayed an approximately 2.5 fold higher ratio of activating

versus inhibitory hits, an inverse of the pattern seen with the Prestwick and Lopac Libraries.

49

Figure 10: Prestwick library screening results. A) Summary of total hits and hit rate

identified through B-score analysis of the 1120 molecules screened from the Prestwick chemical

library. Hits were defined by those points surpassing the 3 standard deviation threshold above

or below the mean. B) Raw data plotted from B-score analysis of the entire screened Prestwick

run is depicted. Lines mark the 3 standard deviation threshold above and below the mean

(middle line).

50

Figure 10

51

Figure 11: Maybridge library screening results. A) Summary of total hits and hit rate

identified through B-score analysis from 10400 molecules screened from the Maybridge

chemical library. Hits were defined by those points surpassing the 3 standard deviation

threshold above or below the mean. B) Raw data plotted from B-score analysis of the entire

screened Maybridge set run is depicted. Lines mark the 3 standard deviation threshold above

and below the mean (middle line)

52

Figure 11

53

3.3.5 Total assay hits from the entire HTP screen

Analysis of Wnt stimulated runs from all three libraries screened identified 120 inhibitors for

follow up work. Of these, 57 were solely detected in the Wnt stimulated runs and an additional

five of these displayed preferential inhibition upon Wnt stimulation. Similarly, there were a

significant number of activators identified, 194 from the unstimulated runs alone. Eighty of these

were detected only in the non-Wnt treated run and are thus of more interest as activators.

Therefore, there are a significant number of compounds which are worthy targets for secondary

analysis (Table 3).

3.4 Investigating selected hits via secondary analyses

3.4.1 Assessing the reproducibility of effects

From the 13,080 screened compounds, we selected nine from Maybridge, three from Prestwick,

and one from Lopac that inhibited β-catenin stability for further analysis. Nine randomly

selected compounds were also used as comparative controls. Lead compound selection was

based on a variety of criteria. Some were chosen for preferential inhibitory activity in the

presence of Wnt stimulation, where others displayed similar affects in both ligand-treated and

untreated screens. While all were hits based on statistical analyses, some compounds displayed

potent destabilizing properties while others displayed more moderate effects.

Compounds were first analyzed using a manual version of the Ff-Luc assay to assess the

reproducibility of hit compounds using the same stable clone 7 cell line but in a 48 well format.

Luciferase activity was measured subsequent to media removal and cell lysis, rather than using a

homogeneous lysis as in the robotics protocol. Results from secondary analyses are summarized

in Table 3. Of the fourteen compounds across three libraries displaying a destabilizing effect on

β-catenin in the Ff-luc assay, only three were reproduced in the duplicate manual Ff-luc (Table

4; Fig. 12), indicating a false positive rate for the Ff-luc of approximately 80%. The apparent

high false positive rate is unexpected given the significant reproducibility seen in the duplicate

runs of the first 4000 Maybridge compounds. However, as only a small number of samples

54

Table 3: Summary of results from all compounds screened. Compounds from the three

libraries screened were identified as inhibitors or activators based on the destabilizing or

stabilizing effect in the Ff-luc assay. Hits are determined as points three standard deviations

above or below the mean using B-score analysis. Data is presented as both totals and percent hit

rate for Wnt-treated (Wnt3A), and untreated (Wo) high-throughput screening runs.

55

Table 4: Summary of secondary assay results for twenty-two selected inhibitors and

random controls. Nine randomly selected controls (compound #10-18) and thirteen inhibitory

compounds were selected from the three libraries screened. Compounds were tested for their

effect in manual repeats of Ff-luc to assess reproducibility. And for effects upon specific

signaling pathway transcriptional reporter assays to ascertain Wnt pathway specific effects. In

signaling assays, HEK293T cells were transfected with the indicated pathway specific reporter

plasmids, and treated overnight with compound and the appropriate ligand. Luciferase activity

was measured and normalized with a β-gal control. Effects in the manual Ff-luc assay are

reported as modulation of Ff-luc-β-catenin levels. Inhibitory or activating effects on pathway

specific transcriptional regulation are indicated with directional arrows. Points in which no

effect was seen are reported with a dashed line.

56

Figure 12: Re-testing of select compounds using the Ff-luc assay. Compound 21H7 was

shown to destabilize the Ff-Luc-b-catenin construct. Other compounds displayed no effect or

stabilized (compound 25A2) Ff-Luc-β-catenin. Manual repeats of the Ff-luc assay were

preformed in 48 well plates at 0.5, 1, 2.5, 5 and 12.5 µM concentrations. Media was removed

and luciferase values were measured from whole cell lysates in a luminometer.

57

(12% of total hits) were tested, this may not be a completely accurate estimate of false-positive

rates. Of the randomly selected compounds not detected as hits during screening, none were

seen to modulate β-catenin stability in the manual Ff-luc repeat, suggesting a low false negative

rate.

3.4.2 Effects of hits on Wnt transcriptional activity and assessing pathway specificity

Next, those compounds scoring positive in the screen were tested for effects on Wnt induced

transcriptional activity in HEK 293T cells. Compound effects were monitored by transfecting

the Wnt pathway specific luciferase reporter plasmid pTopflash. A construct containing a

mutated TCF/LEF binding site, pFopflash, was transfected in parallel as control. Transfection

efficiencies were corrected by cotransfection with a β-galactosidase-encoding reporter plasmid.

Assays were performed using varying compound doses to facilitate determination of the effective

dose range for each compound. β-catenin activated transcriptional activity from the Topflash

reporter assay was abrogated by only five of the fourteen inhibitory compounds tested (Table 4).

Moreover, when comparing to the manual Ff-luc assay, all three of the compounds shown to

reproduce a β-catenin destabilizing effect also demonstrated an inhibitory effect upon Wnt

mediated transcriptional activity (Table 4). Additionally, none of the randomly selected

compounds not identified as inhibitors in the screen modulated the Wnt pathway.

The compounds identified as inhibitors in the screen, manual repeats, or in Topflash assays, were

next assessed for specific or preferential effect upon the Wnt signaling pathway. For this, we

employed multiple pathway specific reporter assays, namely 3TP-lux, IBRE-luc and NFκB

reporter plasmids that are responsive to the TGFβ (Transforming Growth Factor β), BMP (Bone

Morphogenetic Protein) and IL1 (Interleukin 1) ligands, respectively. Only two compounds,

21H7 and 46D10, inhibited Wnt-dependent transcriptional activity, yet had minimal or no effect

on the other pathways tested (Table 4). Therefore, in using these assays, we are able to assess

properties of hit compounds and rule out general cell inhibitors (compounds 23H2, 25A2, and

chelerythrine chloride) which display transcriptional effects on all pathways tested (Table 4).

Compound 21H7 reduced Topflash reporter activity by 60% at 1μΜ concentration (Fig 13).

58

Figure 13: Compound 21H7 preferentially inhibits the Wnt pathway. HEK293T cells were

transfected with the indicated reporter plasmids, and treated overnight with compound and

pathway specific ligand. Luciferase values indicate pathway specific reporter gene expression.

The inhibitory compound (21H7) displays a preferential inhibitory effect upon the Wnt pathway

specific reporter Topflash assay in comparison to TGFβ, IL1 and BMP responsive transcriptional

assays.

59

While only a 20% reduction of NFκB and BMP pathways was observed at this concentration.

Therefore, these results indicate a preferential effect on the Wnt pathway. Although compound

46D10 (Table 4) displayed inhibitory properties, this activity was lost upon subsequent

compound restocking and was not pursued further (data not shown). As the remaining inhibitory

compounds affected other pathways tested, either promoting or inhibiting transcriptional activity

indicative of non-specific effects (Summarized in Table 4), 21H7 was the only compound

selected for further analysis.

3.5 Analysis of 21H7, a selective Wnt pathway inhibitor

3.5.1 Compound 21H7 preferentially inhibits Wnt responsive Topflash activity over the non-responsive Fopflash

We next assessed the effects of a wider range of 21H7 doses in a Topflash luciferase assay using

Fopflash as control. Fopflash as previously reported is a construct identical to Topflash, except

that the LEF/TCF binding sites are mutated and therefore the reporter is non-responsive to Wnt

treatment. Topflash results indicate 21H7 displays a dose-dependent effect, having no activity at

0.1 μM, while at 0.5μM a 30% inhibition of transcriptional activity is observed. The effect levels

out at approximately 80% inhibition at concentrations between 2.5 and 12.5 μM, and the IC50

value is approximately 1 μM (Fig. 14). At inhibitory concentrations (0.5-12.5 μM) in the

Topflash assay, a preferential inhibitory effect in the Wnt treated samples (80%) is observed

compared to the control (45%) treatment. Furthermore, analogous Fopflash experiments (Fig.

14) do not display the same differential effect between the Wnt treated and untreated data sets.

In fact, the Fopflash data appears almost identical in both inhibitory and dose dependent effect to

that of the untreated Topflash data. Analysis of the Fopflash and Topflash data combined

strongly indicates that 21H7 is acting preferentially to block Wnt-dependent transcriptional

activity.

60

Figure 14: 21H7 preferentially inhibits the transcriptional activity of Wnt3A treated cells.

HEK293T cells were transfected with the Wnt responsive Topflash or non-responsive Fopflash

reporter plasmids. One day after transfection, cells were treated with 21H7 or DMSO control

and stimulated overnight with Wnt3A or control ligand. Data is presented as a percent of the

control (DMSO) value. Preferential inhibition is seen in Wnt-treated Topflash data, but not with

the non-Wnt responsive Fopflash reporter.

61

3.5.2 21H7 blocks Wnt-induced β-catenin stabilization

Our screen was designed to identify molecules that effect Wnt-dependent β-catenin stabilization,

thus we next examined the effects of 21H7 on endogenous β-catenin. For this, we utilized

mouse fibroblastic L-cells, a line commonly used to measure Wnt induced β-catenin stabilization

due to their low steady-state β-catenin levels and a pronounced Wnt3A induced stabilization of

β-catenin. In multiple experiments, we found that Wnt3A treatment induces a steady rise in β-

catenin levels for up to four hours at which time levels remained constant up to 16 hours.

Therefore, we examined the effects of the compound at the four hour time point.

As seen in Figure 15, cells stimulated with Wnt3A displayed a robust and dose-dependent

increase in total β-catenin levels. The addition of 21H7 at low concentrations did not affect total

β-catenin levels, but in the range of 5-12.5 μM, dramatic destabilization of β-catenin and no

effect on α-actin levels was observed. Similar results were observed with overnight treatment of

both compound and ligand (data not shown), indicating the compound is effective in both short

term and longer time frames. We noted that even at the highest concentrations, the compound

did not restore β-catenin levels to that observed in controls. Nevertheless, these experiments

indicate that 21H7 can significantly decrease endogenous β-catenin levels induced by the

addition of Wnt ligand.

3.5.3 21H7 inhibits Wnt dependent gene expression in colon cancer cell lines

For a lead compound to have anticancer application, the compound should also demonstrate the

ability to suppress the constitutive Wnt signaling typically observed in cancers. Colorectal

cancer (CRC) cell lines contain a variety of activating mutations in key Wnt pathway

components, typically APC or β-catenin, and in some cases both, that result in constitutively

nuclear β-catenin and activation of Wnt target genes (Ilyas et al., 1997). Thus, we next utilized

the Topflash/Fopflash reporter system to ascertain the ability of compound 21H7 to turn off

constitutive Wnt signaling in CRC cell lines.

62

Figure 15: Compound 21H7 inhibits the Wnt3A induced stabilization of endogenous β-

catenin levels in the presence of Wnt3A and increasing concentrations of compound 21H7. Cell

lysates from mouse L-cells pre-treated with compound for 1 hour then stimulated with Wnt

ligand for a four hour period were immunoblotted with anti-β-catenin and anti-β-actin to

determine protein levels. The Wnt-induced stabilization of β-catenin is inhibited in a dose

dependent manner by 21H7. The final compound concentration ranges from 0.1µM to 12.5 µM,

while control lanes were treated with DMSO only.

63

Common amongst all CRC lines tested is the loss of Wnt induced transcriptional activation of

Topflash signal (Fig.16 B), as a result of mutations that hyperactivate the Wnt pathway. SW620

cells harbor an APC truncation (Zhao et al., 2007), and upon compound treatment we detected a

significant inhibition of Topflash transcriptional activity yet very little inhibition of the Fopflash

construct (Fig. 16A), a pattern of preferential inhibition also seen with HEK293 cells (Fig 14).

Although the pattern of preferential Topflash inhibition in β-catenin mutated Colo205 cells

(Gayet et al., 2001) is seen, the effect was not as dramatic. However, in two additional cancer

cell lines containing mutations in β-catenin (HepG2 cells) and APC (SW480 cells), the inhibitory

effects on neither Topflash nor Fopflash reporters were detected. Together, this set of results

indicates that 21H7 can inhibit Topflash activity in colorectal cancer cell lines harbouring

diverse pathway mutations, however, penetrance of this effect appears to depend on cell

background.

3.5.4 Expression of endogenous Wnt target genes is inhibited by 21H7 in CRC cell lines

We next assessed the inhibitory effects of the compound on the Wnt3A induced expression of

endogenous Wnt target genes in a range of colorectal cancer cell lines. Previous work in the lab

(Labbe et al 2007) and other studies (Shtutman et al., 1999) have demonstrated Wnt-dependent

induction of Axin2 and Cyclin D1, thus, we focused on those genes endogenous targets. Cells

were treated with compounds or with DMSO as control for 16 hours, RNA was isolated and

reverse transcribed, and relative gene expression levels normalized to GAPDH levels were

quantified using Real-Time PCR.

21H7 induced a significant dose-dependent decrease in Axin2 levels in all CRC cell lines tested,

though, the extent of inhibition varied between the lines. In the related cell lines SW480 and

SW620 cells, which are heterozygotic for a truncated APC (Zhao et al., 2007), an 80-90%

reduction in total Axin2 levels was seen (Fig. 17). Analysis of four other cell lines, each

harboring different mutations in Wnt pathway components also revealed significant reductions in

Axin2 mRNA levels. Similar to SW480 cells, LS1034 cells have undergone APC loss of

heterozygosity and possess a truncated form of the remaining allele (Sparks et al., 1998).

64

Figure 16: Differential suppression of Wnt-dependent gene expression in colon cancer cell

lines harboring activating mutations in the Wnt pathway by 21H7. Cells transfected with

individual reporter constructs were treated with the 21H7 and stimulated with Wnt3A or ligand

control overnight. A) In a dose-dependent manner, 21H7 inhibits both Topflash and Fopflash

activity. Data is presented as a percent of the control (DMSO) value. B) All colon cancer cell

lines tested lack ligand responsiveness due to known hyperactivating mutations of the Wnt

pathway.

65

Figure 16

66

Figure 17: Wnt target gene expression is inhibited by 21H7 in colon cancer (CRC) cell

lines irrespective of the differing Wnt pathway mutations. Total cellular RNA was isolated

from prostate cancer cell lines treated with 21H7 or DMSO control for a 16 hour period. The

RNA was reverse transcribed with random hexamers and cDNA’s of established Wnt target

genes were quantified by QPCR (using SYBRGreen in an ABI Prism 7000 sequence detection

system). Levels of GAPDH cDNA were also quantified and used to normalize results by

comparative standard curve analysis. All six CRC cell lines possess activating mutations of key

Wnt pathway components, as shown. Irrespective of their mutational status, all lines tested

display a 21H7 induced decrease in mRNA levels of established Wnt target genes, with differing

magnitudes of effect.

67

Figure 17

68

Despite the similar mutational status, the reduction in Axin2 levels (roughly 50%) was not as

robust as seen in SW480 and SW620 cells. The HCT116 and LS174T cell lines contain wild

type APC, but possess β-catenin mutations at serine 45, a residue phosphorylated in a Wnt

dependent manner (Gayet et al., 2001; Sparks et al., 1998). In HCT116 cells, this mutation

consists of a heterozygous deletion of serine 45, while LS174T cells possess a homozygous

serine to phenylalanine mutation. In addition to possessing similar mutations, HCT116 and

LS174T cells displayed very similar dose-dependency with roughly 70% maximal reduction in

Axin2 mRNA levels (Fig.17). Colo205 cells displayed the lowest reduction of Axin2 mRNA

levels (45%) in the presence of 21H7. The Colo 205 cells possess compound mutations of the

Wnt pathway, whereby neither APC nor β-catenin are wild-type. However, the mutation in β-

catenin is an alanine to serine substitution at amino acid 287, which has not been examined for

effects upon Wnt signaling. From the above data, it is clear that Axin2 mRNA levels are

affected by 21H7 in all cell lines regardless of their Wnt pathway mutational status. Moreover,

the varied effect of inhibition between cell lines displays no clear pattern based the mutational

status of the cells.

CyclinD1 is another established Wnt target gene, though in general, Wnt dependent induction is

not as robust as observed for Axin2 (Jho et al., 2002; Labbe et al., 2007; Tetsu and McCormick,

1999). Levels of CyclinD1 displayed a modest 21H7 induced reduction in most cell lines, for

some lines, no change at was identified (Fig. 17). In addition, the effects on CyclinD1 levels do

not appear to correlate with the mutational status of cells. LS 1034 and Colo 205 cell lines,

which displayed the least reduction in Axin2 mRNA levels, exhibit no significant decrease in

total CyclinD1 mRNA levels. The only cell line in which CyclinD1 and Axin2 levels were

comparably affected, resulting in a maximal reduction of 70% in each case, was the LS174T line.

In all other lines, 21H7 had a significantly more pronounced effect upon Axin2 levels than

CyclinD1. Levels of CyclinD1 are regulated by a variety of other factors including NF- κB

(Guttridge et al., 1999), STAT (Leslie et al., 2006) and Ras-dependent signaling (Gladden and

Diehl, 2005), indicating the balance of CyclinD1 levels in cells is important to or affected by

other factors.

From the combined Axin2 and CyclinD1 data, it is clear that Wnt target gene mRNA levels are

affected by 21H7 in colorectal cancer cell lines regardless of their Wnt pathway mutational

69

status. The inhibitory effects did vary between cell lines and were significantly more pronounced

for Axin2, however, the experiments indicate no clear pattern of inhibition based upon the

mutational status of the cells.

3.5.5 21H7 inhibits expression of endogenous Wnt target genes in prostate cancer cell lines.

Excessive Wnt signaling has been implicated in a variety of cancers. Therefore, we next

assessed the effects of 21H7 on the established prostate cancer cell lines, PC3 and Du145 cells,

in which the mutational status of Wnt pathway components has not been examined by us or

others. In both prostate lines tested, we identified a marked inhibition of the expression of both

the Axin2 and CyclinD1 Wnt target genes (Fig. 18). This demonstrates that 21H7 can suppress

Wnt transcriptional activity in both colon cancer and prostate cell lines. As Wnt pathway

misregulation is prominent in a wide range of cancers including prostate (Yardy and Brewster,

2005), these indicate that 21H7 may have efficacy in a broad range or cancers.

3.5.6 Wnt signaling is inhibited by 21H7 at the level of β-catenin

Having demonstrated the wide ranging and significant inhibitory effect of 21H7 on Wnt-

dependent transcription, we next focused on examining where in the pathway 21H7 acts. While

the Wnt pathway is becoming increasingly complicated, key steps can be viewed as a linear

system, involving ligand-receptor binding, dishevelled activation, destruction complex

dissociation, β-catenin stabilization, nuclear translocation and transcriptional activation (Fig.

19A). Therefore, to determine where 21H7 acts, we performed Topflash assays in the presence

of individual Wnt-activating pathway components, with the goal of identifying whether the

repressive activity of 21H7 occurs in their presence.

Wnt signaling requires a Wnt induced Frizzled-LRP interaction (Holmen et al., 2005; Liu G,

2003). Interestingly, truncated forms of LRP omitting the N-terminal extracellular domain

confers pathway activation by a yet undetermined mechanism (Mi and Johnson, 2005). Our data

indicates that the inhibitory effect of 21H7 is not lost in the presence of LRP6ΔΝ, ruling out any

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Figure 18: 21H7 inhibits Wnt target gene expression in prostate cancer cells. Total cellular

RNA was isolated from prostate cancer cell lines treated with 21H7 or DMSO control for a 16

hour period. The RNA was reverse transcribed with random hexamers and cDNA’s of

established Wnt target genes were quantified by QPCR (using SYBRGreen in an ABI Prism

7000 sequence detection system). Levels of GAPDH cDNA was also quantified and used to

normalize results by comparative standard curve analysis. 21H7 is able to decrease endogenous

mRNA levels of both Wnt target genes in the both prostate lines tested. The mutational status of

APC and β-catenin has not been characterized in either cell line.

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Figure 19: Epistatic analysis indicates 21H7 effects Wnt-dependent signaling at the level of

β-catenin. HEK293T cells were transiently transfected with the Topflash reporter along with

Wnt pathway activators LRP6, Dvl2 and β-catenin. A) Linear schematic of the canonical Wnt

pathway noting the location of overexpressed components. B) 21H7 retains the ability to inhibit

Wnt dependent transcriptional expressions in the presence of upstream activating components

(LRP6 & Dvl2). C) In the presence of β-cateninD91, a constitutively active mutation, 21H7

retains its inhibitory effect on Topflash activity and is therefore not inhibiting the Wnt signaling

cascade.

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effects on ligand binding or receptor interaction (Fig 19B). Likewise, the inhibitory effect

appears to occur downstream of Dishevelled2 (Dvl2) as 21H7 still inhibited Topflash activity in

a Dvl2 overexpression background. Similar results were observed on Topflash when

overexpressing β-catenin (Fig 19B and C). Considering our previous data, that indicated an

effect of 21H7 on β-catenin levels, we next assessed whether this effect was specifically on the

Wnt induced β-catenin stabilization, or on basal levels. The β-cateninΔ91 construct, lacking its

amino terminus and hence the three residues phosphorylated during a Wnt response is a

dominant positive mutant. The ability of our inhibitor to reduce Topflash transcriptional activity

in the presence of overexpressed β-catenin and the mutant construct indicates a mode of action

independent of upstream Wnt pathway activity, but likely promoting β-catenin destruction.

3.5.7 Structurally related compounds mimic the effects of 21H7

To obtain preliminary insights into structure-activity relationships we searched the entire

Maybridge library for compounds displaying substructures, superstructures and compounds with

general similarity to 21H7 using StructureBase software (IDBS). We focused the search to

solely include compounds that had been screened, which resulted in three closely related

compounds; 21A8, 12B11 and 21B8 (Fig. 20). Notably, none of the three compounds displayed

any effect in the Ff-luc assay. We tested the most closely related compound, 21A8 for effects on

Wnt-dependent induction of Axin2 and CyclinD1 expression by Real-time PCR. 21A8, which

has an additional ether moiety, was seen to block Wnt-induced Axin2 and/or CyclinD1

expression similar to 21H7 (Fig. 21) and therefore appears to be a false negative identified

through secondary analysis. Neither of the other two compounds, 21B8 or 12B11, were

available to us at the time of testing and were not assessed for effect.

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Figure 20: Screened Maybridge compounds with significant structural similarity to 21H7.

Compound 21H7 and molecules of structural similarity which were screened are depicted. None

of the additional compounds displayed any effect upon b-catenin levels in the Ff-luc high-

throughput screen.

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Figure 21: Compound 21A8, the molecule of greatest structural similarity to 21H7 inhibits

the expression of Wnt target genes in colon cancer cell lines. RNA isolated from the CRC

cell lines treated with 21A8 or DMSO control was reverse transcribed, and cDNA of established

Wnt target genes was quantified by QPCR. Levels of GAPDH cDNA was also quantified and

used to normalize results. Compound 21A8 displays an effect similar to 21H7 in reducing

endogenous mRNA levels of Wnt target genes in all colon cancer cell lines tested.

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1 Discussion

A large number of cancer therapeutic approaches rely on cytotoxic agents to induce general cell

death. Albeit successful, these methods take an unfocused approach to offset the

hyperproliferative effects of cancers in a nonspecific manner. Future generations of cancer drugs

would ideally be targeted to specific molecular pathways. Targeting known tumorigenic

pathways, or pathways highly correlated to certain cancers presents an ideal opportunity for less

toxic chemopreventive measures. One cancer well suited to a directed approach is colorectal

cancer, for which mutations of key Wnt pathway components appear very early in adenoma

development in approximately 90% of human CRC’s (Giles RH, 2003). Moreover, all such

mutations confer constitutive Wnt pathway activation and the subsequent expression of a variety

of anti-apoptotic and proliferative genes (Polakis, 2000).

In considering how to inhibit the proliferative effect associated with hyperactive Wnt signaling,

several approaches can be envisaged. Studies using antisense (Green et al., 2001) or gene

targeting strategies (Kwong et al., 2002) have been met with considerable obstacles. A more

feasible approach would be to identify small molecules directly targeted to a component of the

signaling pathway. Such studies have already produced molecules which inhibit β-catenin-TCF

interaction (Lepourcelet et al., 2004). Other inhibitors of the pathway that currently exist are

downstream effectors of transcriptional activation (Fig. 2). We therefore sought to identify novel

upstream small molecule inhibitors of Wnt signaling not only for the potential as a therapeutic

agent, but as a research tool to elucidate the mechanisms involved in regulating β-catenin

stability.

To this end we developed and optimized an assay to identify small molecule effectors of β-

catenin stabilization utilizing a high-throughput screen. The cell based screening approach offers

several advantages; any active compound will be cell permeable, and compounds displaying

nonspecific cytotoxicity can be recognized through secondary assays. Moreover, by choosing a

midstream pathway component as readout, the strategy allowed us to identify molecules active at

or upstream of β-catenin in the pathway, independent of any predetermined mechanism of

76

action. Potentially, inhibitors or activators identified may even affect the signaling pathway

through unknown mechanisms or levels of regulation.

The final screening data was analyzed and is presented as B-score values which we believed best

compensated for systemic assay variability. Percent activity measurements, like all control based

measures do not account for systemic sources of error or positional variability (Brideau et al.,

2003). Moreover, B-scores contain adjustments for both row and column positional effects not

present in a simple fold over median measurement. As a result, the B-score proved to perform

the best in our data analysis identifying outliers and minimizing noise (Fig. 8).

Through the screen, we identified a number of effectors of β-catenin stability, hence Wnt

pathway modulators. Hit rates for cell based assays are typically higher than in vitro and in

silico approaches (Zaman, 2004), and our inhibitory hit rate from the Maybridge library (0.6%)

is higher than other Wnt pathway in vitro (Lepourcelet et al., 2004) and in silico (Shan et al.,

2005) screens. However, differences in hit rates are also influenced by the nature of the library

being screened, as noted in the variation between libraries we used for screening. In both the

Lopac and Prestwick libraries, destabilizing compounds were far more frequently identified than

stabilizing molecules. An inverse of the ratio is seen in the Maybridge screening data, and can

be attributed to the nature of the collection. Prestwick and Lopac libraries, which consist of

biologically active small molecules are more likely to possess compounds effecting general cell

viability. The Ff-luc assay is not ideally designed to identify general effectors such as these, and

therefore hit rates are skewed.

Weeding out compounds with general effects was accomplished through the use of manual assay

repeats along with pathway specific reporter assays assessing the effects of these compounds on

the activity of the Wnt pathway as a whole compared to its effects on other developmentally

important signaling pathways (Fig 12, 13, Table 4). Interestingly, the only compound displaying

a repeatable and preferential inhibitory effect upon the Wnt signaling pathway was also the only

molecule (21H7) which preferentially destabilized β-catenin in the Wnt ligand treated run

compared to the control run. We then used immunoblotting to detect changes in the levels of

cytosolic β-catenin and found a concentration dependent decrease in total levels at micromolar

concentrations. Notably, this effect has been seen with other small molecules in cell lines before

77

(Boon et al., 2004; Fujii et al., 2007), but never at low micromolar concentrations as displayed

by compound 21H7.

Because the effects of 21H7 could be occurring at or anywhere upstream of β-catenin, we

utilized a variety of assays to ascertain the molecular target of 21H7. Topflash assays assessing

the epistatic effects of the compound were inconclusive, indicating the compound definitely acts

downstream of the receptor level and dishevelled activation. Moreover, the ability of the

compound to retain its inhibitory effects on Topflash activity in the presence of overexpressed β-

catenin and a β-catenin construct lacking residues phosphorylated during a Wnt response (Fig.

19) points to a mode of action in which β-catenin levels are regulated by a mechanism outside of

the known Wnt pathway effectors. These results, taken together with the immunoblotting data

strongly suggest the inhibitory mechanism is related to β-catenin itself. Due to the inherent

nature of the Ff-luc assay which measures the levels of β-catenin, downstream components can

be ruled out. While β-catenin levels are most profoundly kept in check through Wnt signaling via

the E3 ligase βTrCP (Lustig and Behrens, 2003), the possibility exists of other ligases promoting

β-catenin degradation by a means entirely unrelated to the Wnt pathway. In fact, the E3 ligase,

Siah1 has been shown to work in this manner (Liu et al., 2001). In the future, it will be

important to determine whether 21H7 mediates its effect in a Siah1 or βTrCP dependent manner

through directed knock-down experiments using Topflash as readout. Experiments such as these

could point towards a significantly more complex mechanism regulating β-catenin stability then

what is currently known.

More immediate experiments should be directed towards determining whether the experimental

evidence pointing towards 21H7 in promoting the destruction of β-catenin is in fact correct.

Multiple possibilities can be envisioned whereby a specific pool or subpopulation of β-catenin is

solely affected, or perhaps that the effect of 21H7 is more general than we believe. To this end,

Topflash assays using a combination of 21H7 and general proteosome inhibitors such as LLnL or

MG132 (Xu and Attisano, 2000) would be resistant to the inhibitory effects. Likewise, in

conjunction with β-catenin knock-down Topflash experiments should determine whether the

effect is being mediated solely through β-catenin. The possibility could exist where 21H7 is

acting upon one or more particular forms of β-catenin. Subpopulations of differentially activated

78

forms of β-catenin exist, whereby different phosphorylation states govern the activity of the

molecule (van Noort et al., 2007).

Identifying the cellular components directly or indirectly affected by our inhibitor will

undoubtedly be difficult. Nevertheless, one approach we anticipate using will combine basic

biomolecular techniques with high throughput technologies. We have successfully developed

protocols for screening large siRNA and cDNA libraries for effects in Topflash assays

(Manuscript in preparation). Eventually these screens can be used to identify whether 21H7

retain its inhibitory effect with overexpressed cDNA’s, or whether its effects can be either

mimicked or enhanced by a particular siRNA construct. For these experiments we will initially

utilize a siRNA (Dharmacon, siGenome Smartpool) and cDNA library of known Wnt pathway

components followed by the possibility of larger genome wide libraries.

Constitutive activation of the Wnt pathway is found in nearly every case of colon cancer

(Polakis, 2000). Mutations of key Wnt pathway components lead to a disruption of normal β-

catenin breakdown resulting in increased Wnt/β-catenin target gene expression (Logan and

Nusse, 2004). We therefore set out to explore the effect of 21H7 on a variety of colorectal

cancer (CRC) cell lines possessing hyperactive Wnt signaling due to various mutations by

examining transcript levels of established Wnt target genes (Axin2 and CyclinD1) via real-time

quantitative PCR analysis. Axin2 levels were clearly affected by 21H7 in all colorectal cancer

cell lines regardless of their Wnt pathway mutational status (Fig 17). While the effects did vary

between lines, there was no correlation between degree of effect and mutational status, indicating

21H7 does exert its effect downstream of these mutations, supporting our model of inhibition by

promoting β-catenin degradation. CyclinD1 mRNA levels were not effected to the same extent

as Axin2. In fact, transcript levels in both LS1034 or Colo205 cells displayed no significant

reduction upon treatment with the inhibitor. This is most likely due to the fact that Cyclin D1 is

not as robust a Wnt target, coupled with the fact that its levels are regulated by a variety of other

pathways including STAT (Leslie et al., 2006), NFκB (Guttridge et al., 1999) and Ras (Gladden

and Diehl, 2005).

Interestingly, CyclinD1 it is not the sole Wnt target gene which has its levels influenced by other

pathways. Work done in our lab (Labbe et al., 2007), point towards cross-talk between the Wnt

and TGF-β pathways regulating Axin2 levels and playing a role during intestinal tumorogenesis.

79

It is therefore possible that differences in inhibitory effects displayed between CRC cell lines

may be attributed to synergistic pathway activity. Therefore knowledge of TGF-β activity in all

CRC cell lines assayed would be informative, made more noteworthy because 21H7 displays a

slight inhibitory effect on TGF-β dependent transcriptional activity (Fig 13).

As a lead compound with potential anticancer properties, 21H7 demonstrated the ability to not

only inhibit Wnt transcriptional activity, but also block the effects associated with misregulated

Wnt signaling in CRC cell lines. Thus, we next assessed the breadth of that effect. First, we

determined whether 21H7 can suppress Wnt target gene expression in cell lines derived from

other cancers, by using prostate derived cell lines where misregulated Wnt signaling has been

implicated in prostate cancer (Yardy and Brewster, 2005). We identified a similar inhibitory

effect by 21H7 in two different prostate cancer cell lines via QPCR analysis (Fig. 18). Our

second approach focused on the effects of a structurally related compound. With the exception

of an additional ether moiety, compound 21A8 is structurally identical 21H7 (Fig 20). The fact

that 21A8 displayed matching patterns of inhibition through QPCR analysis in CRC cell lines

signifies a structure-activity relationship. We have yet to acquire additional structurally related

compounds, however, 21H7 will be treated as molecular starting point from which lead

optimization will be pursued. For this purpose we will use StructureBase software (IDBS) to

screen other chemical libraries for substructures, superstructures and compounds with general

similarity. This will allow us to test additional compounds for increased selectivity, potency or

ascertain which chemical moieties are key to the Wnt inhibitory effect.

In considering the soundness of future therapeutic applications, it would be necessary to identify

a positive phenotypic chemopreventive effect managed by 21H7 treatment. Our lead compound

should demonstrate growth inhibitory effects preferential to CRC cell lines as compared to

normal colonic epithelial cells (Emami KH, 2004). Therefore, via cell proliferation (MTT,

ATCC) or soft agar growth assays (Lin et al., 2007), we could ascertain whether the mitogenic

activity of Wnts can be suppressed in CRC cell lines habituated to a constitutively active

pathway. More pertinent experiments should be directed towards animal studies. For this we

could use Min (APC+/-) mice, which harbor a mutation in APC leading to the development of

numerous colonic and small-bowel adenomas (Moser et al., 1995). Quantifying the colonic

polyps, adenomas, or rate of apoptosis in crypts with 21H7 treatment or vehicle alone would

allow us to determine the effectiveness of our inhibitor as a CRC chemopreventive agent.

80

Moreover, future work towards potential therapeutic applications would require ADME/Tox

studies completed in laboratory mice to determine whether patient applications are feasible.

As equally important as an inhibitor would be for its cancer applications, identifying a novel

midstream small molecule inhibitor of Wnt signaling could be of tremendous importance as a

research tool in elucidating the mechanisms involved in regulating Wnt signaling. There are

many unanswered questions within the Wnt signaling field, and if our inhibitor proves to be

suitably selective, it could be utilized in assessing unanswered pathway questions. For instance,

unlike canonical Wnt signaling, other well defined signaling pathways such as TGF-β, ΒΜP and

MAPK exhibit significant amounts of cross-talk (von Bubnoff and Cho, 2001). Therefore,

contingent on the specificity of 21H7 or its derivatives, we could address questions of Wnt

pathway cross-talk in addition to that previously described by our lab (Labbe E, 2000).

In addition to the importance of inhibitors, compounds identified as activators in the screen may

inhibit β-TrCP or GSK3β, two attractive targets for drug intervention. The E3 ligase recognizing

β-catenin also recognizes IκB, the inhibitory subunit of NFκB. Preventing the degradation of the

inhibitory subunit (IκB) will curb the nuclear activities of NFκB associated with a variety of

cancers (Fuchs SY, 2004). Also, GSK3β activation has been shown to increase β-amyloid (Aβ)

production, while its inhibition decrease Aβ levels (Phiel CJ, 2003). Heritable defects of early-

onset Alzheimers are linked to this increase in Aβ production, indicating the potential for

GSK3β inhibitors to reduce Aβ levels (Fuchs SY, 2004). Therefore, while our secondary

analyses have focused entirely on Wnt pathway inhibitors, the activitory hits which are greater in

number may have significant medical and biochemical implications and merit future

consideration.

In summary, we have developed and optimized a high-throughput assay to investigate β-catenin

stability, hence Wnt pathway activity in a human derived cell line using chemiluminescent

readout. This format of Luciferase-tagging a molecule of interest to investigate its stability has

never been reported as a high-throughput technique. Our study demonstrates the viability of this

technique to identify novel pathway small molecule effectors. We provide evidence of an

identified inhibitory compound (21H7) promoting the destruction of β-catenin and repressing

Wnt-dependent gene expression in colorectal cancer cell lines. Furthermore, considering the

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nearly identical effects of structurally similar compounds, these findings support future research

designed to ascertain mechanism of action and to further mine the screening data for additional

Wnt pathway effectors.

82

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Appendices

Appendix 1. Small molecule hits detected from screening the Prestwick chemical library.

Positive and negative effectors of β-catenin stabilization are listed with their common chemical

name, plate/well location and their associated B-score. Negative scores indicate compounds

which destabilize β-catenin, positive values have the opposite effect.

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Appendix 2. Small molecule hits detected from screening the Lopac chemical library. Positive

and negative effectors of β-catenin stabilization are listed with their common chemical name,

plate/well location and their associated B-score. Negative scores indicate compounds which

destabilize β-catenin, positive values have the opposite effect. The known activity of each

compound listed is data provided with the Lopac library (Sigma)