Dissecting the ß-catenin destruction complex: Novel implications of tankyrase inhibitors

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Dissecting the EE-catenin destruction complex: Novel implications of

tankyrase inhibitors

PhD Thesis

Tor Espen Thorvaldsen

Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo Department of Molecular Cell Biology, Institute for Cancer Research, Oslo

University Hospital

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© Tor Espen Thorvaldsen, 2016

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8333-293-3

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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“Science should be fun. If it's not fun, then it's not worth doing.”

-Harald Stenmark-

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Acknowledgements

This work was carried out at the Department of Molecular Cell Biology, Institute for Cancer Research at the Norwegian Radium Hospital in the laboratory of Professor Harald Stenmark between April 2012 and June 2016.

The funding received from Helse Sør-Øst and the Centre for Cancer Biomedicine (CCB) is gratefully appreciated.

First, I would like to thank my supervisor Harald Stenmark for giving me the opportunity to join such an inspiring and talented group of researchers. I am still a bit surprised that you hired a veterinarian without any experience in molecular cell biological research. Thank you for your unlimited support and for always being so positive. I greatly admire your scientific knowledge, dedication and leadership philosophy.

Special thanks also to Nina Marie Pedersen who has been my co-supervisor, closest lab partner and mentor over the last four years. Thank you for all the meaningful (non-)scientific discussions, excellent supervision and for your dedication and hard work. Thank you also for consistently reminding me (and everyone else in the group) to value and celebrate even the smallest

accomplishments (as they do not occur frequently). I further wish to thank Eva Maria Wenzel for joining the WNT team. Your scientific skills, outstanding working capacity and contagious enthusiasm really inspire me. Thank you for extensive contributions to the completion of the thesis and for sharing your knowledge and experience.

Many thanks also to Sebastian Schultz and Andreas Brech. Your scientific and social contributions to the working environment and atmosphere are highly appreciated. Thank you Camilla Raiborg and Lene Malerød for scientific advices, discussions, and for always bringing smiles and laughter into the lab.

Thanks to Fergal O’Farrell and Viola Hélène Lobert for being such great office

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mates and colleagues, and to my two favorite technicians Anne Engen and Eva Rønning for excellent handling of cell cultures and for technical support. A well deserved thanks also to Chema Bassols, our IT expert, for solving every thinkable computer-related challenge. I also would like to acknowledge all the other members in the Stenmark group and at the Department of Molecular Cell Biology for providing such a pleasant and scientifically exciting workplace.

Thank you to all the people in Stefan Krauss´ laboratory at Rikshospitalet for scientific discussions and social events, especially to Jo Waaler for being such a positive and passionate collaborator (and travel companion). I also wish to acknowledge Knut Liestøl for valuable help with statistical analysis.

My deepest gratitude goes to my family and friends, especially my parents and my brother for their love and never-ending support. Also, many thanks to my parents-in-law for substantial efforts to solve the myriad of logistical challenges encountered over the last years. Finally, and above all, I would like to thank Mikkel and Kristine. You are, and always will be, my greatest

achievements! Mikkel, thank you for your unconditional love and trust (and for waking me up in the morning). Kristine, thank you for giving me the most wonderful son and for your endless love and patience – this would not have been possible without you.

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Abbreviations

15R – 15-amino acid repeat domain 20R – 20-amino acid repeat domain APC – Adenomatous polyposis coli ARC – Ankyrin repeat cluster

ARTD – Diphtheria toxin-like ADP-ribosyltransferase AXIN – Axis inhibition protein

BCL9 – B-cell chronic lymphocytic leukemia (CLL)/lymphoma 9 protein E-TrCP – Beta-transducing repeat-containing protein

CBP – Cyclic AMP response element-binding protein CDK – Cyclin-dependent kinase

CID – Catenin inhibitory domain CK1 – Casein kinase 1

COX2 – Cyclooxygenase 2 CRC – Colorectal cancer

CRISPR/CAS - Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR Associated

C-terminal – Carboxyl-terminal

DEP domain – Dishevelled, EGL-10 and Pleckstrin domain DIX domain – Dishevelled and AXIN domain

DKK – Dickkopf

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DNA – Deoxyribonucleic acid DVL – Dishevelled

EGFR – Epidermal growth factor receptor ERK – Extracellular signal-regulated kinase

ESCRT – Endosomal sorting complex required for transport FAP – Familial adenomatous polyposis

FBXW7 – F-box and WD repeat domain containing 7 FoxM1 – Forkhead box protein M1

FOXO – Forkhead box protein O

FRAP – Fluorescence recovery after photobleaching FZD – Frizzled

G1 - S – Growth/ Gap phase 1 - DNA synthesis phase G2 - M – Growth/ Gap phase 2 - Mitosis

GFP – Green fluorescent protein

GLUT4 – Glucose transporter type 4 vesicles GSK3 – Glycogen synthase kinase 3

Ins(1,4,5)P₃

INT-1 – Integration site 1 of the mouse mammary tumor virus – Inositol (1,4,5)-trisphosphate

IRAP - Insulin-responsive aminopeptidase ISC – Intestinal stem cell

IWPs – Inhibitors of WNT production

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JNK – C

KIF1DKinesin family member 1A -Jun N-terminal kinase

KRAS – V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog LEF – Lymphoid enhancer factor

LGR –

LRP – Low density lipoprotein receptor-related protein

Leucine-rich repeat containing G protein-coupled receptor

MEK – Mitogen-activated protein kinase kinase MGMT – O6-methylguanine-DNA methyltransferase MMTV – Mouse mammary tumor virus

mRNA – Messenger ribonucleic acid MVB – Multivesicular body

NAD+ - Nicotinamide adenine dinucleotide NSAIDs – Non-steroidal anti-inflammatory drug N-terminal – Amino-terminal

NuMA – Nuclear mitotic apparatus protein P53 – Tumor protein 53

PARP – Poly(ADP-ribose)polymerase PARsylation – Poly(ADP-ribose)sylation PCP – Planar cell polarity

PDZ domain - Psd-95, Discs large and ZO1 protein domain PGK promoter - Phosphoglycerate kinase 1 promotor

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PI3K – Phosphoinositide 3-kinase PP2A – Protein phosphatase 2A RAC1 –

RAF – Rapidly accelerated fibrosarcoma

Ras-related C3 botulinum toxin substrate 1

RGS domain – Regulation of G-protein signaling domain RNAi – Ribonucleic acid interference

RNF146 – Ring finger protein 146 RSPO – R-spondin

SAM – Sterile alpha motif SAMP –

SAR study – Structure-activity relationship study

Serine, Alanine, Methionine, and Proline motif

sFRPs – Secreted Frizzled-related protein siRNA – Small interfering ribonucleic acid SMAD4 –

TCF – T-cell factor

Mothers against decapentaplegic homolog 4

TLE – TNKS –

Transducin-like enhancer

Telomeric repeat binding factor 1 (TRF1)-interacting ankyrin-related

TNKSi – TNKS inhibitor ADP-ribose polymerase

TRF1 – Telomeric repeat

VPS – Vacuolar protein sorting

binding factor 1

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WIF-1 – WNT-inhibitory factor 1 Wls - Wntless

WNT - Wingless-related integration site WNTs – WNT ligands

WTX -

YAP – Yes-associated protein

Wilms tumor gene on X chromosome

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List of publications included in the thesis

Structure, Dynamics, and Functionality of Tankyrase Inhibitor-Induced Degradasomes

Thorvaldsen TE

Mol Cancer Res., 2015 Nov; 13(11): 1487-501

, Pedersen NM, Wenzel EM, Schultz SW, Brech A, Liestøl K, Waaler J, Krauss S and Stenmark H.

Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity

Pedersen NM, Thorvaldsen TE Submitted for publication

, Wenzel EM and Stenmark H.

Differential Roles of AXIN1 and AXIN2 in Tankyrase Inhibitor-Induced Formation of Degradasomes and EE-catenin Degradation

Thorvaldsen TE Manuscript

, Pedersen NM, Wenzel EM and Stenmark H.

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Introduction

The evolutionarily conserved WNT signaling pathway is one of the predominant signaling pathways during early embryonic development and orchestrates multiple processes in adult life, including cell proliferation, cell polarity, and stem cell maintenance (Logan and Nusse, 2004). The WNT signaling pathway is highly complex and contains numerous components that are subject to various regulatory steps and crosstalk mechanisms. For simplicity, the pathway is often divided into two classes: the canonical and non-canonical pathways, respectively. Most research has focused on the canonical WNT signaling pathway (hereafter referred to as the WNT signaling pathway), which mediates transcriptional output through stabilization and nuclear translocation of the co-transcription factor E-catenin (figure 1

van Amerongen, 2012

). In contrast, the non-ĐĂŶŽŶŝĐĂůɴ-catenin-independent signaling pathways elicit a variety of responses through alternative cascades which still remain poorly understood ( ). The E-catenin destruction complex sits at the heart of the WNT signaling pathway, playing a crucial role to keepE- catenin levels low in the absence of WNT ligands. Extensive research has provided important insight into this large multiprotein complex, but a complete molecular understanding of its structure and function has been elusive. Importantly, due to its wide-ranging implications in various cellular processes, mutations in the WNT signaling pathway are linked to a broad range of human cancers (table 1 Clevers and Nusse, 2012

) and other diseases (

). Although a few clinically approved drugs modulate WNT signaling, no targeted therapies inhibiting the WNT signaling cascade currently exist. Thus far, direct inhibition of WNT activity has been difficult largely due to the lack of pathway-specific targets and the potential redundancy of many pathway components. Intriguingly, recent identification of novel druggable targets has generated substantial efforts on developing small-molecule inhibitors of the

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WNT signaling pathway, some of which have shown promising effects in preclinical studies. In addition, several studies have revealed the potential of these inhibitors as valuable research tools to further dissect the role of the WNT signaling cascade and its components in physiological processes and disease.

Figure 1. WNT/EE-catenin signaling: The biochemical model. In the WNT-off state, the destruction complex resides in the cytoplasm, where it binds to and phosphorylates E- catenin. Phosphorylated E-catenin is ubiquitinated by E-TrCP and subsequently degraded by the proteasome. According to the signalosome hypothesis (Metcalfe and Bienz, 2011), binding of WNT to the Frizzled (FZD) and LRP5/6 receptors triggers the recruitment and DIX- dependent polymerization of Dishevelled (DVL) which enables it to bind to AXIN. GSK3 is recruited together with AXIN and LRP5/6 thus becomes a substrate for GSK3 and CK1 (J- and H-isoforms). Phosphorylated PPPSPxS motifs in the LRP5/6 cytoplasmic tail bind to the catalytic pocket of GSK3, thereby blocking its activity towards E-catenin. Unphosphorylated E-catenin escapes ubiquitination and proteasomal degradation, which allows it to accumulate in the cytoplasm and nucleus.

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Gene Type of mutation Primary tissues

APC Primarily frameshift and deletion mutations leading to a truncated protein with

compromised ability to degrade EE-catenin. The role of APC in E-catenin destruction has not been determined decisively.

Large intestine Stomach Soft tissue Small intestine Pancreas Liver CTNNB1

(E-catenin)

Mutations in E-catenin cluster around the N-terminus and prevent its phosphorylation by GSK3 (S33, S37, T41) and CK1 (S45). This results in impaired degradation of E-catenin.

Liver Soft tissue Endometrium Kidney

Pancreas Ovary

Adrenal gland Pituitary Biliary tract AXIN1 Several mutations prevent AXIN1 from acting

as a scaffold to degrade E-catenin. Biliary tract Liver AXIN2 Loss of heterozygosity and re-arrangements

in a variety of cancers. In addition, somatic point mutations and deletions have been identified. These truncated proteins are likely to be functionally inactive.

Large intestine Liver

Ovary

Endometrium

WTX Predicted to be loss-of-function mutations. Kidney

Large intestine

TCF7L2 Unknown. Large intestine

Table 1. WNT-activating somatic mutations associated with cancer (Anastas and Moon, 2013).

WNT ligand-receptor complexes

The Int-1 gene (now known as WNT1) was identified in 1982 as a frequent target for insertional activation by the mouse mammary tumor virus (MMTV) in mammary carcinomas (Nusse and Varmus, 1982). The segment-polarity gene Wingless in Drosophila Melanogaster was later verified as the homolog

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of Int-1 (Rijsewijk et al., 1987) and the portmanteau WNT (Wingless-related integration site) was established for all genes related to Int-1/Wingless (Nusse et al., 1991). At present, 19 structurally related WNT genes encoding cystein- rich morphogens (WNTs) have been isolated in the human genome (Miller, 2002). The first WNT protein was purified in 2003 and revealed that the WNTs are post-translationally modified by palmitoylation and highly hydrophobic (Willert et al., 2003). Indeed, both glycosylation and palmitoylation appear to be necessary for secretion of active WNTs (Komekado et al., 2007).

Palmitoylation of WNTs is mediated by porcupine (Kadowaki et al., 1996;

Takada et al., 2006), a member of the membrane-bound O-acyl transferase family (Hofmann, 2000), and promotes interaction with, and secretion by, the transmembrane sorting receptor Wntless (Wls) (Bartscherer et al., 2006;

Bänziger et al., 2006; Goodman et al., 2006; Herr and Basler, 2012) which supports transport of WNTs from the Golgi to the plasma membrane. Once WNTs are released from the cell surface, Wls is recycled via endosomes and the retromere complex back to the Golgi (Eaton, 2008). Consequently, mutations in porcupine abolish WNT signaling and result in early embryonic lethality in mice (Barrott et al., 2011; Biechele et al., 2011). In humans, porcupine mutations cause focal dermal hypoplasia, which is a multisystem disorder characterized by skin abnormalities and various developmental malformations and defects (Grzeschik et al., 2007; Wang et al., 2007).

WNT signals are efficiently transduced from WNT ligands to the intracellular pathway by receptors of the Frizzled (FZD) and low density lipoprotein receptor-related protein (LRP) families. Members of the FZD family are seven- transmembrane receptors that interact with WNTs through an N-terminal cystein-rich domain (Hsieh et al., 1999b). The human genome consists of 10 FZD genes, numbered FZD1 through 10 (MacDonald and He, 2012). In contrast, LRP5 and LRP6 are highly homologous single-transmembrane co- receptors that interact with WNTs through the ĨŽƵƌ ƚĂŶĚĞŵ ɴ-

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propeller/epidermal growth factor repeats in their extracellular domain (MacDonald and He, 2012). LRP5/6 are widely co-expressed during embryogenesis and in adult tissues and several studies indicate that they share significant overlapping functions. However, LRP6 seems to play a more crucial role than LRP5, at least during embryogenesis (He et al., 2004b). Unlike FZD, which contributes in multiple WNT pathways (Strutt, 2003), the LRP receptors appear to be specifically required for canonical WNT signaling (He et al., 2004b). The extracellular domains of the LRP5/6 and FZD receptors have been shown to form a complex in vitro in the presence of WNTs (Bourhis et al., 2010; Tamai et al., 2000). Whether LRP5/6 and FZD interact with different domains of the WNT ligands is still an unresolved question. However, 30 years after the isolation of the first WNT gene, a major advance came with the high- resolution structure of Xenopus WNT8 in complex with the cystein-rich domain of mouse FZD8 (Janda et al., 2012).

Natural modulators of WNT signaling

WNT signaling is tightly regulated at the receptor level by a complex network of extracellular agonists and antagonists. Although several aspects of the various modulators remain to be answered, recent progress has provided major insights into the molecular mechanism of this multilayered control system.

Expression of Leucine-rich repeat containing G protein-coupled receptor (LGR)

Barker et al., 2007

5 was initially discovered in cycling cells at the bottom of intestinal crypts, so- called crypt base columnar cells. LGR5-based lineage tracing experiments provided definitive evidence for stemness of the crypt base columnar cells ( ) and the same lineage tracing strategy subsequently revealed stem cells in several other organs and tissues (Barker et al., 2010;

Barker et al., 2012; de Visser et al., 2012; Huch et al., 2013a; Huch et al.,

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2013b; Plaks et al., 2013). LGR4 is co-expressed with LGR5 in all crypt cell types, including crypt base columnar cells (Barker and Clevers, 2010).

Moreover, LGR6, a close homolog of LGR4/5, has been shown to mark a group of stem cells that can give rise to all cell lineages of the skin (Snippert et al., 2010). LGR5 is a direct target gene of WNT signaling, and is consequently expressed in colon cancer and other WNT-dependent tumors (de Lau et al., 2014). The R-Spondins (RSPO1 to -4) constitute a small family of four secreted growth factors that can activate canonical WNT signaling in the presence of WNTs (Binnerts et al., 2007). Intriguingly, LGR4/5/6 have been identified as receptors for RSPO and synergize with RSPO and WNTs in activation of WNT signaling (Carmon et al., 2011; de Lau et al., 2011; Glinka et al., 2011).

Furthermore, two highly homologous WNT target genes, the transmembrane E3 ubiquitin ligase ZNRF3 and its homolog RNF43, were found to be specific to LGR5-positive crypt stem cells (Hao et al., 2012; Koo et al., 2012). ZNRF3- and RNF43-mediated ubiquitination of the FZD receptor promotes endocytosis of the FZD/LRP complex and its destruction in lysosomes. Based on biochemical experiments and crystallographic studies, the following model describes how these molecules collectively control WNT signal strength (de Lau et al., 2014):

LGR4/5/6 receptors recruit RSPO ligands and bring them into position for interaction with RNF43/ZNRF3. This interaction leads to membrane clearance of the RNF43/ZNRF3, and consequently, persistence of surface receptors boosts the WNT signaling output. Norrin is another secreted signaling molecule encoded by the Norrie Disease Pseudoglioma gene (Berger et al., 1992; Chen et al., 1992) that activates WNT signaling by interacting with FZD4, LRP5/6 and the transmembrane protein Tetraspanin-12 (Junge et al., 2009).

The crystal structure of Norrin in complex with the ligand binding domain of FZD4 revealed that Norrin mimics WNT for FZD recognition (Chang et al., 2015; Ke et al., 2013). Intriguingly, recent advances in production of wild-type

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and mutant Norrin proteins might open new avenues for the development of therapeutics to combat abnormal Norrin/WNT signaling.

The dickkopf (DKK) family of secreted proteins consists of four members in vertebrates (DKK1 to -4) and was initially discovered by its ability to block WNT signaling during early Xenopus embryogenesis (Glinka et al., 1998). DKK1 and DKK2 show high affinity-binding to the LRP6 receptor and blocks WNT signal transduction by preventing WNT-induced FZD/LRP6 complex formation (Semënov et al., 2001). A functional interaction with LRP6 has also been demonstrated for DKK4, whereas DKK3 does not affect WNT signaling (Niehrs, 2006). Moreover, WNT-inhibitory factor 1 (WIF-1) has been demonstrated to prevent WNTs from binding to the receptors (Hsieh et al., 1999a), thus affecting both canonical and non-canonical WNT pathways. Although the mechanisms of WIF-1-mediated regulation of WNT signaling is incompletely understood, the observed silencing of WIF-1 in different tumors indicates that it plays an important role in WNT-dependent carcinogenesis and other biological processes (Cruciat and Niehrs, 2013). Finally, the large family of secreted Frizzled-related proteins (sFRP1 to -5), which shows high homology with the ligand-binding cysteine-rich domain of the FZD family of receptors (Kawano and Kypta, 2003), can block WNT signaling either by preventing binding of WNTs to FZD receptors or by forming non-functional complexes with FZD. However, the exact mechanisms of sFRPs activity have not been completely elucidated and some studies have suggested that sFRPs can potentiate WNT signaling rather than inhibiting it (Bovolenta et al., 2008).

The EE-catenin destruction complex

The intricate E-catenin destruction complex has been thoroughly dissected over the last decades. Although its core components have been established (figure 2), their molecular interactions and regulation are not completely

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resolved, nor are their individual contributions ŝŶ ɴ-catenin processing from phosphorylation to ubiquitination and degradation fully understood.

Furthermore, some components may associate only transiently with the complex. Future research will most likely identify additional interaction partners, thereby adding further complexity to this central signal-limiting protein assembly.

EE-catenin was originally identified in adherens junctions as a structural linker- protein between cadherins and the actin cytoskeleton (Hülsken et al., 1994;

Ozawa et al., 1989). In addition to its role in regulating cell adhesion, E-catenin is the key mediator of WNT signaling output. In the WNT-off state, ƚƌĂŶƐĐƌŝƉƚŝŽŶĂůůǇĂĐƚŝǀĞɴ-catenin levels are kept low by CK1D/GSK3-mediated N-terminal phosphorylation and subsequent degradation by the ubiquitin- proteasome system (Liu et al., 2002). Upon WNT activation, E-catenin escapes phosphorylation and proteasomal degradation, translocates to the nucleus and initiates transcription of WNTͬɴ-catenin-responsive genes by complexing predominantly with the TCF/LEF family of transcription factors (MacDonald et al., 2009). The E-catenin protein contains a central structural core of 12 armadillo repeats that constitutes the binding surface for the majority of its binding partners (Huber et al., 1997).

The stability and function of the E-catenin destruction complex is highly dependent on the protein levels of Axis inhibition protein 1 and 2 (AXIN1 and AXIN2). AXIN1 and AXIN2 were both originally identified as negative regulators of WNT signaling and share the key sequence elements (45% amino acid identity) (Behrens et al., 1998; Zeng et al., 1997). AXIN molecules form dynamic puncta by DIX domain-mediated head-to-tail polymerization (Cadigan and Nusse, 1997; Hsu et al., 1999), which are thought to provide high-avidity interaction sites for E-catenin and other destruction complex components (Schwarz-Romond et al., 2005; Schwarz-Romond et al., 2007b; Smalley et al.,

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1999). The N-terminal RGS domain of AXIN binds Adenomatous polyposis coli (APC) through the SAMP motifs in APC (Behrens et al., 1998; Kishida et al., 1998; Spink et al., 2000). In addition, AXIN has two centrally located domains that bind E-catenin and GSK3, respectively (Behrens et al., 1998; Hart et al., 1998; Ikeda et al., 1998; Itoh et al., 1998; Sakanaka et al., 1998). Indeed, GSK3- and CK1-mediated phosphorylation of AXIN (and APC) leads to increased ĂƐƐŽĐŝĂƚŝŽŶǁŝƚŚɴ-catenin and thus enhanced degradation (MacDonald et al., 2009). In contrast to AXIN1, which is constitutively expressed, AXIN2 is a direct target gene of WNT signaling (Leung et al., 2002). This negative feedback mechanism serves to reduce signaling activity by limiting the levels ŽĨ ɴ- catenin (Lustig et al., 2002).

Three Dishevelled (DVL) homologues (DVL-1 to -3) have been identified in humans and mice. The DVL proteins display high sequence homology and contain an amino-terminal DIX domain, a central PDZ domain, and a carboxyl- terminal DEP domain (Gao and Chen, 2010). Head-to-tail polymerization is promoted by the DIX-domain and results in formation of dynamic cytoplasmic puncta with both overexpressed and endogenous DVL proteins. Recruitment of AXIN by DVL positively regulates WNT signaling by blocking the function of AXIN in the E-catenin destruction complex. Of note, the DIX domains of DVL and AXIN do not seem to interact with each other directly (Schwarz-Romond et al., 2007a; Schwarz-Romond et al., 2005; Schwarz-Romond et al., 2007b).

The PDZ domain interacts with FZD and transduces signals to downstream components (Wong et al., 2003). Similarly, the DEP domain is predicted to be crucial for the interaction with various protein partners and to contribute in signal transduction (Wong et al., 2000). In addition, two conserved regions, the proline-rich region and the basic region, have been implicated in protein–

protein interaction and phosphorylation, respectively (Penton et al., 2002).

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Several aspects of the destruction complex function are not completely elucidated, particularly the role of Adenomatous Polyposis Coli (APC). APC contains two types of E-catenin-binding domains: four 15-aa repeat domains and seven 20-aa repeat domains. Phosphorylation of the 20-aa repeats by CK1 and GSK3 has been shown to increase the affinity of APC for E-catenin and this process likely requires AXIN (Stamos and Weis, 2013). The interaction with AXIN is mediated by three SAMP motifs which are interspersed between the 20-aa repeat domains (Behrens et al., 1998; Spink et al., 2000) and AXIN binds in addition to the second 20-aa repeats (20R2) (Schneikert et al., 2014). In addition, the amino-terminal portion of APC contains a dimerization domain (Day and Alber, 2000) and an armadillo repeat domain that interacts with various cytoskeletal regulators (Nelson and Näthke, 2013). Loss of the 20-aa repeat regions (Munemitsu et al., 1995) and/or the SAMP repeats of APC (Behrens et al., 1998) has traditionally been regarded as the mechanism of WNT activation in APC-mutated colorectal cancer (CRC). Although the precise mechanisms and role of APC in E-catenin destruction have not been determined decisively, recent studies demonstrated that ubiquitination of E- catenin by E-TrCP is dependent on the 20R2 and the so-called “catenin inhibitory domain” (CID) in APC (Kohler et al., 2009; Su et al., 2008). The 20R2 and CID domain are commonly deleted in the truncated APC products present in colon cancer cell lines (e.g. SW480), leading to abrogated ubiquitination and ĚĞŐƌĂĚĂƚŝŽŶ ŽĨ ƉŚŽƐƉŚŽƌǇůĂƚĞĚ ɴ-catenin (PBC) (Sadot et al., 2002; Su et al., 2008; Yang et al., 2006). Moreover, ĂƌŽůĞŝŶɴ-catenin degradation has been demonstrated for the 20R3 and 15aa repeats, whereas binding to the 20R1 is neither necessary nor sufficient (Kohler et al., 2010). Of note, a second isoform of the APC protein, designated APC2 (or APCL), is found in most organisms (van Es et al., 1999). APC2 closely resembles APC in overall domain structure and appears to be cooperate with APC for efficient destruction complex activity (Kunttas-Tatli et al., 2012; Schneikert et al., 2013).

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Interestingly, Croy and colleagues (Croy et al., 2016) recently found that tankyrase binds the C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1.

In the WNT-off state, degradation of E-catenin requires phosphorylation by the two kinases Glycogen synthase kinase 3 (GSK3) and Casein kinase 1DD (CK1D). GSK3, which has also been implicated in several other signaling pathways (Forde and Dale, 2007), exists as two homologs encoded by distinct genes, GSK3D and GSK3E. GSK3D and GSK3E share 97% amino acid sequence identity within their catalytic domains, but differ significantly outside the kinase domain (Wu and Pan, 2010). Although the role of GSK3E traditionally has been emphasized, a recent study concluded that both homologs function identically in the WNT signaling pathway (Doble et al., 2007). Like GSK3, ĂƐĞŝŶ ŬŝŶĂƐĞ ϭɲ ;CK1ɲ) is expressed ubiquitously and appears to be constitutively active (Gross and Anderson, 1998). Phosphorylation of S45 in E- catenin ďǇ <ϭɲ proceeds and is required for subsequent GSK3 phosphorylation of T41, S37, and S33. In fact, the two kinases bind to opposite ƐŝĚĞƐ ŽĨ ƚŚĞ ɴ-catenin-binding region in AXIN, thereby promoting effective phosphorylation of ɴ-catenin (Liu et al., 2002; Peifer et al., 1994; Yost et al., 1996). This ultimately leads to its degradation by the proteasome, a process dependent on ubiquitination of PBC by the F-ďŽǆ ĐŽŶƚĂŝŶŝŶŐ ƉƌŽƚĞŝŶ ɴ-TrCP (substrate recognition subunit for the SCF-TrCP E3 ubiquitin ligases) (Hart et al., 1999; Liu et al., 1999).

The poly(ADP-ribose) polymerase (PARP) family of proteins, also known as ADP-ribosyltransferase diphtheria-toxin-like proteins (ARTD), consists of 17 members. The PARPs can be subgrouped into those forming poly(ADP-ribose) chains and those catalyzing mono-ADP-ribosylation (Vyas and Chang, 2014).

Tankyrase 1 (TNKS1/PARP-5a/ARTD5) and tankyrase 2 (TNKS2/ PARP- 5b/ARTD6) are poly(ADP-ribose)polymerases that transfer ADP-ribose

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moieties (poly-ADP-ribosylation) to amino acid side-chains in acceptor proteins. The C-terminal catalytic domain cleaves NAD+ to nicotinamide and ADP-ribose. TNKS1/2 share 82% sequence identity and appear to have largely overlapping functions, as deletion of either gene leads to subtle phenotypes.

However, double knockout of both genes is embryonic lethal (Chiang et al., 2008). The two isoforms contain several ankyrin repeats and a sterile alpha motif (SAM), which are absent in other PARPs. The ankyrin repeats are responsible for target protein interaction (Li et al., 2006), whereas the SAM domain mediates multimerization of the tankyrases (De Rycker and Price, 2004). Importantly, TNKS1/2 are positive regulators of WNT signaling through poly-ADP-ribosylation of AXIN, the concentration-limiting factor for destruction complex stability (Huang et al., 2009). RNF146, a RING-domain E3 ubiquitin ligase, directly interacts with poly(ADP-ribose) through its WWE domain and promotes degradation of PARsylated proteins including AXIN (Callow et al., 2011; Zhang et al., 2011b).

The role of protein phosphatase 2A (PP2A) in the WNT pathway is not completely resolved. In fact, both negative and positive regulatory roles of PP2A have been suggested (Li et al., 2001; Ratcliffe et al., 2000). An appealing model presented by Su an colleagues (Su et al., 2008) suggest a positive regulatory role of PP2A in WNT signaling. They demonstrate that the E-TrCP- binding site created by CK1-/GSK3-ŵĞĚŝĂƚĞĚ ƉŚŽƐƉŚŽƌǇůĂƚŝŽŶ ŽĨ ɴ-catenin (S37 and S33) is highly vulnerable to protein phosphatase 2A (PP2A) and must be protected by APC. Consequently, the N-terminal phosphorylated ƐĞƌŝŶĞͬƚŚƌĞŽŶŝŶĞ ƌĞƐŝĚƵĞƐ ŽĨ ɴ-catenin are exposed to PP2A in APC-mutant ĐĞůůƐůĞĂĚŝŶŐƚŽĚĞƉŚŽƐƉŚŽƌǇůĂƚŝŽŶĂƚƚŚĞƐĞƌĞƐŝĚƵĞƐĂŶĚĂďůŽĐŬŝŶɴ-catenin ubiquitination and degradation. It is plausible that PP2A may have multiple and opposing roles in the WNT pathway depending on the particular associated regulatory subunits and substrates.

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Figure 2. Schematic drawing presenting the main components of the EE-catenin destruction complex. See text for details. BD: binding domain. NLS: nuclear localization signal.

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Transcriptional regulators

The central 12-ĂƌŵĂĚŝůůŽ ƌĞƉĞĂƚƐ ŽĨ ɴ-catenin are the main site of transcription factor interaction, whereas domains in the amino and carboxy termini exhibit transcription-activating functions (Hsu et al., 1998; Huber et al., 1996). The TCF/LEF family of transcription factors complex with E-catenin and are the major end-point mediators of WNT signaling. In the WNT-off state, TCF/LEF proteins function as transcriptional co-repressors through binding to members of the Groucho/TLE family. The switch from transcriptional repression to activation of WNT target genes involves displacement of Groucho through a complex network of interactions including E-catenin, TCF/LEF, BCL9/BCL9-2 and Pygopus (Daniels and Weis, 2005; Jessen et al., 2008; Kramps et al., 2002). Importantly, the TCF/LEF transcription factors have a heterogeneous pattern of activity that enables WNT signals to be interpreted differently. For instance, TCF3 is generally known as a repressor of WNT target genes (Kim et al., 2000), whereas TCF1 and LEF1 are linked to target gene activation (Liu et al., 2005). Differential activities of TCF/LEF raise the possibility that the transcriptional switch promoted by WNT could also involve an exchange of TCF/LEF family members (Cadigan and Waterman, 2012).

Switching off WNT signaling: Three different models

ɴ-catenin levels are normally kept low by CK1a/GSK3-mediated N-terminal phosphorylation of E-catenin and subsequent degradation by the ubiquitin- proteasome system. However, the mechanisms by which destruction complex activity is inhibited in the WNT-on state are currently debated. Extensive research has generated several models, three of which are highlighted here, describing the molecular events following WNT receptor activation.

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WNT activation promotes DVL aggregates at the plasma membrane which co- cluster LRP6 with other pathway components including FZD, AXIN, and GSK3.

Although the mechanisms of formation of these so-called signalosomes are not yet completely understood, several studies have culminated in a biochemical model (figure 1

The cell biological model (

) of GSK3 inhibition through signalosome- mediated phosphorylation of LRP: The multiple conserved PPPSPxS motifs within the cytoplasmic tail of LRP play a crucial role in WNT signal transduction (Mao et al., 2001; Tamai et al., 2004). Following WNT stimulation, the PPPSPxS motifs become sequentially phosphorylated by GSK3 ĂŶĚ<ϭɶͬɸ;ĂǀŝĚƐŽŶĞƚĂů͕͘ϮϬϬϱ͖ĞŶŐĞƚĂů͕͘ϮϬϬϱͿĂŶĚƚŚĞƐĞĞǀĞŶƚƐĚĞƉĞŶĚ on polymerization by the DIX domain of Dishevelled (Bilic et al., 2007;

Metcalfe et al., 2010). Importantly, WNT-dependent inhibition of GSK3 is mediated by direct interaction with phosphorylated PPPSPxS motifs (Cselenyi et al., 2008; Mi et al., 2006; Piao et al., 2008; Wu et al., 2009).

figure 3) (Taelman et al., 2010) on the other hand suggests that phosphorylation of newly synthesized E-catenin is inhibited upon WNT signaling by sequestration of GSK3 and other components of the signalosomes into multivesicular bodies (MVBs). This study displayed that upon WNT stimulation of cells treated with digitonin, a permeabilizing agent which keeps intracellular membranes intact, GSK3 showed resistant to protease treatment. However, protease sensitivity of GSK3 was recovered upon treatment with Triton X-100, which solubilizes all cellular membranes.

Moreover, confocal microscopy revealed that signalosomes colocalized with the late endosomal markers RAB7 and VPS4. Importantly, the presence of GSK3 in MVBs was confirmed by electron microscopy. The authors also demonstrated that WNT signaling depends on the ESCRT (endosomal sorting complex required for transport) machinery, which is required for MVB biogenesis.

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Figure 3. WNT/EE-catenin signaling: The cell biological model. Upon WNT signaling, GSK3 (in orange) is recruited to the cytoplasmic side of WNT receptor complexes and phosphorylates LRP6 and other substrates such as DVL (in pink), AXIN (in blue) and E-catenin (in red).

Although E-catenin protein initially translocates into MVBs together with GSK3, once cytosolic levels of GSK3 are sufficiently depleted, newly translated b-catenin is not phosphorylated. Thus, E-catenin accumulates in the cytoplasm, translocates to the nucleus, and initiates transcription of WNT target genes.

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The same research group later demonstrated that WNT signaling was increased under various conditions generating an accumulation of MVBs (Dobrowolski et al., 2012). Again, these WNT-enhancing effects were dependent on the functional ESCRT machinery and the authors therefore suggested that accumulation of late endosomal structures leads to enhanced canonical WNT signaling through increased sequestration of the WNT- receptor/GSK3 complex. In support of this model, internalization of GSK3- containing WNT-signalosome complexes into MVBs has also been reported in a separate study (Vinyoles et al., 2014).

Figure 4. WNT/EE-catenin signaling: The intact AXIN complex model. WNT activation leaves the destruction complex assembled and does not affect the activity of its kinases. However, the E3 ubiquitin ligase component E-TrCP dissociates from the complex, leading to abolished ubiquitination and degradation of phosphorylated E-catenin. Thus, the intact destruction complex is saturated and thus effectively inactivated. Finally, newly synthesized E-catenin translocates to nucleus and initiates transcription of WNT target genes.

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Finally, the group of Hans Clevers recently presented an opposing model suggesting that the E-catenin destruction complex remains essentially unaltered upon WNT activation (Li et al., 2012). In the WNT-off state, they demonstrate that E-catenin is phosphorylated, ubiquitinated and degraded within an intact AXIN1 complex (figure 4). WNT activation abrogates E-TrCP- mediated ubiquitination, which saturates destruction complexes with PBC molecules, thereby ĂůůŽǁŝŶŐ ŶĞǁůǇ ƐǇŶƚŚĞƐŝǌĞĚ ɴ-catenin to accumulate in a free cytosolic form and engage nuclear TCF transcription factors. In contrast to previously postulated models, they found neither a disassembly of the complex nor an inhibition of phosphorylation of complex-bound E-catenin upon WNT signaling.

Non-canonical WNT signaling

Non-canonical WNT signaling includes pathways that are independent of E- catenin transcriptional activity. The two best-characterised non-canonical pathways are the WNT/Ca²⁺ pathway (Kühl et al., 2000) and the planar cell polarity (PCP) pathway (McEwen and Peifer, 2000). In the WNT/Ca²⁺ pathway, WNT/FZD-mediated activation of G proteins triggers phospholipase C, which in turn stimulates diacylglycerol and inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) production. Ins(1,4,5)P₃-induced Ca²⁺ release from intracellular stores turn on effectors that activate the transcriptional regulator nuclear factor associated with T cells (NFAT). The WNT/Ca²⁺

De, 2011

pathway is involved in cancer, inflammation and neurodegeneration ( ). In PCP signaling, FZD receptors activate a cascade that involves the small GTPases RAC1 and RHOA and JUN-N-terminal kinase (JNK) (Niehrs, 2012).The PCP pathway is involved in regulating cell polarity in morphogenetic processes including cell movement during gastrulation, neural tube closure and the orientation of stereocilia in the inner

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ear (Simons and Mlodzik, 2008). PCP and canonical WNT signaling are known to antagonize each other (Sato et al., 2010).

FoxM1 in WNT signaling and cancer

FoxM1 belongs to a large family of forkhead box (Fox) transcription factors defined by a common DNA-binding domain termed the forkhead box domain.

However, regulation and function of the Fox proteins vary significantly (Myatt and Lam, 2007). The FoxM1 transcription factors (three splice variants in humans) are associated with cell proliferation and predominantly detected in the progenitor and regenerating tissues in adults (Raychaudhuri and Park, 2011). FoxM1 is crucial for G1–S and G2–M cell cycle phase progression and mitotic spindle integrity. It accumulates in the cytoplasm at late G1 and S phases, and nuclear translocation occurs following CDK-cyclin complex (Major et al., 2004) and RAF–MEK–ERK-mediated phosphorylation (Ma et al., 2005).

At the end of M-phase, FoxM1 becomes dephosphorylated, polyubiquitinated and degraded by the proteasome (Park et al., 2008). The expression and transcriptional activity of FoxM1 is also regulated by the tumor suppressor P53 (Barsotti and Prives, 2009; Pandit et al., 2009). Importantly, FoxM1 is overexpressed in most cancer types (Pilarsky et al., 2004) and cross-talk with other signaling pathways appears to play an important role in tumor aggressiveness (Wang et al., 2010).

Indeed, interaction between FoxM1 and the WNT signaling pathway was recently highlighted in two separate studies. Zhang et al. (Zhang et al., 2011a) demonstrated that FoxM1 is required for nuclear translocation of E-catenin in glioblastoma cells. The authors showed that the translocation depends on binding of E-catenin to FoxM1, which is mediated by the armadillo repeats of E-catenin, and the Fox domain of FoxM1. Moreover, this interaction is

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maintained in the nucleus, where both proteins form a complex with TCF transcription factors on WNT target gene promoters. A follow-up study from same group (Chen et al., 2016) displayed that GSK3 phosphorylation mediates ubiquitination of FoxM1 by the E3 ubiquitin ligase FBXW7. Upon WNT stimulation, inhibition of GSK3 activity leads to deubiquitination and stabilization of FoxM1. Thus, FoxM1

Wang et al., 2012

accumulates in the nucleus and recruits ɴ-catenin to WNT target-gene promoters. Soon after, Wang et al. (

) presented an opposing model based on experiments in lung epithelial cells. In this study, depletion of FoxM1 from the respiratory epithelium attenuated the severe lung abnormalities caused by mutant KRAS, supporting the concept that FoxM1 is a downstream target of KRAS signaling during lung development. Importantly, the authors demonstrated that KRAS/FoxM1 signaling inhibited the activity of canonical WNT signaling. In line with this, siRNA-mediated depletion of FoxM1 increased the nuclear localization of E-catenin and expression of E-catenin target genes.

Surprisingly, they demonstrated that AXIN2 mRNA and protein levels were reduced after activation of canonical WNT signaling in FoxM1-deficient distal lung epithelium. Indeed, FoxM1 was shown to directly bind to and increase transcriptional activity of the AXIN2 promoter region. Together, this study suggests that FoxM1 is a more potent transcriptional activator of AXIN2 than E-catenin. The reasons for the discrepancy between these two studies remain elusive. One explanation could be that regulation of AXIN2 by either FoxM1 or E-catenin is dependent on cell specificity and the biological context.

Targeting the WNT signaling pathway

Extensive research has pointed out a crucial role for hyperactivated WNT signaling in colorectal cancer. In addition to mutations in the APC gene, which are found in the majority of CRCs, mutations in additional tumor suppressors

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and oncogenes (e.g. KRAS, BRAF, P53 and SMAD4) are required to ensure progression into malignant colorectal tumors (carcinomas) (Fearon and Vogelstein, 1990; Luo et al., 2011; Phelps et al., 2009). Moreover, sequencing studies of colorectal tumor samples from patients have identified mutations in additional WNT pathway genes such as transcription factor 7-like 2 (TCF7L2;

previously known as TCF4), CTNNB1 (E-catenin), AXIN and WTX (Cancer Genome Atlas Network, 2012). Of note, inactivating mutations in

adenomatous polyposis coli (APC) cause familial adenomatous polyposis (FAP), an inherited condition that can progress to colorectal carcinomas following concomitant mutations in KRAS and P53 Korinek et al., 1997 ( ; Morin et al., 1997). WNT pathway mutations are also regularly identified in other types of cancer. Finally, altered expression of WNT receptors or modulators may lead to deregulated signaling output. Although several compounds are being explored in preclinical settings, therapeutic agents specifically targeting the WNT pathway have only recently entered clinical trials and none has yet been approved (table 2).

Non-steroidal anti-inflammatory drugs (NSAIDs) and the selective COX2 inhibitor celecoxib have been shown to inhibitE-catenin-dependent

transcription in colorectal cells and FAP patients. Their precise mechanism of action is complex and is likely to be unique for each NSAID class (Barker and Clevers, 2006). Inhibitors of WNT Production (IWPs) is another class of small molecules that abrogates the actions of WNT proteins through inhibition of porcupine (Chen et al., 2009a; Lum and Clevers, 2012; Wang et al., 2013).

Porcupine catalyzes palmitoylation of WNT ligands, which is required for their secretion and signaling activity. The porcupine inhibitor LGK974 is currently in a phase 1 clinical trial. In 2009, Huang and colleagues discovered the first small molecule tankyrase inhibitor (TNKSi) XAV939 (Huang et al., 2009).

Furthermore, the previously published compound IWR-1 (Chen et al., 2009a) was verified as an antagonist of the same target. Subsequently, similar large-

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scale screening programs have identified several other TNKSi (James et al., 2012; Okada-Iwasaki et al., 2016; Shultz et al., 2013; Shultz et al., 2012;

Voronkov et al., 2013; Waaler et al., 2012; Waaler et al., 2011). The TNKSi JW67, JW74 and JW55 were identified as primary hits in a reporter-based high-throughput screen using a small-molecule library consisting of 37,000 compounds (Waaler et al., 2012; Waaler et al., 2011). Further characterization revealed that all three chemotypes acted as TNKS1/2 inhibitors that

orchestrate degradation of E-catenin through stabilization of AXIN.

Importantly, they were also shown to induce cell cycle arrest in colon cancer cells and reduce tumor development in various mouse models of CRC. The effects of the JW74 chemotype were further explored in several osteosarcoma cell lines that displayed increased WNT activity. Intriguingly, JW74 stabilized AXIN protein levels in all the tested osteosarcoma cell lines, accompanied by a reduction in nuclear E-catenin, WNT reporter activity and AXIN2 mRNA levels (Wessel Stratford et al., 2014). However, the initial chemotypes were rapidly degraded in microsomal stability assays and affected canonical WNT signaling only to a limited extent. To further enhance the potency against TNKS1/2 and to improve the uptake and stability in vivo, an extensive structure-activity relationship (SAR) study based on the JW74 chemotype was initiated. This study resulted in the optimized compound G007-LK (Voronkov et al., 2013), which was stable in microsomal stability assays and displayed an excellent pharmacokinetic profile in mice. Moreover, crystallographic studies of G007- LK bound to the PARP domain of TNKS2 revealed that G007-LK interacts with unique structural features in the extended adenosine binding pocket, which forms the structural basis for its high target selectivity and specificity. In

contrast to XAV939, which targets several PARP family members (Huang et al., 2009), only TNKS1/2 were found to be displaced by G007-LK, confirming the high selectivity of G007-LK towards the TNKS enzymes (Voronkov et al., 2013).

Furthermore, G007-LK inhibits only TNKS1 and TNKS2 in biochemical assays

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(Lau et al., 2013). The TNKSi have emerged as a promising new cancer therapeutic approach and can be divided into sub-groups based on their binding mode to the donor NAD+ binding groove in the catalytic domain of TNKS. Inhibitors such as XAV939 and PJ34 (Kirby et al., 2012) bind to the nicotinamide pocket and inhibit both PARP1 and TNKS1/2, whereas JW55, JW74, G007-LK, IWR1, WIKI and K-756 bind to the adjacent adenosine binding pocket and are specific inhibitors of TNKS1/2. RNF146, a RING-domain E3 ubiquitin ligase, mediates TNKS-dependent degradation of AXIN by directly interacting with poly(ADP-ribose) through its WWE domain. RNF146 also destabilizes tankyrase itself and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels (Callow et al., 2011; Zhang et al., 2011b).

Inhibition of the catalytic activity of TNKS1/2 increases the levels of AXIN, which is the rate-limiting factor for destruction complex stability, and consequently abƌŽŐĂƚĞƐtEdͬɴ-catenin signaling (figure 5

Chen et al., 2009b

). Several other small-molecule drugs have also been shown to modulate the WNT signaling pathway by different means: The anti-helminthic niclosamide appears to serve as a negative modulator of WNT signaling by depleting the up-stream signaling molecules FZD and DVL ( ), while NSC668036 has been shown to interrupt the FZD-DVL interaction (Shan et al., 2005). In addition, Pyrvinium inhibits WNT signaling through activation of CK1D thus ůĞĂĚŝŶŐƚŽĞŶŚĂŶĐĞĚɴ-catenin degradation (Thorne et al., 2010), whereas a number of inhibitors abrogate the binding of EE-catenin to various

transcriptional activators (Kahn, 2014). For instance, the small molecule ICG- 001 specifically targets the transcriptional co-activator cyclic AMP response element-binding protein (CBP) (Emami et al., 2004). The natural compound carnosate, on the other hand, attenuates WNT signaling by promoting

aggregation of active E-catenin, thereby preventing its binding to the co-factor BCL9 (de la Roche et al., 2014; de la Roche et al., 2012).

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Figure 5. Mechanisms of tankyrase inhibition. The E3 ubiquitin ligase RNF146 mediates tankyrase-dependent degradation of AXIN by directly interacting with poly-ADP-ribose (PAR).

RNF146 also destabilizes tankyrase itself and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. Inhibition of the catalytic activity of TNKS1/2 increases the levels of AXIN, which is the rate-limiting factor for destruction complex stability.

Consequently, E-catenin destruction complexes accumulate in the cytoplasm, which promotes ŝŶĐƌĞĂƐĞĚĚĞŐƌĂĚĂƚŝŽŶŽĨɴ-catenin and reduced WNT signaling output.

Antibody-based targeting of ligands and receptors that are over-expressed in tumors may potentially be an attractive strategy to reduce aberrant WNT signaling. Indeed, several WNT-blocking antibodies have been shown to inhibit proliferation and induce apoptosis in certain cancer types (He et al., 2004a;

Mikami et al., 2005; Rhee et al., 2002). Other studies have used blocking

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antibodies targeting WNT receptors to inhibit cell growth and to induce apoptosis in cancer cells (Ettenberg et al., 2010; Pode-Shakked et al., 2011).

Moreover, a few studies have suggested the utility of WNT-modulatory peptides in WNT-dependent cancers (Lavergne et al., 2011; Zhang et al., 2009). The recent elucidation of the crystal structure of a WNT/FZD complex (Janda et al., 2012) may facilitate future design of such signal-modulating agents.

The binding promiscuity of TNKS

The functional consequences of TNKS inhibition remain incompletely resolved due to the binding promiscuity of TNKS. The ankyrin repeats of tankyrase can be divided into five domains called ankyrin repeat clusters (ARCs) and each of these ARCs consist of five stacked ankyrin repeats. The tankyrase-binding motif of the substrate proteins usually contains a consensus sequence RXXPDG (Sbodio and Chi, 2002) of which the Arginine and Glycine at positions 1 and 6 have been verified as the most critical amino acids for substrate binding (Guettler et al., 2011). Tankyrases interact with a variety of potential target proteins (Haikarainen et al., 2014) and have been implicated in a wide range of cellular functions: (1) TNKS1/2 were originally identified as a TRF1- binding protein at human telomeres. ADP-ribosylation of TRF1, which is a negative regulator of telomere length maintenance, diminishes its ability to bind to telomeric DNA, thereby allowing access of telomerase to the telomeres (Kaminker et al., 2001; Smith and de Lange, 2000; Smith et al., 1998). (2) TNKS has also been implicated as a negative regulator in insulin- dependent transport of Glucose transporter type 4 vesicles (GLUT4) from the Golgi apparatus to the cell surface (Chi and Lodish, 2000; Yeh et al., 2007).

This process is suggested to depend on a protein complex consisting of AXIN, KIF1D and TNKS (Guo et al., 2012). Furthermore, TNKS interacts with insulin-

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responsive aminopeptidase (IRAP) which is one of the integral membrane proteins in GLUT4 vesicles (Chi and Lodish, 2000). (3) RNA interference (RNAi) depletion studies have shown that TNKS1 is required for resolution of sister telomere cohesion at mitosis (Dynek and Smith, 2004). Moreover, TNKS appears to be a key component of mitotic spindle assembly and structure through interaction with the nuclear mitotic apparatus (NuMA) protein at the spindle poles during mitosis (Chang et al., 2005a, 2009; Chang et al., 2005b).

Of note, TNKS was also recently implicated in regulation of directional cell locomotion (Lupo et al., 2016). (4) Finally, assembly of the 26S proteasome is regulated by TNKS (Cho-Park and Steller, 2013). In addition, the role of TNKS in various cellular signaling pathways has not been completely elucidated. For instance, tankyrase inhibitors were recently shown to suppress YAP activity, the key effector in Hippo signaling (Wang et al., 2015).

Taken together, future studies will need to elucidate the consequences of inhibiting TNKS in the abovementioned biological processes. Although such a plethora of functions could potentially result in side effects, it also opens up for novel TNKSi-based therapeutic approaches. For instance, in certain types of cancer inhibiting tankyrase may delineate a chemical approach for disabling two cancer-associated cellular processes (e.g. telomere length and WNT signaling) with a single agent (Kulak et al., 2015).

The WNT signaling pathway as target in multimodal cancer therapy

Colon cancer is a leading cause of cancer-related deaths, with approximately 1.4 million new cases and 700.000 deaths estimated to have occurred in 2012 (Torre et al., 2015). Although surgical resection combined with adjuvant therapy is efficient at the early stages of disease, subsequent relapse and metastasis often occur at advanced stages due to drug resistance and

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ineffective treatment (Gustavsson et al., 2015). APC mutations are regarded as the initiating event for intestinal tumor formation. In line with this, mice and humans carrying different mutations in the APC gene

Galiatsatos and Foulkes, 2006

develop numerous

adenomatous colorectal polyps ( ; Su et al.,

1992). However, mutations in additional tumor suppressors and oncogenes (eg.KRAS, BRAF, P53 and SMAD4) are needed for progression into malignant colorectal tumors (carcinoma) (Fearon and Vogelstein, 1990; Luo et al., 2011;

Phelps et al., 2009). Moreover, crosstalk between different signaling pathways has been demonstrated during oncogenesis (Hu and Li, 2010; Lemieux et al., 2015). Given that deregulation of WNT signaling alone is not sufficient to induce malignant tumor formation, it is likely that inhibition of several signaling pathways will be necessary to curb cancer progression. Indeed, recent studies have illustrated promising therapeutic effects upon combined WNT/KRAS inhibition in colorectal cancer cells (Mologni et al., 2012; Mologni et al., 2010). The PI3K-AKT-FOXO3a pathway also plays a central role in colorectal cancer. AKT negatively regulates FOXO3a-mediated apoptosis by relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. However, in the presence of high nuclear E-catenin content, these inhibitors seem to promote metastasis of cells. Interestingly, the TNKSi XAV939 was shown to reverse the negative effect of PI3K and AKT inhibitors, thereby promoting apoptosis in WNT-dependent colorectal cancers (Arques et al., 2016; Tenbaum et al., 2012). The porcupine inhibitors (Lum and Clevers, 2012) antagonize secretion of WNT ligands and such treatment may potentially add to the effect of downstream TNKSi. Moreover, it has been demonstrated that reduced WNT signaling can sensitize cancer cells to chemotherapeutic agents (Ma et al., 2013; Wu et al., 2016). The WNT pathway has also been shown to contribute to the maintenance of lung cancer cells during EGFR inhibition, and a combination of EGFR and WNT inhibitors may therefore improve the clinical outcome in patients with EGFR-dependent

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cancers (Casás-Selves et al., 2012; Okada-Iwasaki et al., 2016). Finally, Wickstrøm and colleagues recently revealed that activation of WNT signaling induces the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which is commonly overexpressed in cancers and is implicated in the development of chemoresistance. Consequently, inhibition of WNT signaling was shown to augment the effects of alkylating drugs and restore chemosensitivity in different cancers (Wickström et al., 2015).

Collectively, these studies demonstrate that inhibiting the WNT signaling pathway in conjunction with other cancer therapeutics results in cooperative inhibition of tumor growth in certain cancer types.

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Aims of the thesis

Tankyrase inhibitors (TNKSi) have evolved as promising candidate drugs targeting the WNT signaling pathway. Upon tankyrase inhibition, the formation of cytoplasmic puncta containing destruction complex components was demonstrated and those structures were termed degradasomes. They represent most likely the morphological correlate of the endogenous E- catenin destruction complex. Our overall aim of this study was to increase our knowledge about the structure, function and regulation of TNKSi-induced degradasomes, as this would further elucidate the potential of TNKSi in cancer treatment and as research tools to further dissect the role of the WNT signaling cascade and its components in physiological processes and disease.

WĂƉĞƌȻ

Several studies showed the formation of degradasomes upon treatment with TNKSi in various colorectal cancer cell lines. However, extensive structural and functional studies of the protein assemblies were lacking. Our aim was to provide a direct mechanistic link between degradasome formation and Ecatenin degradation by taking advantage of live-cell imaging, high- resolution microscopy, and photobleaching approaches.

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WĂƉĞƌȻȻ

Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity

As part of our work in paper Ȼ, we had coincidentally observed that prolonged incubation with the proteasome inhibitor MG132 and G007-LK counteracted the formation of degradasomes. In this paper, we sought to characterize the mechanisms underlying this observation.

WĂƉĞƌȻȻȻ

Differential Roles of AXIN1 and AXIN2 in Tankyrase Inhibitor-Induced Formation of Degradasomes and EE-catenin degradation

Our findings in paper Ȼ and ȻȻ highlighted the AXIN proteins as key components in TNKSi-induced degradasomes. Since the role of AXIN1 versus AXIN2 as scaffolding proteins in the WNT signaling pathway still remains incompletely understood, we set out elucidate their relative contribution in formation of degradasomes and degradation of E-catenin.

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Summary of papers

WĂƉĞƌȻ

The tankyrase enzymes have recently emerged as promising drug targets in WNT-dependent tumors. Inhibition of the catalytic activity of tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) increases the levels of AXIN, which is a rate- limiting factor for ɴ-catenin destruction complex stability, and consequently ĂďƌŽŐĂƚĞƐtEdͬɴ-catenin signaling. Intriguingly, immunofluorescence imaging of various CRC cells has demonstrated puncta of colocalized destruction

complex components, referred to as degradasomes, upon TNKSi treatment.

Since extensive studies on these central inhibitor-induced protein complexes were lacking, we set out to characterize their structural composition and functional involvement in E-catenin degradation. Firstly, live-cell imaging demonstrated that these multiprotein assemblies were highly mobile and structurally dynamic. Moreover, high-resolution microscopy revealed that the degradasomes consisted of a filamentous assembly of high electron densities and discrete subdomains of various destruction complex components. Finally, ǁĞĨŽƵŶĚƚŚĂƚƉŚŽƐƉŚŽƌǇůĂƚĞĚɴ-catenin, ubiquitin, ĂŶĚɴ-TrCP localized to the degradasomes, ŝŶĚŝĐĂƚŝŶŐƚŚĂƚɴ-catenin gets both phosphorylated and ubiquitinated in degradasomes. This indication was corroborated by

photobleaching experiments which further demonstrated a rapid turnover of ɴ-catenin. We conclude that TNKS inhibition promotes highly dynamic

assemblies of functionally active destruction complexes, thereby providing a direct mechanistic link between degradasome formation and reduced WNT signaling in colorectal cancer cells. Importantly, our findings also highlight the potential of TNKSi as a valuable research tool to study the signal-ůŝŵŝƚŝŶŐɴ- catenin destruction complex.

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WĂƉĞƌȻȻ

Formation of Tankyrase Inhibitor-induced Degradasomes Requires Proteasome Activity

We observed by coincidence that prolonged incubation with the proteasome inhibitor MG132 counteracted the G007-LK-induced formation of

degradasomes. Furthermore, the protein levels of AXIN2 were substantially reduced in the same experimental setup. Quantitative real-time PCR

demonstrated a considerable decline in AXIN2 mRNA levels upon treatment with MG132 and this was not changed in the presence of both MG132 and G007-LK, thus confirming that the lack of AXIN2 stabilization originated from altered mRNA levels and not due to a regulation on the protein level. E- catenin has traditionally been regarded as the main activator of AXIN2 transcription. However, as both immunofluorescence imaging and

ďŝŽĐŚĞŵŝĐĂůĨƌĂĐƚŝŽŶĂƚŝŽŶĞǆƉĞƌŝŵĞŶƚƐƌĞǀĞĂůĞĚĂŶĂĐĐƵŵƵůĂƚŝŽŶŽĨɴ-catenin in the nucleus upon MG132 treatment, we concluded that E-catenin could not be responsible for the decreased transcription of AXIN2 mRNA. Forkhead box M1 (FoxM1) was recently implicated as a positive regulator of AXIN2 mRNA levels by two different means: Besides binding to and increasing the

transcriptional activity of the AXIN2 promotor region, FoxM1 was reported to proŵŽƚĞƚŚĞŶƵĐůĞĂƌůŽĐĂůŝǌĂƚŝŽŶŽĨɴ-ĐĂƚĞŶŝŶĂŶĚƐƵƉƉŽƌƚɴ-catenin-mediated transcription. Interestingly, proteasome inhibitors, including MG132, were reported to negatively regulate the transcriptional activity and protein expression of FoxM1. We found that 6hr treatment with MG132 drastically reduced FoxM1 mRNA levels as monitored by quantitative real-time PCR analysis, while protein levels increased. Moreover, siRNA-mediated depletion of FoxM1 led to a reduction in both AXIN2 mRNA levels and the TNKSi-induced stabilization of AXIN2 protein levels. Finally, FoxM1 protein lysates revealed a higher molecular weight band in the DMSO-treated conditions, which was

(49)

absent in the lysates of the MG132 treated cells and which was sensitive to phosphatase activity. We therefore suggest that despite FoxM1 localizing to the nucleus after MG132 treatment, it is dephosphorylated and thus

transcriptionally less active, which can explain the decreased protein levels of AXIN2 after MG132 treatment.

WĂƉĞƌȻȻȻ

Differential Roles of AXIN1 and AXIN2 in Tankyrase Inhibitor-Induced Formation of Degradasomes and EE-catenin Degradation

We previously showed that tankyrase inhibition induces highly dynamic

assemblies of destruction complexes, so-called degradasomes, which promote degradation of E-catenin and reduced WNT signaling activity in CRC cells (paper Ȼ). In the current study, we sought to elucidate the role of AXIN1 versus AXIN2 in the formation of degradasomes, as their relative contribution as scaffolding proteins in the WNT signaling pathway still remains incompletely understood. Surprisingly, we found that AXIN1 was not required for

degradasome formation. In contrast, the stability and function of

degradasomes was considerably impaired in G007-LK-treated cells depleted of AXIN2. We also showed that new synthesis of AXIN2 was required for

degradasome formation, which implies that a free pool of AXIN is necessary to serve as a structural scaffold for degradasome formation. When investigating the contribution of other destruction complex components, we further demonstrated that TNKSi-ŝŶĚƵĐĞĚĚĞŐƌĂĚĂƚŝŽŶŽĨɴ-catenin was severely impaired by depletion of GSK3 alpha, but not GSK3 beta, and efficient degradation appeared to require the presence of truncated APC proteins.

Taken together, these results give novel insights into the role of the key players in degradasome-ŵĞĚŝĂƚĞĚĚĞƐƚƌƵĐƚŝŽŶŽĨɴ-catenin.