Wnt Signalling and Prostate Cancer

GW Yardy1,2* & SF Brewster2 1Cancer & Immunogenetics Laboratory, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK; and 2Department of Urology, Churchill Hospital, Oxford, UK

The Wnt signalling pathway plays a role in the direction of embryological development and maintenance of stem cell populations. Heritable alterations in genes encoding molecules of the Wnt pathway, including mutation and epigenetic events, have been demonstrated in a variety of cancers. It has been proposed that disruption of this pathway is a significant step in the development of many tumours. Interactions between b-catenin—the effector molecule of the Wnt pathway—and the androgen receptor highlight the pathway’s relevance to urological malignancy. Mutation or altered expression of Wnt genes in tumours may give prognostic information and treatments are being developed which target this pathway. Prostate Cancer and Prostatic Diseases (2005) 8, 119–126. doi:10.1038/sj.pcan.4500794 Published online 5 April 2005

Keywords: Wnt signalling; b-catenin

Introduction Figure 1 illustrates the interaction of the components of the Wnt signalling pathway. The central molecule of The Wnt pathway directs embryonic growth, governing the pathway is b-catenin, which exists in three cellular processes such as cell fate specification, proliferation, pools10—at the membrane (associated with E-cadherin, polarity and migration. It is also implicated in main- a-catenin and other molecules, involved in cell adhe- tenance of stem cell populations. It is conserved in sion), cytoplasmic and nuclear. In the left-hand part of organisms from worms to mammals and its mechanism diagram, in the absence of a Wnt signal, a multiprotein was first elucidated through analysis of Wingless complex including APC, GSK3b and the scaffold protein signalling, its Drosophila homolog.1 Wnt proteins—a axin phosphorylates free cytoplasmic b-catenin, marking large family of cysteine-rich secreted ligands—bind to it for degradation by ubiquitin.11,12 The kinetics of the a class of seven-pass transmembrane receptors encoded protein–protein interactions of the constituents of this by the frizzled genes2 transducing a signal to the complex have been represented by a mathematical cytoplasmic protein Dishevelled (Dvl),3 which is re- model.13 The presence of a Wnt ligand at the transmem- cruited to the membrane, forms a complex with Axin4 brane receptor Frizzled activates the cytoplasmic protein and induces its dephosphorylation.5 Axin (product of the Dishevelled, which dephosphorylates axin. This de- gene AXIN1) acts as a scaffold protein maintaining the creases the capacity of axin to form complexes with configuration of a complex involving APC (encoded by APC and b-catenin. Conductin, a protein with structural the Adenomatous Polyposis Coli gene6) and b-catenin homology to axin and encoded by the gene AXIN2,14 can (encoded by CTNNB17) and facilitating phosphorylation function in a similar way to axin in this protein of both APC8 and b-catenin9 by glycogen synthase kinase complex.15 Phosphorylation of b-catenin by GSK3b, and 3b (GSK3b). hence degradation of b-catenin by ubiquitin, is de- creased. Cytoplasmic b-catenin thus accumulates and is translocated to the nucleus16 where it associates with *Correspondence: GW Yardy, Cancer & Immunogenetics Laboratory, members of the T-cell factor (TCF) and lymphoid Weatherall Insitute of Molecular Medicine, John Radcliffe Hospital, enhancer factor (LEF) family of transcriptional factors.17 Oxford, UK. The b-catenin–TCF/LEF complex activates transcription E-mail: [email protected] 18 Received 13 November 2004; revised 23 January 2005; accepted 30 of target genes including c-MYC, c-jun and fra-1 January 2005; published online 5 April 2005 (components of the AP-1 transcription complex) and Wnt signalling and prostate cancer GW Yardy and SF Brewster 120 Wnt Frizzled receptor

DVL

GSK3β n i A t c P actin u n C i d x β n o α GSK3 APC A -cat C

n E cadherin i

x β -catenin A β-catenin Conductin β-catenin

β -catenin

β-catenin degradation β-catenin TCF/ LEF

β -catenin

TCF/ Transcription LEF

target genes inc. c-myc, c-jun, fra-1, matrilysin, cyclin D1

Figure 1 The Wnt signalling pathway. DVL—dishevelled, GSK3b—glycogen synthase kinase b, APC—adenomatous polyposis coli, a-cat—a-catenin, TCF/LEF—T-cell factor/lymphoid enhancer factor transcription factors.

uPAR (the urokinase-type plasminogen activator recep- Phosphatidylinositol 3-kinase/Akt pathway effector tor),19 the metalloproteinases matrilysin20 and MMP-2621 molecules, through inhibition of GSK3b, control cyto- and cyclin D1.22 plasmic b-catenin levels.29 Siah-1 (the human homolog of Although mainly understood to be active in the Drosophila seven in absentia), a p53-inducible mediator of cytoplasm in the protein complex described above, axin cell cycle arrest, tumour suppression and apoptosis, is also shuttled in and out of the nucleus23 and appears to interacts with APC and promotes degradation of shuttle b-catenin back to the cytoplasm with it,24 further cytoplasmic b-catenin via a mechanism independent of limiting the effects of Wnt pathway activation. GSK3b.30–32 b-catenin binds to and inhibits the activity of Closer analysis of conductin’s function is equally the transcription factor NFkB in breast and colon cancer intriguing. As well as participating in the b-catenin cells.33,34 Ozz-E3, a muscle-specific ubiquitin ligase destruction complex, its gene, AXIN2, is a target of TCF/ adaptor, regulates b-catenin levels at the cell membrane LEF transcription—acting as a negative feedback me- in muscle cells.35 Protein phosphatase 2A also inhibits chanism to control Wnt pathway activation.25,26 the axin/APC/GSK3bb-catenin destruction complex36 In summary, activation of the pathway by the presence and the structural basis for this interaction has been of a Wnt molecule on the cell surface receptor decreases elucidated.37 phosphorylation of b-catenin by the APC/axin/GSK3b Retinoid receptors have an influence. Retinoic acid complex. b-catenin is thus not degraded, and accumu- receptor (RAR) decreases the activity of b-catenin–TCF/ lates in the nucleus, where it stimulates transcription of a LEF transcription independently of the APC pathway.38–40 variety of cancer-associated genes. Retinoid X receptor (RXR) facilitates degradation of b-catenin by another pathway independent of APC.41 Gene transcription by TCF-4 is also under the control of Interactions, Wnt signalling disruption the p53 signalling network42 and a feedback loop between and oncogenesis the actions of deregulated b-catenin, Axin and p53 has been demonstrated, which may be protective against Intracellular signalling pathways are frequently found to oncogenesis.43 Integrin-linked kinase and cyclin-depen- be interconnected; the Wnt pathway is not an exception. dent kinase 2 also influence the Wnt pathway at multiple Some interacting pathways are displayed in Figure 2. points.44–46 The pathway interaction, which may be most Insulin-like growth factor (IGF) type 1 receptor stimula- relevant to prostatic tumorigenesis, is the recently tion facilitates dissociation of b-catenin at the cell observed function of b-catenin as a coactivator of the membrane into the cytoplasmic pool in colorectal cells27 androgen receptor. This will be described in detail below. and potentiates b-catenin–TCF/LEF transcription in Abnormal activation of the pathway, such as muta- hepatoma cells.28 tions that lock the pathway into a ligand-independent

Prostate Cancer and Prostatic Diseases Wnt signalling and prostate cancer GW Yardy and SF Brewster 121 Wnt Frizzled receptor

PI3K/Akt/PTEN pathway DVL PP2A inhibit IGFs GSK3β n i A t c P actin u n C Siah-1 i d x β n o GSK3 APC activates A α-cat C

n E cadherin i

x β-catenin A β-catenin Conductin β-catenin RXR degrades

β-catenin Ozz-E3 IGFs β -catenin β-catenin degradation β-catenin AR RAR TCF/ LEF

β-catenin

AR NFκB β-catenin TCF/ Transcription LEF

target genes inc. c-myc, c-jun, fra-1, matrilysin, cyclin D1

Figure 2 Some influences of other signalling pathways and factors on the Wnt pathway. PP2A—protein phosphatase 2A, IGFs—insulin-like growth factors, Siah-1—human homolog of Drosophila seven in absentia, AR—androgen receptor, RAR—retinoic acid receptor A, RXR—retinoid X receptor, NFkB—nuclear factor kappa B transcription factor. state of constitutive activation, result in mis-specification mechanisms other that CTNNB1 mutation were also of cells towards stem cell or stem cell-like fates, which activating Wnt signalling abnormally.59 may give rise to neoplasia. Hepatocellular carcinoma (HCC) also showed nuclear APC mutation is a common occurrence in carcinoma of b-catenin more frequently than CTNNB1 mutation. This the colon.47 The gene is somatically altered in at least prompted a search for AXIN1 mutations in six HCC cell 60% of sporadic tumours48 and familial adenomatous lines and 100 primary HCCs. Among the four lines and polyposis is caused by germline mutations of the 87 HCCs without CTNNB1 mutations, AXIN1 mutations gene.6,49 In colon cancer cell lines containing only mutant were detected in three lines and six mutations in five of APC, a stable b-catenin-hTcf-4 complex was found that the primary HCCs.60 Another study of 73 HCCs and 27 was constitutively active (hTcf-4 is a Tcf family member hepatoblastomas (HBs) found b-catenin mutations in that is expressed in colonic epithelium). Reintroduction around 20% of HCCs and 80% of HBs, and AXIN1 and of wild-type APC removed b-catenin from hTcf-4 and AXIN2 mutations in an additional 10% of HCCs and abrogated the transcriptional activation.50 Constitutive HBs.61 transcription of Tcf target genes, caused by loss of APC Analysis of 17 samples of adenocarcinomas of the function, may be a crucial event in the early transforma- gastro-oesophageal junction with nuclear accumulation tion of colonic epithelium. of b-catenin showed loss of heterozygosity at the AXIN1 b-catenin mutations have been detected in a variety of locus in 10 cases, but no mutations of the AXIN1 gene.62 tumours such as those of the colon,47,51 including five of A total of 86 sporadic cerebellar medulloblastomas (MBs) 21 colorectal cancer cell lines,52 uterine endometrium,53 and 11 MB cell lines were found to harbour seven large ovary,54 stomach,55 hepatocellular carcinoma56 and lung deletions and a single somatic point mutation of the adenocarcinoma.57 AXIN1 gene.63 However, another study of 39 MBs found In a study of malignant melanoma, activation of the two AXIN1 mutations but no deletions.64 Wnt pathway was found to be due to a stabilising AXIN2 (encoding conductin—an alternate component mutation of CTNNB1, rendering b-catenin invulnerable of the APC/GSK3b complex which phosphorylates to phosphorylation by the APC/GSK3b complex and b-catenin in response to a Wnt signal14) was found allowing it to accumulate.58 In another melanoma study, mutant in eleven of 45 colorectal cancer specimens with the proportion of samples demonstrating nuclear accu- defective mismatch repair.65 mulation of b-catenin outweighed the number of In all, 45 primary ovarian endometroid adenocarcino- samples with b-catenin gene mutations, suggesting that mas and two cell lines included 14 b-catenin mutations,

Prostate Cancer and Prostatic Diseases Wnt signalling and prostate cancer GW Yardy and SF Brewster

122 biallelic inactivation of the APC gene in another, one prostatectomies and all of nine metastatic specimens specimen with a mutation in AXIN1 and one with provided by one autopsy. mutant AXIN2.66 In a subsequent study, Chesire et al78 found a nuclear Wnt-independent phosphorylation of GSK3b invol- immunohistochemical staining pattern for b-catenin (a ving p5367 and participation of GSK3b in pathways that hallmark of Wnt activation) in a heterogeneous fashion influence vascular endothelial cell migration, growth and in 24% of metastatic specimens from 21 patients, but survival have been investigated.68 CTNNB1 exon 3 mutations in only 5% of the same samples. This suggests that alterations in the function of Wnt members other than b-catenin may play a role in The Wnt pathway and prostate cancer prostatic carcinogenesis. In another immunohistochemical study, b-catenin Loss of heterozygosity69,70 and mutation71 of the APC staining was abnormal in 23% of 122 (hormone-naı¨ve) gene have been detected in some tumour samples, but radical prostatectomy specimens, compared to 38% of 90 another group could find no APC mutations.72 The specimens from transurethral prostatectomies performed expression of E-cadherin, b-catenin and GSK3b is to treat bladder outflow obstruction in patients with hor- 79 diminished in prostate cancer cell lines of greater mone-refractory prostate cancer. This supports the hypo- invasive potential.73 thesis that alterations in the Wnt pathway may contribute Expression of b-catenin in bone metastases appears to progression of prostate cancer to androgen indepen- downregulated, compared with that seen in correspond- dence. A more recent study used digital image analysis to ing primary tumours in patients with untreated prostate quantify b-catenin expression, which was correlated with 80 cancer.74 tumour grade and stage. In all, 83% of advanced tumours Alterations in expression and mutations of b-catenin showed abnormal staining for b-catenin. documented in prostate cancer are summarised in Dysfunction of the wnt pathway in prostate cancer has Table 1. b-catenin mutations were found in five out of been assessed in other laboratory studies. Expression of a 100 prostate cancer specimens by Voeller et al,75 but they mutant form of b-catenin, which is less susceptible to studied only radical prostatectomy specimens (pre- degradation in a mouse model, produced lesions with sumed early tumours) and only looked at exon 3 of the histological appearances similar to prostatic intraepithe- b-catenin gene CTNNB1. This exon corresponds with the lial neoplasia (PIN), the putative precursor of prostate site of phosphorylation of b-catenin by GSK3b so such cancer, as early as 10 weeks of age, but these lesions did mutations would make b-catenin invulnerable to degra- not progress to invasion or metastasis in animals up to 5 81 dation, allowing it to accumulate and effect gene months of age. transcription. The authors demonstrated by analysis of Sublines of apoptosis-resistant LNCaP cells were multiple areas of some tumours that b-catenin mutations developed by repeated brief exposure to apoptotic occurs focally, and suggested that mutation frequency stimuli. These showed increased nuclear b-catenin may have been underestimated because detection of expression and increased TCF/LEF transcription com- 79 mutations in small foci was beyond the sensitivity of pared to the parent lines. their technique. The search for AXIN1 and 2 mutations in prostate In a study of three prostate cancer cell lines, 13 cancer has not yet been documented and seems worth- xenografts and six pT3 radical prostatectomy specimens, while, considering the discovery of such mutations in three APC mutations were found in the xenografts and other tumours with more frequent nuclear b-catenin two CTNNB1 mutations were found in the prostatec- accumulation than b-catenin mutation, and the Wnt tomies.76 All mutations were mutually exclusive, con- pathway negative feedback functions that axin and 10 sistent with their equivalent effects on b-catenin stability. conductin serve. Also, mutations of the b-catenin gene Chesire et al77 studied 81 radical prostatectomy speci- outside exon 3 are currently being pursued. Mutations mens and samples, including metastatic deposits, from elsewhere, such as the sequence encoding the section of 19 autopsies. Again, only exon 3 of CTNNB1 was b-catenin which interacts with Siah-1, may result in studied, but mutants were found in five of the radical augmented Wnt pathway activation.

Table 1 b-catenin mutations and abnormal immunohistochemistry in clinical prostate cancer tissue Specimens Exon 3 mutations Abnormal immunohistochemistry

Voeller et al75 100 RPs 5 (5%) 4 LN metastases 0 Chesire et al77 81 RPs 5 (6.2%) 22 LN metastases 0 19 patients’ other metastatic tissue 1 (5.3%) Gerstein et al76 6 T3 RPs 2 (33.3%) Chesire et al78 21 patients’ post-mortem metastatic tissue 1 (4.8%) 5 (23.8%) nuclear staining De la Taille et al79 122 RPs 28 (23.0%) nuclear or cytoplasmic 90 HRPC TURs 35 (38.9%) 8 HRPC TURs 0 Chen et al80 49 early 25 (51.0%) nuclear or cytoplasmic 18 metastatic 15 (83.3%)

RP: radical prostatectomy specimen; LN: lymph node; HRPC TUR: transurethral prostatectomy specimen from patient with hormone refractory prostate cancer.

Prostate Cancer and Prostatic Diseases Wnt signalling and prostate cancer GW Yardy and SF Brewster b-Catenin and the androgen receptor of expression of these proteins in cancers may be 123 prognostically valuable or indicate specific treatments. Studies using prostate cancer cells have demonstrated The reintroduction of functional genes to replace those another function of nuclear b-catenin as an activator of found to be altered is one therapeutic option, but other the androgen receptor (AR). Along with mutation and strategies to influence disrupted pathways are more amplification of the AR gene, changes in the influences of likely to be successful. AR coactivators are considered important in the conver- 82 Putative cancer chemopreventive agents have an effect sion to lethal androgen-independent disease. In cell on the Wnt pathway. A variety of dietary factors lines expressing the androgen receptor, b-catenin sig- influence carcinogenesis93 and part of the chemopreven- nificantly enhances androgen-stimulated transcriptional tive effect of folic acid may be due to inhibition of activity by the AR and diminishes its antagonism by b 94 83 shuttling of -catenin into the nucleus. Chemopreven- bicalutamide (AR antagonist). A further study has tion strategies involving cyclooxygenase inhibitors have confirmed this observation, noting enhanced androgen- also received attention.95 However, nonsteroidal anti- mediated transcription on overexpression of b-catenin in 78 inflammatory drugs are promiscuous with respect to the prostate cancer cells. This suggests that the role of b- enzymes they inhibit: their antineoplastic properties are catenin in prostate oncogenesis may not be limited to not necessarily due to their effect on cyclooxygenases.96 transcriptional activation of TCF/LEF and further links There is evidence that they also inhibit nuclear accumu- b-catenin with development of androgen insensitivity. lation of b-catenin and subsequent oncogene transcrip- A specific protein–protein interaction has been demon- tion.97–99 strated between b-catenin and AR in both prostatic84 and 85 Compounds that specifically target the Wnt pathway neuronal cells. A proportion of the b-catenin translo- have been sought: two groups have recently screened cated from the cytoplasm to the nucleus may be shuttled libraries of naturally occurring substances and molecules bound to AR, stimulated by androgen, independent of b 86 that inhibit the -catenin–TCF/LEF transcription com- Wnt molecules. plex have been identified.100,101 Via a similar approach, a b-catenin/TCF-related transcription in prostate cancer 87 small molecule which inhibits the Wnt pathway by cell lines is inhibited by androgen treatment. This mimicking the function of APC has been identified.102 A inhibition is AR-dependent, as it only occurs in cells gene therapy technique using a recombinant adenovirus expressing AR endogenously or transiently, and is bearing a gene under the control of a b-catenin/Tcf- abrogated by AR antagonists. Androgen-induced sup- response promoter produced an anti-tumour effect in pression of TCF/LEF transcription may be due to animal models with Wnt pathway activation.103 competition between AR and TCF/LEF for b-catenin. There is hope that our current therapeutic impotence Such competition has been demonstrated in prostate cells in advanced prostate cancer may be addressed by and colorectal cells transiently transfected with an AR 88 targeting pathways disrupted specifically in cancer cells, expression construct. such as Wnt signalling, avoiding toxicity associated with However in a study of a panel of AR regulators in treatments with more general actions on all dividing prostate cancer cell lines, xenografts and clinical speci- cells. mens using quantitative real-time RT-PCR, overexpression of b-catenin was not found.89 Crosstalk between the Wnt pathway, the AR and the phosphatidylinositol 3-kinase/Akt pathway has been Acknowledgements demonstrated in prostate cancer cell lines. The tumour suppressor PTEN, which is frequently mutated in GWY is funded by a grant from the Prostate Research prostate cancer, inhibits the PI3K/Akt pathway. PTEN Campaign, UK. We are grateful to Sir Walter Bodmer for has been shown to modulate androgen-induced prostate his critique of this manuscript. cancer cell growth and AR-mediated transcription.90,91 Work using a synthetic PI3K inhibitor, and re-expression of PTEN in a PTEN-null prostate cancer cell line has shown that the mechanism of this involves the Wnt References pathway: GSK3b, a downstream effector of PI3K/Akt, also participates in the Wnt pathway; PTEN phosphor- 1 Wodarz A, Nusse R. Mechanisms of Wnt signaling in develop- ylates and inactivates GSK3b via PI3K/Akt; GSK3b- ment. Annu Rev Cell Dev Biol 1998; 14: 59–88. dependent inactivation of cytoplasmic b-catenin is 2 Yang-Snyder J et al. A frizzled homolog functions in a vertebrate attenuated; b-catenin shuttles to the nucleus and aug- Wnt signaling pathway. 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