Wnt signalling and prostate cancer - Nature

Prostate Cancer and Prostatic Diseases (2005) 8, 119–126 & 2005 Nature Publishing Group All rights reserved 1365-7852/05 $30.00 www.nature.com/pcan

Review

Wnt signalling and prostate cancer GW Yardy1,2* & SF Brewster2 1

Cancer & 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 The Wnt pathway directs embryonic growth, governing processes such as cell fate specification, proliferation, polarity and migration. It is also implicated in maintenance of stem cell populations. It is conserved in organisms from worms to mammals and its mechanism was first elucidated through analysis of Wingless signalling, its Drosophila homolog.1 Wnt proteins—a large family of cysteine-rich secreted ligands—bind to a class of seven-pass transmembrane receptors encoded by the frizzled genes2 transducing a signal to the cytoplasmic protein Dishevelled (Dvl),3 which is recruited to the membrane, forms a complex with Axin4 and induces its dephosphorylation.5 Axin (product of the gene AXIN1) acts as a scaffold protein maintaining the configuration of a complex involving APC (encoded by the Adenomatous Polyposis Coli gene6) and b-catenin (encoded by CTNNB17) and facilitating phosphorylation of both APC8 and b-catenin9 by glycogen synthase kinase 3b (GSK3b). *Correspondence: GW Yardy, Cancer & Immunogenetics Laboratory, Weatherall Insitute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK. E-mail: [email protected] Received 13 November 2004; revised 23 January 2005; accepted 30 January 2005; published online 5 April 2005

Figure 1 illustrates the interaction of the components of the Wnt signalling pathway. The central molecule of the pathway is b-catenin, which exists in three cellular pools10 —at the membrane (associated with E-cadherin, a-catenin and other molecules, involved in cell adhesion), cytoplasmic and nuclear. In the left-hand part of diagram, in the absence of a Wnt signal, a multiprotein complex including APC, GSK3b and the scaffold protein axin phosphorylates free cytoplasmic b-catenin, marking it for degradation by ubiquitin.11,12 The kinetics of the protein–protein interactions of the constituents of this complex have been represented by a mathematical model.13 The presence of a Wnt ligand at the transmembrane receptor Frizzled activates the cytoplasmic protein Dishevelled, which dephosphorylates axin. This decreases the capacity of axin to form complexes with APC and b-catenin. Conductin, a protein with structural homology to axin and encoded by the gene AXIN2,14 can function in a similar way to axin in this protein complex.15 Phosphorylation of b-catenin by GSK3b, and hence degradation of b-catenin by ubiquitin, is decreased. Cytoplasmic b-catenin thus accumulates and is translocated to the nucleus16 where it associates with members of the T-cell factor (TCF) and lymphoid enhancer factor (LEF) family of transcriptional factors.17 The b-catenin–TCF/LEF complex activates transcription of target genes including c-MYC,18 c-jun and fra-1 (components of the AP-1 transcription complex) and

Wnt signalling and prostate cancer GW Yardy and SF Brewster

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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 receptor),19 the metalloproteinases matrilysin20 and MMP-2621 and cyclin D1.22 Although mainly understood to be active in the cytoplasm in the protein complex described above, axin is also shuttled in and out of the nucleus23 and appears to shuttle b-catenin back to the cytoplasm with it,24 further limiting the effects of Wnt pathway activation. Closer analysis of conductin’s function is equally intriguing. As well as participating in the b-catenin destruction complex, its gene, AXIN2, is a target of TCF/ LEF transcription—acting as a negative feedback mechanism to control Wnt pathway activation.25,26 In summary, activation of the pathway by the presence of a Wnt molecule on the cell surface receptor decreases phosphorylation of b-catenin by the APC/axin/GSK3b complex. b-catenin is thus not degraded, and accumulates in the nucleus, where it stimulates transcription of a variety of cancer-associated genes.

Interactions, Wnt signalling disruption and oncogenesis Intracellular signalling pathways are frequently found to be interconnected; the Wnt pathway is not an exception. Some interacting pathways are displayed in Figure 2. Insulin-like growth factor (IGF) type 1 receptor stimulation facilitates dissociation of b-catenin at the cell membrane into the cytoplasmic pool in colorectal cells27 and potentiates b-catenin–TCF/LEF transcription in hepatoma cells.28 Prostate Cancer and Prostatic Diseases

Phosphatidylinositol 3-kinase/Akt pathway effector molecules, through inhibition of GSK3b, control cytoplasmic b-catenin levels.29 Siah-1 (the human homolog of Drosophila seven in absentia), a p53-inducible mediator of cell cycle arrest, tumour suppression and apoptosis, interacts with APC and promotes degradation of cytoplasmic b-catenin via a mechanism independent of GSK3b.30–32 b-catenin binds to and inhibits the activity of the transcription factor NFkB in breast and colon cancer cells.33,34 Ozz-E3, a muscle-specific ubiquitin ligase adaptor, regulates b-catenin levels at the cell membrane in muscle cells.35 Protein phosphatase 2A also inhibits the axin/APC/GSK3b b-catenin destruction complex36 and the structural basis for this interaction has been elucidated.37 Retinoid receptors have an influence. Retinoic acid receptor (RAR) decreases the activity of b-catenin–TCF/ LEF transcription independently of the APC pathway.38–40 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 the p53 signalling network42 and a feedback loop between the actions of deregulated b-catenin, Axin and p53 has been demonstrated, which may be protective against oncogenesis.43 Integrin-linked kinase and cyclin-dependent kinase 2 also influence the Wnt pathway at multiple points.44–46 The pathway interaction, which may be most relevant to prostatic tumorigenesis, is the recently observed function of b-catenin as a coactivator of the androgen receptor. This will be described in detail below. Abnormal activation of the pathway, such as mutations that lock the pathway into a ligand-independent


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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 of cells towards stem cell or stem cell-like fates, which may give rise to neoplasia. APC mutation is a common occurrence in carcinoma of the colon.47 The gene is somatically altered in at least 60% of sporadic tumours48 and familial adenomatous polyposis is caused by germline mutations of the gene.6,49 In colon cancer cell lines containing only mutant APC, a stable b-catenin-hTcf-4 complex was found that was constitutively active (hTcf-4 is a Tcf family member that is expressed in colonic epithelium). Reintroduction of wild-type APC removed b-catenin from hTcf-4 and abrogated the transcriptional activation.50 Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium. b-catenin mutations have been detected in a variety of tumours such as those of the colon,47,51 including five of 21 colorectal cancer cell lines,52 uterine endometrium,53 ovary,54 stomach,55 hepatocellular carcinoma56 and lung adenocarcinoma.57 In a study of malignant melanoma, activation of the Wnt pathway was found to be due to a stabilising mutation of CTNNB1, rendering b-catenin invulnerable to phosphorylation by the APC/GSK3b complex and allowing it to accumulate.58 In another melanoma study, the proportion of samples demonstrating nuclear accumulation of b-catenin outweighed the number of samples with b-catenin gene mutations, suggesting that

mechanisms other that CTNNB1 mutation were also activating Wnt signalling abnormally.59 Hepatocellular carcinoma (HCC) also showed nuclear b-catenin more frequently than CTNNB1 mutation. This prompted a search for AXIN1 mutations in six HCC cell lines and 100 primary HCCs. Among the four lines and 87 HCCs without CTNNB1 mutations, AXIN1 mutations were detected in three lines and six mutations in five of the primary HCCs.60 Another study of 73 HCCs and 27 hepatoblastomas (HBs) found b-catenin mutations in around 20% of HCCs and 80% of HBs, and AXIN1 and AXIN2 mutations in an additional 10% of HCCs and HBs.61 Analysis of 17 samples of adenocarcinomas of the gastro-oesophageal junction with nuclear accumulation of b-catenin showed loss of heterozygosity at the AXIN1 locus in 10 cases, but no mutations of the AXIN1 gene.62 A total of 86 sporadic cerebellar medulloblastomas (MBs) and 11 MB cell lines were found to harbour seven large deletions and a single somatic point mutation of the AXIN1 gene.63 However, another study of 39 MBs found two AXIN1 mutations but no deletions.64 AXIN2 (encoding conductin—an alternate component of the APC/GSK3b complex which phosphorylates b-catenin in response to a Wnt signal14) was found mutant in eleven of 45 colorectal cancer specimens with defective mismatch repair.65 In all, 45 primary ovarian endometroid adenocarcinomas and two cell lines included 14 b-catenin mutations, Prostate Cancer and Prostatic Diseases


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biallelic inactivation of the APC gene in another, one specimen with a mutation in AXIN1 and one with mutant AXIN2.66 Wnt-independent phosphorylation of GSK3b involving p5367 and participation of GSK3b in pathways that influence vascular endothelial cell migration, growth and survival have been investigated.68

The Wnt pathway and prostate cancer Loss of heterozygosity69,70 and mutation71 of the APC gene have been detected in some tumour samples, but another group could find no APC mutations.72 The expression of E-cadherin, b-catenin and GSK3b is diminished in prostate cancer cell lines of greater invasive potential.73 Expression of b-catenin in bone metastases appears downregulated, compared with that seen in corresponding primary tumours in patients with untreated prostate cancer.74 Alterations in expression and mutations of b-catenin documented in prostate cancer are summarised in Table 1. b-catenin mutations were found in five out of 100 prostate cancer specimens by Voeller et al,75 but they studied only radical prostatectomy specimens (presumed early tumours) and only looked at exon 3 of the b-catenin gene CTNNB1. This exon corresponds with the site of phosphorylation of b-catenin by GSK3b so such mutations would make b-catenin invulnerable to degradation, allowing it to accumulate and effect gene transcription. The authors demonstrated by analysis of multiple areas of some tumours that b-catenin mutations occurs focally, and suggested that mutation frequency may have been underestimated because detection of mutations in small foci was beyond the sensitivity of their technique. In a study of three prostate cancer cell lines, 13 xenografts and six pT3 radical prostatectomy specimens, three APC mutations were found in the xenografts and two CTNNB1 mutations were found in the prostatectomies.76 All mutations were mutually exclusive, consistent with their equivalent effects on b-catenin stability. Chesire et al77 studied 81 radical prostatectomy specimens and samples, including metastatic deposits, from 19 autopsies. Again, only exon 3 of CTNNB1 was studied, but mutants were found in five of the radical

prostatectomies and all of nine metastatic specimens provided by one autopsy. In a subsequent study, Chesire et al78 found a nuclear immunohistochemical staining pattern for b-catenin (a hallmark of Wnt activation) in a heterogeneous fashion in 24% of metastatic specimens from 21 patients, but 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 prostatic carcinogenesis. In another immunohistochemical study, b-catenin staining was abnormal in 23% of 122 (hormone-naı¨ve) radical prostatectomy specimens, compared to 38% of 90 specimens from transurethral prostatectomies performed to treat bladder outflow obstruction in patients with hormone-refractory prostate cancer.79 This supports the hypothesis that alterations in the Wnt pathway may contribute to progression of prostate cancer to androgen independence. A more recent study used digital image analysis to quantify b-catenin expression, which was correlated with tumour grade and stage.80 In all, 83% of advanced tumours showed abnormal staining for b-catenin. Dysfunction of the wnt pathway in prostate cancer has been assessed in other laboratory studies. Expression of a mutant form of b-catenin, which is less susceptible to degradation in a mouse model, produced lesions with histological appearances similar to prostatic intraepithelial neoplasia (PIN), the putative precursor of prostate cancer, as early as 10 weeks of age, but these lesions did not progress to invasion or metastasis in animals up to 5 months of age.81 Sublines of apoptosis-resistant LNCaP cells were developed by repeated brief exposure to apoptotic stimuli. These showed increased nuclear b-catenin expression and increased TCF/LEF transcription compared to the parent lines.79 The search for AXIN1 and 2 mutations in prostate cancer has not yet been documented and seems worthwhile, considering the discovery of such mutations in other tumours with more frequent nuclear b-catenin accumulation than b-catenin mutation, and the Wnt pathway negative feedback functions that axin and conductin serve.10 Also, mutations of the b-catenin gene outside exon 3 are currently being pursued. Mutations elsewhere, such as the sequence encoding the section of b-catenin which interacts with Siah-1, may result in augmented Wnt pathway activation.

Table 1 b-catenin mutations and abnormal immunohistochemistry in clinical prostate cancer tissue Specimens 75

Voeller et al

100 RPs 4 LN metastases 81 RPs 22 LN metastases 19 patients’ other metastatic tissue 6 T3 RPs 21 patients’ post-mortem metastatic tissue

Chesire et al77 Gerstein et al76 Chesire et al78 79

De la Taille et al Chen et al80

122 RPs 90 HRPC TURs 8 HRPC TURs 49 early 18 metastatic

Exon 3 mutations 5 0 5 0 1 2 1

Abnormal immunohistochemistry

(5%) (6.2%) (5.3%) (33.3%) (4.8%)

5 (23.8%) 28 (23.0%) 35 (38.9%)

0 25 (51.0%) 15 (83.3%)

nuclear staining nuclear or cytoplasmic nuclear or cytoplasmic

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

Prostate Cancer and Prostatic Diseases


b-Catenin and the androgen receptor Studies using prostate cancer cells have demonstrated another function of nuclear b-catenin as an activator of the androgen receptor (AR). Along with mutation and amplification of the AR gene, changes in the influences of AR coactivators are considered important in the conversion to lethal androgen-independent disease.82 In cell lines expressing the androgen receptor, b-catenin significantly enhances androgen-stimulated transcriptional activity by the AR and diminishes its antagonism by bicalutamide (AR antagonist).83 A further study has confirmed this observation, noting enhanced androgenmediated transcription on overexpression of b-catenin in prostate cancer cells.78 This suggests that the role of bcatenin in prostate oncogenesis may not be limited to transcriptional activation of TCF/LEF and further links b-catenin with development of androgen insensitivity. A specific protein–protein interaction has been demonstrated between b-catenin and AR in both prostatic84 and neuronal85 cells. A proportion of the b-catenin translocated from the cytoplasm to the nucleus may be shuttled bound to AR, stimulated by androgen, independent of Wnt molecules.86 b-catenin/TCF-related transcription in prostate cancer cell lines is inhibited by androgen treatment.87 This inhibition is AR-dependent, as it only occurs in cells expressing AR endogenously or transiently, and is abrogated by AR antagonists. Androgen-induced suppression of TCF/LEF transcription may be due to competition between AR and TCF/LEF for b-catenin. Such competition has been demonstrated in prostate cells and colorectal cells transiently transfected with an AR expression construct.88 However in a study of a panel of AR regulators in prostate cancer cell lines, xenografts and clinical specimens 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 demonstrated in prostate cancer cell lines. The tumour suppressor PTEN, which is frequently mutated in prostate cancer, inhibits the PI3K/Akt pathway. PTEN has been shown to modulate androgen-induced prostate 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 pathway: GSK3b, a downstream effector of PI3K/Akt, also participates in the Wnt pathway; PTEN phosphorylates and inactivates GSK3b via PI3K/Akt; GSK3bdependent inactivation of cytoplasmic b-catenin is attenuated; b-catenin shuttles to the nucleus and augments ligand-stimulated transcription by AR.92

Prostate cancer management and the Wnt pathway Wnt pathway dysfunction is an important component of prostatic tumorigenesis and offers a variety of treatment targets. In the future, knowledge of an individual tumour’s genotype will influence management decisions. Status of genes encoding Wnt molecules or levels

of expression of these proteins in cancers may be prognostically valuable or indicate specific treatments. The reintroduction of functional genes to replace those found to be altered is one therapeutic option, but other strategies to influence disrupted pathways are more likely to be successful. Putative cancer chemopreventive agents have an effect on the Wnt pathway. A variety of dietary factors influence carcinogenesis93 and part of the chemopreventive effect of folic acid may be due to inhibition of shuttling of b-catenin into the nucleus.94 Chemoprevention strategies involving cyclooxygenase inhibitors have also received attention.95 However, nonsteroidal antiinflammatory drugs are promiscuous with respect to the enzymes they inhibit: their antineoplastic properties are not necessarily due to their effect on cyclooxygenases.96 There is evidence that they also inhibit nuclear accumulation of b-catenin and subsequent oncogene transcription.97–99 Compounds that specifically target the Wnt pathway have been sought: two groups have recently screened libraries of naturally occurring substances and molecules that inhibit the b-catenin–TCF/LEF transcription complex have been identified.100,101 Via a similar approach, a small molecule which inhibits the Wnt pathway by mimicking the function of APC has been identified.102 A gene therapy technique using a recombinant adenovirus bearing a gene under the control of a b-catenin/Tcfresponse promoter produced an anti-tumour effect in animal models with Wnt pathway activation.103 There is hope that our current therapeutic impotence in advanced prostate cancer may be addressed by targeting pathways disrupted specifically in cancer cells, such as Wnt signalling, avoiding toxicity associated with treatments with more general actions on all dividing cells.

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Acknowledgements GWY is funded by a grant from the Prostate Research Campaign, UK. We are grateful to Sir Walter Bodmer for his critique of this manuscript.

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