Understanding the molecular etiology of prostate cancer (CaP) progression is paramount for broadening current diagnostic and therapeutic modalities. Current interest in the role of wnt pathway signaling in prostate tumorigenesis was generated with the finding of β-catenin mutation and corresponding nuclear localization in primary lesions. The recent finding of β-catenin-induced enhancement of androgen receptor (AR) function potentially ties β-catenin to key regulatory steps of prostate cell growth, differentiation, and transformation. By immunohistological analysis of metastatic tumors, we detected nuclear β-catenin in 20% of lethal CaP cases, suggesting a more common role for β-catenin in advanced disease than would be predicted by its mutation rate. Interestingly, β-catenin nuclear localization was found to occur concomitantly with androgen-induced regrowth of normal rat prostate. These in vivo observations likely implicate β-catenin involvement in both normal and neoplastic prostate physiology, thus prompting our interest in further characterizing modes of β-catenin signaling in prostate cells. Extending our previous findings, we demonstrate that transient β-catenin over-expression stimulates T cell factor (TCF) signaling in most CaP cell lines. Further, this activity is not subject to cross-regulation by phosphoinositide-3-kinase (PI3-K)/Akt signaling, a stimulatory pathway often upregulated in CaP upon PTEN inactivation. Consistent with a previous report, we observed that transient β-catenin over-expression enhances AR-mediated transcription off two natural target gene promoters. However, we were unable to recapitulate β-catenin-induced stimulation of ectopically expressed AR in AR-negative cells, suggesting that other AR-associated factors are required for this activity. Although LNCaP cells are capable of this mode of AR co-stimulation, stable expression of mutant β-catenin did not alter their proliferative response to androgen. In total, our characterization of β-catenin signaling in CaP reveals the complex nature of its activity in prostate tissue, indicating that β-catenin potentially contributes to multiple stimulatory inputs required for disease progression.
Prostate carcinoma is the most common non-cutaneous cancer affecting American men (Tindall and Scardino, 1999). Although our ability to detect early stage tumors has increased, predictive diagnosis and effective tumor therapy remain limited, thereby incurring greater morbidity rates. Advanced CaP brought to remission with hormonal ablative therapy often relapses, leading to androgen-independent tumors for which only experimental treatments exist (Culig et al., 1998). Urgently needed is a better understanding of CaP progression at the molecular level in order to reveal new targets for novel therapy design.
Several mechanisms underlying CaP progression have been described in molecular detail (Elo and Visakorpi, 2001). Previous work and data presented herein implicate the wnt pathway and its effector signaling molecule, β-catenin, in this disease (Chesire et al., 2000; Voeller et al., 1998). β-catenin mutations were detected at a rate of ∼5–7% and shown to result in its accumulation and nuclear localization. β-catenin was originally identified to participate in adherens-mediated cell/cell adhesion, linking the cytoplasmic domain of E-cadherin to the actin binding protein α-catenin (Takeichi, 1991). Whereas E-cadherin and α-catenin often exhibit altered expression in human cancer, β-catenin expression is rarely lost, implying that its signaling functions are important for cell viability. Soluble (cytosolic) β-catenin is subject to phosphorylation at its amino terminus by glycogen synthase kinase-3β (GSK-3β), prompting its ubiquitination and subsequent proteasomal degradation (Orford et al., 1997; Aberle et al., 1997).
Binding of secreted wnt factors to the frizzled family of cell surface receptors initiates a signaling cascade leading to GSK-3β inhibition and alleviation of β-catenin repression (Miller and Moon, 1996). Although growth factor-induced Akt activity can abrogate GSK-3β function (Cross et al., 1995), it is not clear whether or not β-catenin stabilization results (Ding et al., 2000; Yuan et al., 1999). Under wnt signaling, soluble β-catenin levels accrue, eventually leading to its nuclear import and interaction with the TCF family of transcription factors. β-catenin de-represses TCF target gene expression, via recruitment of transcription factors such as Creb binding protein (CBP) and TATA binding protein (TBP) to its amino and carboxy terminal transactivation domains (Takemaru and Moon, 2000; Hecht et al., 1999). Targets of the β-catenin/TCF bipartite transcription factor include the proto-oncogenes c-myc, cyclin D1 and matrix metalloproteinase (He et al., 1998; Tetsu and McCormick, 1999; Crawford et al., 1999). Despite being less well characterized, β-catenin has also been shown to play a role in gene expression mediated by other transcription factors besides TCF (Easwaran et al., 1999; Truica et al., 2000; Palmer et al., 2001; Zorn et al., 1999). To encompass this breadth of β-catenin involvement in gene transactivation, we employ the term CRT (β-catenin-related transcription), which was originally defined by Morin et al. (1997) to describe only β-catenin-TCF-regulated transcription.
Wnt signaling was originally discovered as a key regulator of tissue development and patterning in Drosophila, but more recently has been shown to play a role in several human cancers (Morin, 1999). Indeed, CRT is associated with cell proliferation, survival, potency, and maintenance of undifferentiated phenotypes (Korinek et al., 1998; Zhu and Watt, 1999; Orford et al., 1999; Ross et al., 2000). Importantly, however, implications of its involvement in tissue differentiation indicate that various inputs (molecular, temporal, morphological) shape the ultimate outcome of CRT signaling (DasGupta and Fuchs, 1999; Park et al., 1999; Hsu et al., 2001). Although wnt factor over-expression is not clearly associated with tumorigenesis in humans, aberrant signaling can result from changes in β-catenin turnover. Mutation of the adenomatous polyposis coli (APC) gene product and axin, proteins required for GSK-3β recognition and phosphorylation of β-catenin, lead to β-catenin stabilization (Korinek et al., 1997; Satoh et al., 2000). Similarly, direct mutation of β-catenin at Ser/Thr phosphorylation sites, or neighboring residues, results in its accumulation (Morin et al., 1997). Likely, β-catenin hyperactivation can result from alterations in other pathway regulators, such as the FRAT family of proteins and the F-box protein β-TrCP (Yost et al., 1998; Hart et al., 1999). One hallmark of β-catenin signaling in both normal and neoplastic tissue is nuclear staining. However, the effects thereof may vary due to differential expression of necessary cofactors and potential cross-regulation by other pathways.
Central to processes of prostate growth, differentiation, and tumorigenesis is androgen receptor (AR) signaling (Culig et al., 1998; Cude et al., 1999). Current models place inactive AR in the cytosol where, upon binding its ligand dihydrotestosterone, it undergoes a conformational change accompanied by nuclear translocation. In a homodimeric fashion, ligand-bound AR binds specific DNA elements in target gene promoter and enhancer regions, upregulating expression via recruitment of other transcription factors (e.g. p160 proteins) to its amino terminus (Cude et al., 1999). Target genes include those related to both cell cycle progression (CDK2) and secretory function (kallikrein expression). Little is known to explain the functional dichotomy that exists with AR stimulation of cellular proliferation and transformation versus quiescence and differentiation.
At present, blockade of AR activity through androgen ablation is one of the only effective treatments for managing advanced CaP. As relapse to androgen-independent disease is very common, attention has focused on coincident changes in AR expression and function. Several mechanisms, all of which are founded on resumption of AR signaling, have been proposed to explain progression to androgen-independence. Missense mutations in the ligand binding domain can impart promiscuous behavior to AR, often resulting in its nuclear induction by female steroids and certain anti-androgens used for ablative therapy (Cude et al., 1999). Such mutations, and increased AR levels resulting from gene amplification (Linja et al., 2001), may restore androgen sensitivity to metastatic prostate tumor cells in an otherwise androgen-scarce milieu. Phosphorylation of AR may result in ligand-independent signaling, essentially bypassing the classical mode of activation. Extracellular signaling factors, such as insulin-like growth factor-I (IGF-I) and interleukin-6 (IL-6), have been clinically and functionally linked to this form of AR modification in cancer (Culig et al., 1994; Lin et al., 2001). Likewise, intracellular signaling mediated by protein kinase A (PKA) and Akt have been shown to upregulate ligand-independent AR activity in this fashion (Sadar, 1999; Wen et al., 2000).
Distinguishing between the effects of signaling pathways on CaP progression mediated by either AR-dependent or AR-independent mechanisms is of great importance for future therapy design. It is possible that some forms of AR-independent signaling elicit the same response as an AR-dependent signal (e.g. gene activity, growth phenotype). Such redundant downstream events may render androgen ablative therapies more tenuous, and therefore more focus should be drawn to inhibition of upstream oncogenic activities. For example, are the oncogenic effects of PTEN inactivation derived from consequent Akt-mediated AR phosphorylation, or rather from Akt phosphorylation of other targets leading to increased cell survival? With these ideas in mind, we wished to examine facets of β-catenin signaling in CaP cells to evaluate its contribution to disease progression in vivo. Results shown here, along with previous work (Chesire et al., 2000), directly implicate β-catenin as a potential nuclear signaling factor in both neoplastic and rapidly dividing normal prostate epithelium. The function of β-catenin as a transactivator of TCF target gene activity to drive cell proliferation is well established. However, more recent evidence points to a role for β-catenin in ligand-dependent co-stimulation of AR activity in prostate cells (Truica et al., 2000). Further understanding of these two modes of β-catenin signaling in CaP cells may not only lend support to existing models of tumorigenesis, but also reveal previously unrecognized strategies for growth selection.
Nuclear localization of β-catenin in prostate cells
Previously, we had performed a mutation screen of β-catenin exon 3 and found activating mutations in primary cases (∼5%) and one metastatic case (Chesire et al., 2000). Immunostaining of these tumors demonstrated nuclear localization of β-catenin, suggesting that these mutations imparted greater signaling and CRT. Despite having found only one advanced case with mutation and positive nuclear staining (A17), other data revealing nuclear staining in melanoma tumors wild type for β-catenin prompted our further inquiry into the potential role of β-catenin in advanced CaP (Rimm et al., 1999). To this end, we performed immunohistological analysis on our entire panel of metastatic tumors obtained at autopsy from individuals who failed hormonal ablation therapy. In addition to case A17, four other cases (A2, A5, A11, A15; Figure 1) were positive for nuclear localization, resulting in a positivity rate of ∼24% (5/21). In these cases, tumors at other metastatic sites likewise contained positive nuclei. Nuclear staining was heterogeneous, suggesting that β-catenin signaling may be subject to multiple inputs despite potential pathway aberrations. Additional mutation analysis of genomic DNA by direct sequencing of PCR products ruled out activating mutations in exon 3 as accounting for the observed nuclear β-catenin expression. Since amplified fragments were of the predicted size (∼950 bp, primers located in exons 2 and 4), we concluded that no large interstitial deletions had occurred which would have precluded successful sequencing of all amplified fragments with internal primers.
β-catenin signaling plays a key role in tissue growth and homeostasis through maintenance of proliferative potential in stem cell compartments (Korinek et al., 1998; Zhu and Watt, 1999). Because of these observations and our discovery of putative CRT upregulation in both primary and advanced stages of CaP, we investigated β-catenin activity in normal prostate growth. To address this question, we took advantage of the well characterized model of involution and regrowth of the rat ventral prostate (Sandford et al., 1984; Kerr and Searle, 1973). Using this model, adult rats were castrated to commence a 2 week period of prostate involution followed by testosterone-induced gland restoration. As expected, glands from intact animals contained tall columnar epithelia while glands of castrated animals exhibited marked atrophy (Figure 2a). Although β-catenin was localized predominantly to cell/cell borders in glands of intact animals, it was distributed between both cell/cell borders and cytoplasm in glands of castrated animals. Most interestingly, β-catenin displayed nuclear localization in glands of testosterone-restored animals (>=24 h treatment). This staining profile paralled increased Ki67 staining, suggesting that β-catenin may be involved in androgen-induced prostate regrowth (Figure 2a). Androgen receptor was markedly upregulated by 12 h post testosterone treatment (data not shown).
To better understand the mechanism of androgen-induced β-catenin upregulation in vivo, we examined the effects of androgen on β-catenin metabolism in AR-positive CaP cell lines (CWR22-Rv1, LAPC-4) by immunofluorescence microscopy (Figure 2b). As expected, androgen treatment elicited increased AR nuclear accumulation, consistent with our observation of androgen-induced promoter upregulation (see Figure 5a,b), but did not alter β-catenin localization. By Western blot analysis, we observed ready induction of PSA, but not of β-catenin, with extended androgen treatment of the AR-positive cell line LNCaP (Figure 2c). These results (Figure 2b,c) likely indicate an indirect effect of androgen on β-catenin localization in prostate epithelial cells in vivo.
β-catenin signaling through TCF transactivation
Our observations of nuclear β-catenin in prostate growth and carcinoma implicate its signaling as necessary for prostate cell growth and/or survival. To better understand the mode(s) of signaling β-catenin orchestrates, we analysed its activity in the contexts of either TCF- or AR-based target gene expression (Morin et al., 1997; Truica et al., 2000). Certain CaP cell lines are capable of supporting β-catenin nuclear translocation and TCF-based CRT, as shown by immunofluorescence and reporter studies (Chesire et al., 2000). We determined the fold activation of a synthetic β-catenin/TCF-responsive luciferase reporter (pOT) upon ectopic expression of wild type or mutant (Del, Δ24-47) β-catenin in a broader array of CaP cell lines (Figure 3a). As expected from previous work, the mutant form was more potent in reporter activation compared to wild type protein, suggesting that mechanisms of β-catenin downregulation are intact in these cells.
Others have shown that the cyclin D1 promoter contains TCF-binding elements that endow its partial regulation by β-catenin (Tetsu and McCormick, 1999). Similar to results obtained with the artificial promoter construct (pOT), our experiments demonstrate cyclin D1 promoter (−1009 bp) stimulation by β-catenin (data not shown), further arguing that wnt pathway genes can be targeted in CaP.
The variation in induction among the cell lines may result from involvement of β-catenin in alternative pathways or from differences in requisite signaling molecules. Despite reports of TCF4 expression being restricted to gastrointestinal and mammary tissues (Barker et al., 1999), we have observed its expression in a panel of normal and neoplastic specimens by cDNA microarray analysis (Luo et al., 2001). Therefore, we examined properties of TCF4 expression in CaP cell lines, as such may greatly impact CRT. Western blot analysis demonstrated moderate expression of TCF4 in TSU, DU145, PC-3, CWR22-Rv1, and LAPC-4 cells (Figure 3b, data not shown). However, TCF4 expression in LNCaP cells was low, perhaps explaining their poor β-catenin transactivation potential. We observed two TCF4 isoforms (∼86 and 64 kD), perhaps resulting from either alternative splicing events (Roose and Clevers, 1999; Duval et al., 2000) or from post-translational modification (Ishitani et al., 1999; Sachdev et al., 2001). The latter possibility seems more plausible, since the ORF of the longest TCF4 splice form in the GenBank database (accession CAA72166) encodes a peptide of ∼68 kD, far short of 86 kD. Calf intestinal phosphatase treatment (CIAP) of immunoprecipitates verified that TCF4 is phosphorylated, but does not account for the molecular weight difference between the two isoforms (Figure 3c). This observation is interesting, as the only known mechanism of TCF4 phosphorylation, and its consequent inhibition, is through MAP kinase signaling to TGF-β-activated kinase (TAK) (Ishitani et al., 1999). Sachdev et al. (2001) have recently shown that lymphoid enhancer factor-1 (LEF-1), a TCF4 homologue, is inhibited by covalent attachment of small ubiquitin-related modifier (SUMO) proteins. Given that both TCF4 and LEF-1 share the same two sumoylation sites, and that SUMO protein (∼11–12 kD) addition would account for the molecular weight difference between the TCF4 isoforms, there is a possibility that TCF4 is SUMO-modified. In any case, it is important to note that SW480 (APC−/−) colon cancer cells, which harbor constitutively high levels of TCF-based CRT (Korinek et al., 1997), display the same TCF4 expression profile as CaP cells (Figure 3c, data not shown). Since TCF4 is the only TCF family member appreciably expressed in SW480 cells (Korinek et al., 1997), we infer that the 86 kD TCF4 isoform in CaP cells likely contributes significantly to CRT.
These cells exhibit low basal β-catenin/TCF4 interaction (Figure 3d, columns 2, 6, 10 and 14), consistent with our observation of low basal nuclear activity (Figure 3a, pOT + pCDNA only). Ectopic expression of mutant β-catenin (Del) led to increased TCF4 (86 kD) co-precipitation with β-catenin antibody (Figure 3d, columns 4, 8 and 12). Although difficult to detect, a very small amount of TCF4 (86 kD) was pulled down with β-catenin antibody in LNCaP-del-β-cat clone 12 (Figure 3d, column 16). We repeatedly observed somewhat lower levels of TCF4 (86 kD) precipitating with β-catenin in PC-3 (Figure 3d, column 12) compared to that in TSU and DU145 (columns 4 and 8, respectively). Perhaps this discrepancy affects β-catenin transactivation potential in PC-3 cells, but cannot itself explain their unresponsiveness to exogenous β-catenin. We are currently investigating expression of other TCF family members, as they could potentially shape β-catenin signaling. For example, we have found little or no expression of LEF-1 in all cell lines compared to that of TCF4 (data not shown).
The above variation in signaling may result from differential expression of other factors required for β-catenin transactivation. The chromatin remodeling protein, Creb binding protein (CBP), is recruited by β-catenin to upregulate target gene expression in mammalian cells (Takemaru and Moon, 2000). Interestingly, we found that DU145 cells express low levels of CBP (Figure 3b), possibly explaining their modest induction.
Cross-regulation of β-catenin signal transduction
Since β-catenin nuclear localization and its consequent effect on gene expression may be selected in a subset of CaPs (Figure 1) (Chesire et al., 2000), we were interested in whether or not other pathways commonly altered in this disease affect β-catenin signaling. E-cadherin (E-cad) expression is often downregulated in prostate tumorigenesis, yielding a more invasive phenotype (Umbas et al., 1992). E-cad-mediated cell/cell adhesion potentially modulates CRT by complexing β-catenin at the cell border (Sadot et al., 1998). Although this mode of sequestration may account for lower (or total lack of) CRT induction in E-cad positive cells (PC-3, DU145, LNCaP) in comparison to TSU (E-cad negative), it cannot account for the robust CRT induction displayed by E-cad-expressing CWR22-Rv1 and LAPC-4 cells (Figure 3a). These results are in line with our observation of nuclear staining in cells transiently transfected with β-catenin (LAPC-4 and CWR22-Rv1, Figure 2b).
The PTEN tumor suppressor gene is commonly deleted in CaP (Ali et al., 1999). As PTEN is a negative regulator of the phosphoinositide-3-kinase (PI3-K) pathway, it is surmised that downstream effects of PI3-K are important for CaP cell growth and survival. Growth factor signaling induces PI3-K activation, which indirectly upregulates Akt, a serine/threonine kinase that modulates several protein substrates including GSK3-β (Figure 4a) (Cross et al., 1995). Since PTEN inactivation renders enhanced Akt activity in CaP cells (Wu et al., 1998), we wished to test if Akt upregulates β-catenin signaling, presumably through alleviation of GSK3-β suppression. To provide a framework in which to ask this question, we demonstrated that TSU cells in either depleted or serum-free medium had no activated Akt (Pi-473S; Figure 4b, columns 1 and 2), but serum-starved cells treated for 30 min with either fresh medium or 3.5 μM insulin did show activation (Figure 4b, columns 3 and 4). The PI3-K inhibitor Ly294002 (50 μM) substantially dampened Akt activation in cells treated with fresh medium (Figure 4b, column 5), but marginally for cells treated with insulin (Figure 4b, column 6). With this data in mind, we tested the effects of serum-induced PI3-K/Akt activity on TCF-based CRT in TSU and CWR22-Rv1 cells (Figure 4c). As various treatments did not alter basal activity (+pCDNA filler), we examined cells transiently transfected with wild type β-catenin (+WT β-catenin). Ectopic β-catenin expression may yield a more robust read-out for effects of Akt activation on CRT, if any. We observed no real change in signaling with addition of fresh medium on cells in depleted medium (Figure 4c, compare columns 1 with 5). Indeed, the trend here hints of signaling inhibition, which is opposite of what would be predicted with serum-induced Akt activation. Inhibition of the PI3-K pathway with either PTEN (WT) over-expression or Ly294002 did not cause a significant drop in CRT (Figure 4c; compare column 1 with 2 and 4, 5 with 6 and 8). Finally, despite its reported ability to inhibit wild type PTEN in a dominant-negative fashion, over-expression of inactivated PTEN (C124S) did not elicit an increase in CRT (Figure 4c; compare columns 1 with 3, 5 with 7) (Wu et al., 1998). These data, along with evidence that biochemical compartmentalization of PI3-K and wnt stimulatory events renders pathway-specific GSK-3β downregulation (Ding et al., 2000), suggest that these pathways elicit distinct outcomes. It is interesting that fresh serum did not stimulate CRT, given that growth factor-induced tyrosine phosphorylation of junctional β-catenin increases its soluble levels (Playford et al., 2000; Muller et al., 1999).
AR signaling enhancement by β-catenin
Aside from its putative role in upregulating TCF-based gene expression, β-catenin may contribute to prostate growth and tumorigenesis through stimulating other pathways. β-Catenin has recently been shown to enhance AR target gene expression in LNCaP and AR-transfected TSU cells, reportedly through its involvement in AR complexes (Truica et al., 2000). Aberrant AR co-activation, along with AR gene amplification and mutation, is theorized as an important event leading to conversion of androgen-dependent disease to the lethal, androgen-independent form (Culig et al., 1998). Since this phenomenon potentially constitutes a major role for β-catenin in CaP, we wished to examine this interaction further. Upon transient expression of β-catenin in the AR-positive cells CWR22-Rv1 and LAPC-4, we observed ligand-dependent enhancement of AR activity on a PSA enhancer/probasin promoter-driven luciferase reporter (pBK-PSE-PB-luc, Figure 5a). This increase was AR-dependent, as no reporter response to androgen was observed in AR-negative cells (TSU and DU145, Figure 5a). Compared to wild type, mutant β-catenin (Del) was more potent in co-activation (CWR22-Rv1, Figure 5a), suggesting that stabilized forms have a greater impact on AR signaling in vivo. Interestingly, we consistently saw that β-catenin-induced augmentation of AR function was greater in CWR22-Rv1 cells compared to LAPC-4 cells. Perhaps the AR mutation in CWR22-Rv1 cells, which may account for their greater fold response to androgen, predisposes to amplified co-activation events. Indeed, this mutation, although present in the ligand binding domain, is theorized to enhance recruitment of auxiliary transcription factors to the AR amino terminus (McDonald et al., 2000). On the other hand, expression differences in factors that contribute to AR signaling may exist between CWR22-Rv1 and LAPC-4 cells. As charcoal-stripping may remove other signaling factors besides steroids, it is important to note that fold β-catenin co-activation was not affected when experiments were performed in a complete medium context (data not shown). This observation suggests that the mechanism for β-catenin-mediated AR co-activation is not subject to extracellular signaling events known to alter AR function.
We wished to understand the prevalence of β-catenin co-activation of AR function; is it a global phenomenon, or rather limited to a few particular target promoters? Thus far, the only prostate-specific AR target promoter tested is that for probasin. Therefore, we examined the effects of β-catenin expression on androgen induction of a human kallikrein-2 (HK-2) promoter/enhancer reporter construct. Interestingly, we detected a ligand-independent rise in reporter activity in CWR22-Rv1 cells (Figure 5b). This rise may not require AR, as we observed a ligand-independent increase of reporter function in TSU cells (Figure 5b). However, in dose response to androgen, β-catenin over-expression was associated with a greater incremental rise in R1881-induced activity in CWR22-Rv1 cells. These results are consistent with the proposal that β-catenin affects AR transcriptional activity in a universal fashion. Our observation of ligand-independent HK2 promoter/enhancer stimulation by ectopic β-catenin suggests that certain AR target genes are subject to multiple forms of regulation. As such, constitutive β-catenin nuclear function may sensitize certain target genes to androgen induction.
AR transient expression and activation
We next asked whether or not β-catenin stimulation of AR function could be recapitulated in AR-negative cells. Similar to AR in LNCaP, CWR22-Rv1, and LAPC-4 cells, we saw that transiently expressed wild type human AR in TSU and DU145 cells accrued upon androgen stimulation (Figure 6a, data not shown). By luciferase assay, we demonstrated that ectopic expression of AR permitted androgen induction of pBK-PSE-PB-luc reporter activity (Figure 6b, AR + pCDNA). In contrast to the AR-positive cells already tested, we did not observe AR enhancement with co-expression of mutant β-catenin (Figure 6b, AR + Del). Comparable results were also obtained with the AR-negative cell line HEK-293 (data not shown). Although TSU and DU145 cells manifest androgen sensitivity upon transient AR expression, these cells might have lost those features required for β-catenin co-activation during the course of their original transformation. Our results obtained with TSU contrast with those of Truica et al. (2000), potentially highlighting the intrinsic difficulties of studying complex signaling events.
Proliferation of cells stably expressing mutant β-catenin
Thus far, we have demonstrated that β-catenin signaling, whether via TCF- or AR-based mechanisms, can occur in cultured CaP cells. Given our observations of nuclear β-catenin in prostate tumors and in growing prostate, we wished to test the effects of β-catenin signaling on cell growth. LNCaP cells most likely would not adequately model the effects of constitutive β-catenin/TCF target gene expression (see Figure 3). However, because LNCaP cells do exhibit AR signaling enhancement by β-catenin (Truica et al., 2000), they could potentially model the effect of β-catenin on androgen-regulated growth. We had previously generated stable LNCaP clones that constitutively express mutant β-catenin (Del) (Figure 7a, LNCaP-1, and -12). Interestingly, neither LNCaP-1 or 12 manifests clear nuclear accumulation compared to parental cells or non-expressing clone LNCaP-4 (Figure 7b). Since TCF expression may be a requirement for β-catenin nuclear localization, LNCaP cells may be deficient here due to their low TCF4 levels (Figure 3b). Alternatively, the robust expression of E-cadherin in these cells may abrogate soluble levels of mutant β-catenin. Although this negative staining is in accordance with low TCF-based CRT in LNCaP cells, it is important to bear in mind that positive staining is not requisite for nuclear activity, as HCT-116 colon cancer cells (Δ45S-β-catenin) (Morin et al., 1997) have constitutively high CRT despite absence of observable nuclear staining (Chesire et al., 2000; Shih et al., 2000).
To test the effect of mutant β-catenin expression on LNCaP growth response to androgen, we performed MTS growth assays on cells treated with R1881 (Figure 7c, data not shown). For each dose (0.1, 1, 10 nM), we observed relatively equal growth responses between non-mutant expressers (parental and LNCaP-4 cells) and mutant-expressing clone LNCaP-12. However, mutant-expressing clone LNCaP-1 exhibited reduced growth in comparison. Similar proliferation assays demonstrated that low expressers of mutant β-catenin (clones LNCaP-5 and 8) exhibit the same growth profile as parental cells (data not shown). Altogether, these data suggest that mutant β-catenin expression in LNCaP cells does not alter their growth response to androgen in vitro. An explanation may be that β-catenin enhances only a subset of AR target genes, presumably those not affecting cell growth. Or perhaps a clonal phenomenon exists in which certain positive clones were lost during selection due to unstable growth characteristics. In the absence of R1881, growth of all clones was greatly abrogated (Figure 7d, data not shown), suggesting that mutant β-catenin expression may not affect androgen-independent modes of proliferation in LNCaP cells. Finally, clone growth in complete medium (RPMI/10% FCS) remained unaltered, indicating that factors otherwise absent in charcoal-stripped serum are not required for β-catenin-induced changes in growth (Figure 7e, data not shown).
We have uncovered evidence that β-catenin may demonstrate a more pervasive role in both normal and neoplastic prostate cell physiology than previously recognized. Our immunohistological data predict that processes of β-catenin signaling contribute to prostate cell growth and survival. That nuclear β-catenin can exist under very different physiological contexts (normal and neoplastic tissues) may reflect its eclectic signaling capacity. Therefore, one focus of this report was to further characterize two known modes of β-catenin signaling in CaP, that via TCF or AR, in order to better understand their respective contribution to disease progression. To accomplish this goal, we analysed these signaling events in more cell lines (only a limited number are available to CaP researchers), considered potential cross-regulatory effects of a prominent CaP pathway (PI3-K/Akt/PTEN), and tested the mitogenic effects of β-catenin over-expression.
Our study is the first to demonstrate positive nuclear β-catenin staining in metastatic CaP, linking the wnt pathway to androgen deprivation therapy failure and consequent lethality. Previously, β-catenin was shown to contain putative activating mutations in approximately 5–7% of all CaPs and to exhibit nuclear localization therein (Chesire et al., 2000; Voeller et al., 1998). The mutations were discovered almost exclusively in primary lesions, except for a single metastatic case in which the mutation was likely derived from the primary tumor. Despite absence of detectable mutations in our panel of metastatic cases (except A17), we found nuclear localization of β-catenin at an unexpected rate of 20%. This result is not unprecedented, as others have observed nuclear staining in cancers bearing no activating mutations in β-catenin exon 3 (Rimm et al., 1999). Little evidence for APC mutation in CaP exists, suggesting that alteration of other pathway components (e.g. axin, β-TrCP) and heretofore uncharacterized interacting factors may account for our observation. We propose that, given the putative discrepancy in the molecular etiology of β-catenin upregulation between primary and secondary CaPs, β-catenin may mediate distinct modes of signaling at different stages of tumor progression.
The finding of nuclear β-catenin at any stage of CaP tumorigenesis may link its signaling function to cell survival and proliferation. That nuclear translocation of β-catenin occurs during androgen-induced regrowth of rat prostate corroborates this notion. The mechanism underlying this phenomenon remains unknown, but may not result from direct effects of androgen on epithelia. Currently, there exists intense interest in defining stromal/epithelial cell interactions, since neoplastic development may rely on certain intercellular signaling networks. From such studies, Planz et al. (1998) proposed that, in response to androgen, AR-positive stromal cells may secrete keratinocyte growth factor (KGF) leading to epithelial cell stimulation. In a similar fashion, androgen may induce stromal cell secretion of wnt factor(s) which presumably would effect β-catenin upregulation in adjacent epithelia. Indeed, we did observe AR-positive stromal cells directly bordering epithelia (data not shown). On a separate note, it will be of interest to understand if β-catenin is involved not only in androgen-induced prostate regrowth, but also in the eventual growth quiescence presumably attained upon complete gland restoration. Underscoring this possibility is work demonstrating β-catenin involvement in growth/differentiation cycles and a nearly exclusive role in the well differentiated, glandular form of gastric cancer (DasGupta and Fuchs, 1999; Hsu et al., 2001; Park et al., 1999; Miyazawa et al., 2000).
Several ‘pathway-specific’ transcription factors are now known to act in multiple, seemingly distinct signaling cascades. For example, the SMAD family of proteins plays a central role in transducing TGF-β signals (Heldin et al., 1997), but recent data demonstrate a broader signaling spectrum ranging from vitamin D receptor and TCF co-activation to AR repression (Yanagisawa et al., 1999; Nishita et al., 2000; Labbe et al., 2000; Hayes et al., 2001). These findings carry substantial ramifications: signaling molecules execute a diverse array of events impacting both physiological and pathological mechanisms. Thus, the discovery of AR stimulation by β-catenin, on top of its already well characterized role in TCF transactivation, piqued our interest. Does β-catenin function in different capacities depending on when and where it signals in prostate (normal vs transformed)? In order to answer this question in a meaningful way, we further characterized these two modes of β-catenin signaling in widely utilized CaP cell lines.
Based on immunofluorescence work and luciferase reporter assays, we conclude that most CaP cell lines are able to exhibit β-catenin nuclear translocation and subsequent TCF target gene upregulation. Although not described in this report, the cyclin D1 promoter (−1009) manifested a similar response to ectopic β-catenin as that observed with the artificial promoter. Therefore, we postulate that nuclear β-catenin may orchestrate expression of known TCF target genes in prostate cells in vivo, prompting cell division and survival. If the predominant function of nuclear β-catenin is to activate such gene expression, then it may feed into alternate physiologic pathways depending on tumor stage. For example, potential myc upregulation by β-catenin in low grade tumors may usually trigger p53-dependent apoptosis, yet may drive proliferation in more advanced tumors as already suggested by studies of myc gene amplification (Bubendorf et al., 1999). Such context-dependent events may explain the disparate rates of putative β-catenin activation between localized and metastatic disease.
Certain pathways independent of wnt may impact on β-catenin metabolism leading to increased target gene activity (Yost et al., 1998; Muller et al., 1999; Playford et al., 2000). Inactivation of the PTEN tumor suppressor protein occurs frequently during CaP pathogenesis by chromosomal deletion and/or direct mutation (Ali et al., 1999). Wu et al. (1998) have shown that, in prostate cells, loss of PTEN activity results in unchecked Akt activity. Despite the attractive hypothesis of β-catenin upregulation by Akt-mediated GSK-3β inhibition, our direct analysis indicates that these two pathways may be distinct from one another. Recent evidence supports this conclusion (Ding et al., 2000), thus arguing for the importance of other Akt-mediated events in CaP such as AR phosphorylation (Wen et al., 2000).
Investigators over the last decade have recognized the necessity to regard cancers as tissue-specific diseases, thus bearing unique pathways of growth selection. Perhaps the finding of ligand-dependent AR stimulation by β-catenin reflects this assessment (Truica et al., 2000). We have extended this body of work by demonstrating the same activity in CWR22-Rv1 (mutant AR) and LAPC-4 (wild type AR) cell lines, and that it likely functions globally on AR target promoters. However, we were unable to recapitulate AR co-activation by β-catenin in AR-negative cell lines. Although not described in this study, we were also unable to show specific β-catenin/AR interaction by IP analysis utilizing various antibody and detergent combinations, including those detailed by Truica et al. (2000). In spite of these observations, multiple mechanisms may account for this novel form of AR stimulation. For example, β-catenin signaling via another DNA binding factor (e.g. TCFs) may heighten expression of a factor(s) capable of the observed activity. As myc has been shown to play an intimate role in expression of the AR gene itself (Grad et al., 1999), its induction by β-catenin/TCF may impact on AR protein output. However, this idea is inconsistent with the observed lack of β-catenin/TCF signaling in LNCaP cells, given their reported ability to support β-catenin-enhanced AR function (Truica et al., 2000). The apparent conflict of our results with those of Truica et al. (2000), along with the implications posed by a report showing β-catenin co-activation of another nuclear receptor (RAR) (Easwaran et al., 1999), mandates further inquiry of this potentially significant mode of AR pathway cross-regulation.
The observation that β-catenin augments AR signaling in an androgen-dependent manner has important consequences with respect to interpreting nuclear β-catenin function in vivo. For example, under conditions imposed by androgen ablation therapy of metastatic disease, AR enhancement by β-catenin may not occur. Although upregulated β-catenin was shown to mollify the inhibitory effects of the anti-androgen bicalutamide on AR activity and to increase such activity elicited by an adrenal androgen (Truica et al., 2000), β-catenin's contribution to metastatic tumors may lie solely in TCF target gene transactivation. This idea that alternate pathways can successfully substitute the growth/survival cues of AR signaling is plausible, as a certain percentage of advanced cases exhibit heterogeneous or even total absence of AR expression (Kinoshita et al., 2000). In considering normal prostate and primary cancer, a likewise complex, but different set of events may govern the modes and consequences (proliferation versus differentiation) of β-catenin signaling. To conclude, we have shown positive nuclear β-catenin staining in normally proliferating prostate and 20–25% of androgen-independent CaP metastases. In addition, we have examined the signaling qualities of β-catenin on TCF- and AR-dependent transcription in prostate cells. Future tumorigenesis and transgenesis experiments should further elucidate the potentially complex role of β-catenin signaling in various contexts of prostate cell physiology.
Materials and methods
Cell lines, human tumor specimens, plasmids and antibodies
Cells were acquired from the American Type Culture Collection (Manassas, VA, USA; DU145, PC-3, LNCaP, SW480) and from Dr John Isaacs (Johns Hopkins University; TSU, CWR22-Rv1, LAPC-4) and maintained at 37°C/5% CO2 in RPMI/10% FCS (Life Technologies, Grand Island, NY, USA) or Iscove's Modified DMEM/10% FCS (LAPC-4). CWR22-Rv1 and LNCaP cells express AR that is mutated in the ligand binding domain (McDonald et al., 2000), whereas LAPC-4 expresses wild type AR (Klein et al., 1997). TSU, DU145, and PC-3 cells are highly proliferative, AR-negative prostate cancer cells. It is important to note here that, during preparation of this manuscript, a report placing doubt on the origin of the TSU cell line was published (van Bokhoven et al., 2001). Synthetic androgen (R1881) and PI3-K inhibitor Ly294002 were purchased from New England Nuclear (Boston, MA, USA) and Calbiochem (La Jolla, CA, USA), respectively. Insulin was purchased from Life Technologies. In all androgen treatment procedures, cells were incubated in media containing 10% charcoal-stripped FCS (CS-FCS, Cocalico Biologicals, Reamstown, PA, USA) for 48 h prior to addition of the same media (fresh) including R1881.
Metastatic tumors were removed from various anatomical sites of autopsy cases and fixed in phosphate-buffered formalin. The deceased patients had failed hormonal ablation therapy and therefore these tumors represent the lethal, androgen-independent stage of advanced disease.
Control plasmids pGL3-Basic, pGL3-Control, pRL-CMV were from Promega (Madison, WI, USA) and utilized for both luciferase assay optimization and normalization (see below). The wild type β-catenin expression vector (pCI-wt-β-catenin) and β-catenin/TCF reporter constructs (pGL3-OT, pGL3-OF) were provided by Dr Ken Kinzler (Johns Hopkins University). pGL3-OT (herein referred to as pOT) consists of three optimal TCF binding sites upstream a basic promoter (TATA box) that drives firefly luciferase expression. The β-catenin/TCF control reporter, pGL3-OF, which is comprised of pOT with mutated TCF binding sites, was used to judge background levels (data not shown). pCI-Del-β-catenin was used for expression of a mutant form (Δ24–47) of β-catenin that lacks the GSK-3β phosphorylation target sites and exhibits constitutive nuclear activity (Chesire et al., 2000). PTEN expression vectors (pCDNA-PTEN WT and C134S) were furnished by Dr Charles Sawyers (UCLA). Human AR expression was achieved with use of pCDNA-hAR. Briefly, a 3.2 kb fragment encoding hAR cDNA (Luke and Coffey, 1994) was cloned into the multiple cloning site (NheI/BamHI) of pCDNA 3.1(−). Empty pCDNA 3.1(−) was purchased from InVitrogen (Carlsbad, CA, USA) and used to compensate for unequal CMV promoter levels in luciferase assays. Androgen-responsive firefly luciferase reporter constructs (pBK-PSE-PB-Luc, pBK-HK-2-enhancer/promoter-Luc) were provided by Dr Ron Rodriguez (Johns Hopkins University).
Antibodies were obtained from Transduction Laboratories (Lexington, KY, USA; β-catenin), Pharmingen (San Diego, CA, USA; AR), DAKO (Carpinteria, CA, USA; PSA), Calbiochem (α-tubulin), Upstate Biotechnology (Lake Placid, NY, USA; TCF4, CBP), New England BioLabs (Beverly, MA, USA; Akt, Phospho-Ser474-Akt), Zymed (South San Francisco, CA, USA; Ki67), Jackson ImmunoResearch Laboratories (West Grove, PA, USA; rhodamine-conjugated donkey anti-mouse IgG), and Pierce (Rockford, IL, USA; HRP-conjugated goat anti-mouse and anti-rabbit IgG).
Rat prostate regression and restoration
Male Copenhagen rats were obtained from Harlan Sprague Dawley (Indianapolis, IN, USA) and castrated as previously reported (Kyprianou and Isaacs, 1987). Prostate involution, a consequence of both glandular cell apoptosis and atrophy (Kerr and Searle, 1973), was allowed to progress for 2 weeks at which time testosterone-releasing implants were inserted subcutaneously (Kyprianou and Isaacs, 1987). Time points of 0, 12, 24, 36, 48, 60 and 72 h were taken post testosterone restoration upon which pairs of animals were terminated and prostates removed. Tissue was fixed in phosphate-buffered formalin.
Immunohistochemistry and immunocytochemistry
Formalin-fixed tissues were embedded in paraffin and sectioned at 5 microns. Slides deparaffinized in xylene (twice for 5 min) were hydrated through a graded ethanol series (2×absolute, 2×95%, 1×70% dipped until clear) and placed in deionized water followed by 1% Tween-20 (Sigma Chemical, St. Louis, MO, USA). Heat induced epitope retrieval (HIER) was utilized in unmasking antigen sites with the use of citrate buffer (Vector, Burlingame, CA, USA). Slides were paired to form a capillary gap and placed into a steaming basket for 14 min. Slides were then air dried for 5 min and placed into PBS with Tween (PBST, Sigma). The detection of β-catenin and Ki67 was performed with the EnVision Plus System (DAKO) for monoclonal antibodies. The EnVision System provides biotin-free detection that utilizes an HRP labeled polymer eliminating the staining of endogenous biotin. Slides were incubated with hydrogen peroxide according to the EnVision kit instructions and blotted dry. Antibodies for β-catenin and Ki67, diluted 1 : 500 and 1 : 50, respectively, were incubated on slides for 30 min at room temperature. The slides were then rinsed with Tris buffered saline with Tween-20 (TBST, Sigma) and incubated with the HRP conjugated secondary anti-mouse IgG for 30 min. As per the EnVision kit protocol, the chromagen diaminobenzidine was incubated on slides for 5 min. Slides were then rinsed in TBST, counterstained with hematoxylin, dehydrated through graded alcohols, cleared in xylene, and mounted with Cytoseal 60 mounting media (Stephens Scientific, Riverdale, NJ, USA).
Immunocytochemistry was performed essentially as reported (Chesire et al., 2000). Diluted antibodies (PBS/3.75% BSA) were incubated with fixed and permeabilized cells on two-chamber glass slides (Nunc, Naperville, IL, USA) at 1 : 250 (β-catenin) and 1 : 100 (AR). Slides were then treated with rhodamine-conjugated anti-mouse IgG (1 : 400), washed, mounted, and viewed under fluorescence microscopy (AxioScope, Carl Zeiss, Thornwood, NY, USA). Images were documented using IP-Lab image-capturing software. For both staining procedures, non-specific background was monitored by treating specimens with secondary antibody only.
Immunoprecipitation, dephosphorylation and Western blot analyses
Cell lysates were prepared from subconfluent cultures by first washing cells twice with cold PBS and then extracting with either RIPA (50 mM Tris 7.4, 0.5% DOC, 0.1% SDS, 1.0% NP-40, 150 mM NaCl, 1 mM EDTA) or NP-40 (50 mM Tris 7.4, 1.0% NP-40, 150 mM NaCl, 1 mM EDTA) detergent solutions. Protein concentrations of lysates were determined using a BCA kit (Pierce). All cell lysates and IP mixes contained a protease inhibitor cocktail obtained from Roche (Indianapolis, IN, USA). For IP analysis, equal amounts of protein in 300 μl total volume were incubated with antibody (concentrations: β-catenin, 1.5 μg/ml; TCF4, 3 μg/ml) or mouse serum (1 : 1000, Sigma) for at least 4 h at 4°C. RIPA buffer was used in IP of TCF4 for the dephosphorylation study (Figure 3c), whereas NP-40 buffer was used for TCF4/ β-catenin co-immunoprecipitation (Figure 3d). Immuno-complexes were pulled down by addition of 75 μl buffer-washed protein G+/A agarose beads (Oncogene Research Products, Cambridge, MA, USA), incubated at 4°C for at least 2 h, and washed four times in the appropriate extraction buffer. Prior to gel loading, SDS–PAGE gel loading buffer (Sambrook et al., 1989) was added to the beads and heated at 95°C for 4 min.
For dephosphorylation assay, TCF4 immunoprecipitates (complexes on beads) were washed as above and then three times in CIAP reaction buffer (50 mM Tris 8.5, 0.1 mM EDTA). After washing, beads were resuspended in 170 μl of the same buffer to which 25 μl CIAP (1 u/μl, Roche) was added as appropriate. All reactions (+/− CIAP) were incubated at 37°C for 1.5 h after which the complexes were washed twice with RIPA buffer and prepared for gel loading.
Techniques for running SDS–PAGE and Western blots were as described (Sambrook et al., 1989). Pre-stained molecular weight standards were purchased from Bio-Rad (Hercules, CA, USA). Upon completion of electrophoresis, proteins were transferred by Western blot to ECL nitrocellulose (Amersham-Pharmacia, Piscataway, NJ, USA) and blocked in 5% non fat-dried milk/PBS/0.1% Tween-20. Membranes were probed in blocking buffer with antibodies at 0.5 μg/ml, washed, and probed with HRP-conjugated secondary antibody (anti-IgG). For appropriate blots, α-tubulin levels were also determined to verify equal loading. Enhanced chemiluminescence was performed with the ECL kit (Amersham-Pharmacia) followed by autoradiography.
Transfection, luciferase and proliferation assays
For luciferase assay experiments, cells were plated at medium density (10 000–20 000 cells) to clear bottom, opaque-walled 96 well plates (Isoplate TC, Perkin Elmer Wallac, Gaithersburg, MD, USA). With those experiments involving androgen treatment, cells were given media/10% CS-FCS 24 h after plating (day 2), transfected (day 3), treated with R1881 (day 4), and analysed for luciferase activity (day 5). Transfections were performed using Fugene 6 Transfection Reagent (Roche) at 1 μl/0.2 μg DNA. On transfection day, media was replaced with 25 μl of the appropriate media, to which was added DNA/Fugene complexes in a total volume of ∼25 μl. Complexes were prepared such that every well received 50 ng reporter plasmid (encoding firefly luciferase), 50 ng each appropriate expression plasmid and 10 ng pRL-CMV. To compensate for unequal DNA amounts between certain transfection groups, pCDNA 3.1 was included as appropriate. pRL-CMV, which encodes Renilla luciferase, was included in all mixes to allow for transfection normalization. For transient transfection with pCI-Del-β-catenin (Figure 3d) and pCDNA-hAR (Figure 6a), cells were transfected in 10 cm and six-well plates (Falcon, Franklin Lakes, NJ, USA), respectively. Media was replaced and cell lysates were prepared 1 and 2 days post transfection, respectively.
Luciferase assays were performed essentially as described using non-proprietary substrate/buffer mixes (Dyer et al., 2000). The substrates for firefly and Renilla luciferase enzymes, luciferin and coelenterazine, respectively, were purchased from Biosynth AG (Staad, Switzerland). Cells were washed once in 100 μl PBS and then lysed in 30 μl 1×Passive Lysis Buffer (Promega), which permits optimal enzyme activity. Using the Wallac 1450 Microbeta Jet luminescence reader, firefly and Renilla luciferase substrate mixes (100 μl) were injected sequentially, allowing 10 s activity readout for each enzyme. In this dual luciferase assay, firefly luciferase activity is first monitored under basic conditions. Then, an acidic, high salt buffer is added that abolishes firefly luciferase function, but permits Renilla luciferase activity. All experiments were repeated at least three times, and all samples were performed in quadruplicate. For normalizing each well, the firefly luciferase value was divided by the Renilla luciferase value.
Cell proliferation assays were performed with the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega), which utilizes MTS instead of MTT. The number of live cells is proportional to the level of formazan-like compound produced from reduction of MTS by NAD(P)H. LNCaP cells (5000) were plated in clear 96-well plates (Falcon). Media was replaced 24 h later with that containing 10% CS-FCS and incubated with cells for 2 days. Then, media containing R1881 was given to the cells (100 μl) to commence androgen treatment. Measurements of relative cell number were taken by adding MTS reagent to wells, incubating cells for 1 h at 37°C, and reading absorbance at 490 nm. Day 0 measurements were taken immediately following R1881 addition (single plate), and all measurements thereafter were taken at 24 or 48 h intervals (one plate for each day). No obvious cell death occurred throughout these experiments, implying that our analysis did not underestimate proliferation. These assays were duplicated and all samples were performed in 6–7 wells each.
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This study was supported by SPORE grant CA58236 from the National Cancer Institute. We thank Dr John Isaacs (JHU), Dr Ken Kinzler (JHU), Dr Ron Rodriguez (JHU), and Dr Charles Sawyers (UCLA) for supplying reagents used in this study.
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