Identification of aryl hydrocarbon receptor as a putative Wnt/beta-catenin pathway target gene in prostate cancer cells.

Recent genetic and functional analyses have implicated the wnt/beta-catenin signaling pathway in prostate cancer (CaP) pathogenesis. Thus, there is much interest in understanding the consequences of wnt signaling in CaP; target gene expression is one important area of inquiry and is the focus of this report. Adenoviral-mediated overexpression of a mutant, hyperactive form of beta-catenin in CWR22-Rv1 CaP cells led to increased aryl hydrocarbon receptor (AhR, or dioxin receptor) and transmembrane protein 2 RNA transcript expression, as detected by cDNA-microarray analyses. Validating these results, reverse transcription-PCR assays demonstrated that in CWR22-Rv1 cells as well as in LAPC-4 CaP cells, increased putative target gene RNA expression occurs with transient overexpression of mutant beta-catenin, treatment of cells with lithium chloride, or with wnt3a-conditioned medium, three distinct modes of experimental wnt/beta-catenin pathway activation. This beta-catenin-associated expression of AhR and transmembrane protein 2 does not require de novo protein synthesis and may only involve a certain subset of CaP cell lines. Western and immunofluorescence analyses were undertaken to assess the relationship between the wnt/beta-catenin-stimulated increase in AhR transcripts and AhR protein expression; we provide evidence that an association exists whereby up-regulation of AhR RNA by wnt or beta-catenin is coupled with augmented AhR protein levels. Intriguingly, these studies also demonstrated that nuclear beta-catenin staining may not be a sole deciding factor when predicting the status of wnt/beta-catenin signaling in CaP cells. Finally, the extent to which wnt signaling may synergize with an environmental agonist of AhR (2,3,7,8-tetrachlorodibenzo-p-dioxin) to potentiate AhR transcriptional activity was examined. Considering previous work linking AhR to processes of development and carcinogenesis, our data may highlight one particular role for wnt/beta-catenin signaling in prostate tumor biology.

Ligand-dependent inhibition of beta-catenin/TCF signaling by androgen receptor.

Beta-catenin signaling may contribute to prostate cancer (CaP) progression. Although beta-catenin is known to upregulate T cell factor (TCF) target gene expression in CaP cells, recent evidence demonstrates its capacity to enhance ligand-dependent androgen receptor (AR) function. Thus, we wished to further understand the interaction between these two pathways. We find in both CaP cells (CWR22-Rv1, LAPC-4, DU145) and non-CaP cells (HEK-293, TSU, SW480, HCT-116) that beta-catenin/TCF-related transcription (CRT), as measured by activation of a synthetic promoter and that of cyclin D1, is inhibited by androgen treatment. This inhibition is AR-dependent, as it only occurs in cells expressing AR endogenously or transiently, and is abrogated by AR antagonists. Additional analyses convey that the ligand-dependent nature of CRT suppression depends on transactivation-competent AR in the nucleus, but not on indirect effects stemming from AR target gene expression. Given the recent work identifying an AR/beta-catenin interaction, and from our finding that liganded AR does not prompt gross changes in the constitutive nuclear localization of TCF4 or mutant beta-catenin, we hypothesized that transcription factor (i.e. AR and TCF) competition for beta-catenin recruitment may explain, in part, androgen-induced suppression of CRT. To address this idea, we expressed an AR mutant lacking its DNA-binding domain (DBD). This receptor could not orchestrate ligand-dependent CRT repression, thereby providing support for those recent data implicating the AR DBD/LBD as necessary for beta-catenin interaction. Further supporting this hypothesis, TCF/LEF over-expression counteracts androgen-induced suppression of CRT, and requires beta-catenin binding activity to do so. Interestingly, TCF4 over-expression potently antagonizes AR function; however, this inhibition may occur independently of beta-catenin/TCF4 interaction. These results from TCF4 over-expression analyses, taken together, provide further evidence that AR-mediated suppression of CRT is a consequence of limiting amounts of beta-catenin, and not AR target gene expression. Our analyses point to a reciprocal balance between AR and CRT function that may shape critical processes during normal prostate development and tumor progression.

In vitro evidence for complex modes of nuclear beta-catenin signaling during prostate growth and tumorigenesis.

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 beta-catenin mutation and corresponding nuclear localization in primary lesions. The recent finding of beta-catenin-induced enhancement of androgen receptor (AR) function potentially ties beta-catenin to key regulatory steps of prostate cell growth, differentiation, and transformation. By immunohistological analysis of metastatic tumors, we detected nuclear beta-catenin in 20% of lethal CaP cases, suggesting a more common role for beta-catenin in advanced disease than would be predicted by its mutation rate. Interestingly, beta-catenin nuclear localization was found to occur concomitantly with androgen-induced regrowth of normal rat prostate. These in vivo observations likely implicate beta-catenin involvement in both normal and neoplastic prostate physiology, thus prompting our interest in further characterizing modes of beta-catenin signaling in prostate cells. Extending our previous findings, we demonstrate that transient beta-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 beta-catenin over-expression enhances AR-mediated transcription off two natural target gene promoters. However, we were unable to recapitulate beta-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 beta-catenin did not alter their proliferative response to androgen. In total, our characterization of beta-catenin signaling in CaP reveals the complex nature of its activity in prostate tissue, indicating that beta-catenin potentially contributes to multiple stimulatory inputs required for disease progression.