The results of these studies showed that the AR-negative PCa cell line MDA PCa 118b expresses D32G-mutant beta-catenin, which results at least partially in nuclear beta-catenin accumulation and active beta-catenin–TCF signaling. We also showed—for the first time, to our knowledge—that D32G-mutant beta-catenin induces expression of HAS2, a membrane-bound synthase of HA, a major constituent of the ECM (32
). Our results concur with those previously published that beta-catenin stimulates HA production and also lend support to previous observations that the transforming effects of beta-catenin depend on HA production and HA–cell interactions (33
). Earlier studies also showed that increased HA production promotes anchorage-independent growth and cell invasiveness and that HA expression in PCa biopsy specimens correlates with biochemical recurrence after radical prostatectomy (29
). Our findings add to these by suggesting that beta-catenin–induced HAS2 plays an important role in the progression of PCa.
Abundant evidence also exists showing that activation of downstream target genes by beta-catenin–TCF complex leads to increased proliferation and decreased differentiation of epithelial cells (34
). Exon 3 is the hotspot for mutation in various malignant human tumors because it contains not only the consensus sequences for multiple kinases but also a ubiquitination consensus sequence. At that exon, the most common mutations occur at serine and threonine residues Ser37, Thr41, and Ser45. Additionally, the nonphosphorylatable residues Asp32 and Gly34 rank within the top 6 mutations identified in human tumors: beta-catenin residues in the destruction motif Asp32, Ser33, Gly34, and Ser37 directly bind beta-TrCP1 (35
), and mutation of Asp32 greatly reduces the ability of beta-catenin to be ubiquitinated by beta-TrCP1 without altering the ability to act as a substrate for GSK3b (36
). Mutated Asp32 also mediates a transforming phenotype for growth and migration in the stable nontransformed epithelial cell line MDCK (23
Further, the Wnt signaling pathway has previously been implicated in the progression of PCa (19
). Various of the amino acid–substitution mutations located at exon 3 of beta-catenin have been found in about 5% of PCa specimens tested by several groups of investigators (24
). For example, in 1 previous study of a codon-32 mutant of beta-catenin, D32A, in which aspartate is mutated to alanine, expression of the D32A mutant in a nontransformed epithelial cell line increased oncogenic cellular transformation. In addition, a slightly different codon-32 mutant of beta-catenin, D32G, in which aspartate is mutated to glycine, was identified in MDA PCa 118b cells and, as our results have confirmed, that mutation is associated with beta-catenin nuclear accumulation in PCa (24
), suggesting that the Wnt canonical pathway is activated. Thus, although mutations in beta-catenin have been reported in only 5% of cases of PCa, the high transcription activity that results from the D32G mutant is an important tool for identifying downstream target genes in PCa.
Moreover, beta-catenin may act as a coactivator of the AR (18
), and it has been proposed that the AR competes with TCF/LEF for beta-catenin, thus interfering with beta-catenin–TCF/LEF signaling (19
). Our finding an inverse association between beta-catenin nuclear localization and AR expression in human PCa bone metastases supports the concept that the beta-catenin:AR ratio may help to identify different subpopulations of patients with PCa who may require different management of the disease.
It has also been reported that beta-catenin–LEF-1 signaling is activated after epithelial-to-mesenchymal transition (EMT; thought to mediate invasive and metastatic behavior during cancer progression) and that beta-catenin signaling contributes to the maintenance of EMT (39
). Recent reports indicate that androgens induce EMT in PCa epithelial cells and that expression levels of AR correlate inversely with androgen-mediated EMT in those cells (41
). Thus, low AR content may be required for the EMT phenotype by enabling beta-catenin–LEF-1 signaling. Together, this evidence and our results suggest that activation of beta-catenin by androgen signaling serves as an alternative mechanism of androgen-induced EMT in PCa epithelial cells.
We conclude that our identification of a previously unknown downstream target gene of activated Wnt–beta-catenin, HAS2, combined with high nuclear localization of beta-catenin in PCa cells with little or no AR expression may define a subpopulation of men with bone-metastatic PCa whose disease requires a different form of treatment than those used in other men with PCas. This pattern of findings may thus help clinicians provide indivualized therapy for some men with advanced PCa.