A Human- and Male-Specific Protocadherin that Acts through the Wnt Signaling Pathway to Induce Neuroendocrine Transdifferentiation of Prostate Cancer Cells

Abstract

Protocadherin-PC (PCDH-PC) is a gene on the human Y chromosome that is selectively expressed in apoptosis-and hormone-resistant human prostate cancer cells. The protein encoded by PCDH-PC is cytoplasmically localized and has a small serine-rich domain in its COOH terminus that is homologous to the B-catenin binding site of classical cadherins. Variants of prostate cancer cells that express PCDH-PC have high levels of nuclear B-catenin protein and increased wnt-signaling. In this study, we show that transfection of human prostate cancer cells (LNCaP) with PCDH-PC or culture of these cells in androgen-free medium (a condition that up-regulates PCDH-PC expression) activates wnt signaling as assessed by nuclear accumulation of B-catenin, increased expression of luciferase from a reporter vector promoted by Tcf binding elements and increased expression of wnt target genes. Moreover, LNCaP cells transfected with PCDH-PC or grown in androgen-free medium transdifferentiate to neuroendocrine-like cells marked by elevated expression of neuron-specific enolase and chromogranin-A. Neuroendocrine transdifferentiation was also observed when LNCaP cells were transfected by stabilized B-catenin. Increased wnt signaling and neuroendocrine transdifferentiation of LNCaP cells induced by culture in androgen-free medium was suppressed by short interfering RNAs that target PCDH-PC as well as by dominant-negative Tcf or short interfering RNA against B-catenin, supporting the hypothesis that increased expression of PCDH-PC is driving neuroendocrine transdifferentiation by activating wnt signaling. These findings have significant implications for the process through which prostate cancers progress to hormone resistance in humans. (Cancer Res 2005; 65(12): 5263-71)

Complex regulation of human androgen receptor expression by Wnt signaling in prostate cancer cells

“…A CHIP assay was used to determine whether any of these potential binding elements were occupied by a protein complex that contained b-catenin in control LNCaP cells (transfected with empty vector) or in LNCaP cells with Wnt signaling activated either by transfection with a mutated (stabilized) b-catenin or with protocadherin-PC (PCDH-PC), another gene product known to stimulate LEF-1/TCF-mediated transcription in these cells (Yang et al, 2005). Fixed, sheared chromatin was immunoprecipitated using antib-catenin antibody and the immunoprecipitated chromatin was PCR amplified using primer sets that distinguished the various potential binding sites as described in Figure 1a.…”

“…Mutations in both APC (Watanabe et al, 1996) and b-catenin (Voeller et al, 1998;Chesire et al, 2000) have been described in human PCa specimens, however, their apparent occurrence is at too low a frequency to account for the evidence for more frequent activation of Wnt signaling in this tumor system. Recently, we provided evidence that a novel member of the protocadherin gene family, protocadherin-PC (PCDH-PC) is upregulated in apoptosis-and hormone-resistant human PCa cells and that a major effect of this gene product is the upregulation of Wnt signaling (Yang et al, 2005). In our prior study, Wnt signaling mediated by PCDH-PC expression or by expression of mutated b-catenin was shown to confer neuroendocrine-like characteristics on PCa cells and this phenotype is often described in association with aggressive PCa cells in vivo.…”

“…Cell lines, plasmids and siRNAs LNCaP, CWR22rv-1, PC-3 and DU145 cells were obtained from ATCC and were passaged in medium (for LNCaP, CWR22rv-1 and DU145 RPMI 1640 or for PC-3, F-12k with 10% fetal calf serum and supplements) or androgen-free maintained as previously described (Yang et al, 2005). A defective adenovirus that expresses Wnt-1 protein (Ad-Wnt-1) and control, Lac Z expressing adenovirus (Ad-lac Z) were previously described (Young et al, 1998).…”

“…A defective adenovirus that expresses Wnt-1 protein (Ad-Wnt-1) and control, Lac Z expressing adenovirus (Ad-lac Z) were previously described (Young et al, 1998). These viruses were applied at 20 particles/cell in low serum (2%) medium for 1 h. Expression plasmids containing mutated (stabilized) human b-catenin (Tetsu and McCormick, 1999), dominant negative TCF-4 (Chen et al, 2001) or PCDH-PC cDNA were transfected into cells as previously described (Yang et al, 2005). Small interfering (si) RNAs targeting b-catenin or lamin were purchased from Dharmacon Inc. siRNA targeting human MDM2 was purchased from Qiagen Inc (Valencia, CA).…”

“…Cells were harvested and protein extracts prepared, quantified and used to prepare Western blots as previously described (Yang et al, 2005). Western blots were probed with mouse monoclonal antibodies against human Akt protein, phospho-MDM2 (ser 166), hAR (Santa Cruz Biotechnology Inc., Santa Cruz, CA), actin (Sigma Chemical Co., St Louis, MO) or with rabbit polyclonal antibodies against phospho-Akt (ser 473) or human MDM2 (Cell Signaling Technology, Beverly, MA).…”