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

Abstract

beta-Catenin, a component of the Wnt signaling pathway, is a coactivator of human androgen receptor (hAR) transcriptional activity. Here, we show that Wnt signaling also influences androgen-mediated signaling through its ability to regulate hAR mRNA and protein in prostate cancer (PCa) cells. Three functional LEF-1/TCF binding sites lie within the promoter of the hAR gene as shown by CHIP assays that captured beta-catenin-bound chromatin from Wnt-activated LNCaP cells. Chimeric reporter vectors that use the hAR gene promoter to drive luciferase expression confirmed that these LEF-1/TCF binding elements are able to confer robust upregulation of luciferase expression when stimulated by Wnt-1 or by transfection with beta-catenin and that dominant-negative TCF or mutations within the dominant TCF-binding element abrogated the response. Semi-quantitative and real time RT-PCR assays confirmed that Wnt activation upregulates hAR mRNA in PCa cells. In contrast, hAR protein expression was strongly suppressed by Wnt activation. The reduction of hAR protein is consistent with evidence that Wnt signaling increased phosphorylation of Akt and its downstream target, MDM2 that promotes degradation of hAR protein through a proteasomal pathway. These data indicate that the hAR gene is a direct target of LEF-1/TCF transcriptional regulation in PCa cells but also show that the expression of the hAR protein is suppressed by a degradation pathway regulated by cross-talk of Wnt to Akt that is likely mediated by Wnt-directed degradation of the B regulatory subunit of protein phosphatase, PP2A.

Figure 1

A CHIP Assay identifies functional LEF-1/TCF binding sites within the proximal promoter of the hAR gene. (a) Scheme identifies relative sites of potential LEF-1/TCF binding sites within the first 2000 bp 5′ upstream of the start of transcription (TSS) of the hAR gene and sites of primer amplification products used to analyse DNA extracted from immunoprecipitated chromatin from cell specimens. (b) Ethidium bromide-stained agarose gel profiles of PCR reaction products from input control DNA (In), β-catenin antibody immunoprecipitated control transfected (empty vector) LNCaP cell chromatin DNA (Con), β-catenin transfected LNCaP cell DNA (Cat) or PCDH-PC transfected LNCaP cell DNA (PCDH). (c) Ethidium bromide-stained agarose gel profiles of PCR reactions products from input control LNCaP DNA (In), β-catenin antibody immunoprecipitated chromatin from 48 h Ad-lac Z transduced LNCaP cells (Con) or from 48 h Ad-Wnt-1 transduced LNCaP cells (Wnt-1). Results show that sheared chromatin within three regions of the hAR promoter were immunoprecipitated by the antibody in β-catenin and PCDH-PC transfected cells as well as the known LEF-1/TCF binding elements within the promoters of the cyclin D1 and c-myc gene but these regions were not immunoprecipitated in control transfected cells.

Figure 2

The promoter of the human androgen receptor gene contains β-catenin sensitive elements that upregulate luciferase expression in chimeric reporter vectors. (a) Chimeric hAR promoter/luciferase reporter vectors with varying amounts of upstream hAR promoter (left) were co-transfected into LNCaP cells along with empty vector (pcDNA3) or β-catenin and normalized luciferase activity was measured after 48 h (right). Results show progressive increase in luciferase as promoter element length is increased. (b) Comparison of normalized luciferase expression from vector 5, above with wild type, deleted (A at −1162) or mutated (G instead of A at −1162) LEF-1/TCF binding site (−1158 to −1164) when co-transfected with empty vector or β-catenin, as indicated. (c) Semiquantitative RT-PCR analysis of hAR (Top) or G3PDH (Bottom) mRNA expression in LNCaP cells or Wnt-activated LNCaP cells (grown in androgen-free medium for 3, 6 or 9 days or transfected with PCDH-PC or β-catenin) or in LNCaP-E-T6 cells (stably transfected with ecdysterone-inducible PCDH-PC expression vector) with or without ponasterone (Pon).

Figure 3

Expression of hAR protein is downregulated in Wnt-activated LNCaP cells by a proteasomal degradation pathway. (a) Western blot shows relative expression of hAR or actin in control LNCaP cells (Control) or in LNCaP cells transfected with β-catenin or PCDH-PC or LNCaP cells grown in androgen-free medium for 7 days. (b) Western blot shows hAR protein is likewise downregulated in LNCaP cells transduced for 48 h with Ad-Wnt-1 but not from cells transduced with Ad-Lac Z. (c) Expression of hAR protein in Wnt-activated cells (β-catenin transfected or cultured in androgen-free medium for 7 days) is restored to levels commensurate with elevated hAR mRNA levels when Wnt-stimulated cells were treated with proteasome inhibitors, MG132 or lactacystin.

Figure 4

Phosphorylation of Akt and its downstream target, MDM2, is increased in LNCaP cells with increased Wnt signaling. (a) Western blot shows that transfection of LNCaP cells with β-catenin or PCDH-PC increases pAKT and pMDM2 levels approximately 50-fold compared to control cells (untransfected or transfected with empty vector). (b) Western blot shows that agents that suppress Wnt signaling in LNCaP cells grown in androgen-free medium (transfection with siRNA against β-catenin or PCDH-PC or dominant-negative (DN-) TCF suppress pAkt and pMDM2 levels compared to control (untransfected or transfected with siRNA against lamin) LNCaP cells.

Figure 5

Suppression of MDM2 expression or direct Akt activity relieves Wnt-mediated suppression of hAR protein expression. (a) Western blot (top) shows that MDM2 protein expression is suppressed by greater than 88% by an siRNA that targets the gene and this siRNA relieves the Wnt-mediated suppression of hAR expression induced by transfection with β-catenin or PCDH-PC. (middle). Actin control (bottom). (b) Western blot shows that direct suppression of Akt signaling by inhibitor 5233705 but not by PI3-kinase inhibitor LY294002 relieves Wnt-mediated suppression of hAR protein (top) and Wnt-mediated upregulation in phosphorylation of MDM2 (middle) in β-catenin transfected LNCaP cells. Actin control (bottom).

Figure 6

Proteasome inhibitors block the suppression of MDM2 phosphorylation and suppress degradation of PP2A B subunit protein in Wnt-activated LNCaP cells. Western blots shows that Wnt-activation (by β-catenin transfection or culture of LNCaP cells for 7 days in androgen-free medium) upregulates phosphorylation of MDM2 (top) that is blocked by proteasome inhibitors MG132 or lactacystin and this activity corresponds with loss of the regulatory subunit (b) of PP2A that is blocked by proteasome inhibitors (middle). There was no change in the PP2A catalytic C subunit levels in Wnt-activated or proteasome-inhibitor treated cells (bottom).