Cyclin D1b variant influences prostate cancer growth through aberrant androgen receptor regulation

Abstract

Cyclin D1 is a multifaceted regulator of both transcription and cell-cycle progression that exists in two distinct isoforms, cyclin D1a and D1b. In the prostate, cyclin D1a acts through discrete mechanisms to negatively regulate androgen receptor (AR) activity and thus limit androgen-dependent proliferation. Accordingly, cyclin D1a is rarely overexpressed in prostatic adenocarcinoma and holds little prognostic value in this tumor type. However, a common polymorphism (A870) known to facilitate production of cyclin D1b is associated with increased prostate cancer risk. Here we show that cyclin D1b is expressed at high frequency in prostate cancer and is up-regulated in neoplastic disease. Furthermore, our data demonstrate that, although cyclin D1b retains AR association, it is selectively compromised for AR regulation. The altered ability of cyclin D1b to regulate the AR was observed by using both in vitro and in vivo assays and was associated with compromised regulation of AR-dependent proliferation. Consistent with previous reports, expression of cyclin D1a inhibited cell-cycle progression in AR-dependent prostate cancer cells. Strikingly, cyclin D1b significantly stimulated proliferation in this cell type. AR-negative prostate cancer cells were nonresponsive to cyclin D1 (a or b) expression, indicating that defects in AR corepressor function yield a growth advantage specifically in AR-dependent cells. In summary, these studies indicate that the altered AR regulatory capacity of cyclin D1b contributes to its association with increased prostate cancer risk and provide evidence of cyclin D1b-mediated transcriptional regulation.

Fig. 1.

The AA genotype and transcript b are prevalent in PCa cells. (A) DNA was harvested and analyzed by using the strategy outlined in Fig. 7A. Data are representative of three independent experiments, and determined genotypes are shown. (B) RT-PCR detection of cyclin D1 isoform mRNA in PCa cell lines and tumor samples. A duplicate sample of 22Rv1 RNA, incubated in the absence of reverse transcriptase, was included as a control (lane 2). For each PCR, generated cDNA or cyclin D1b-encoding plasmid (lanes 3 and 4) was used in combination with the specific primers. (C) mRNA was isolated from tumor and normal or PIN lesions from the same patient (lanes 1–8), a prostatic metastatic tumor sample (lane 9), and HUNC-E cells (lane 10). RT-PCR analysis for D1b and GAPDH expression was performed as in B.

Fig. 2.

Cyclin D1b associates with the AR in PCa cells. (A) Immunoblot of cyclin D1b in transfected C33A cells or endogenous expression in LNCaP and 22Rv1 cells. (B) Lysates from LNCaP and 22RV1 cells were immunoprecipitated with antibodies specific for AR, cyclin D1b, or a nonspecific control. The precipitated complex was immunoblotted for AR expression.

Fig. 3.

Cyclin D1b is compromised for AR corepressor activity. (A) Impact on AR N/C-terminal interactions was assessed in CV1 cells transfected with Gal4-LUC, β-gal, the depicted constructs encoding the mammalian-2-hybrid system (Top), and cyclin D1a, cyclin D1b, or vector. After transfection, cells were treated for 24 h with either vehicle or 5th-dihydrotestosterone (DHT) as indicated. After stimulation, cells were analyzed for relative luciferase activity. Results shown represent the average induction with standard deviations (+, P > 0.05). (B) COS7 cells transfected with HDAC3 and cyclin D1b were analyzed as in Fig. 2. (C) CV1 cells were transfected as indicated and analyzed in A. Relative DNA used is shown. (Inset) Relative expression of AR- and HA-tagged proteins transfected at a 1:3 ratio. (D) LNCaP cells were transfected with pBABE-Puro and pCDNA, cyclin D1a, cyclin D1b, or cyclin D1-ΔRD. mRNA from puromycin resistant cells was harvested and subjected to quantitative real-time RT-PCR. Data represent the average of at least three independent points performed in triplicate with standard deviations.

Fig. 4.

Cyclin D1b displays promoter-specific regulation of the AR. (A) Reporter assays were performed as in Fig. 3C by using ARE-containing promoters (∗, P < 0.05; ∗∗∗, P < 0.001). (B) CV1 cells were transfected as in A by using the MMTV-LUC reporter (+, P > 0.05).

Fig. 5.

Cyclin D1b demonstrates promoter-specific repression of AR activity in vitro. (A) Xenopus oocytes were injected and treated as shown here and as described in Material and Methods. (B) Injected oocytes containing increasing amounts of flag-cyclin D1a, flag-cyclin D1b, or vector were lysed and analyzed for AR or cyclin expression by anti-flag immunoblot. (C and D) Oocytes were injected and treated as depicted in A with either MMTV-CAT or PSA-61-LUC. Primer extension to detect control and experimental mRNAs was conducted as described.

Fig. 6.

Regulation of androgen-dependent growth is compromised by cyclin D1 polymorphism. (A) LNCaP cells were transfected with H2B-GFP alone, H2B-GFP and ΔRD, GFP-cyclin D1a, or GFP-cyclin D1b. After labeling and fixation, cells were stained for BrdUrd incorporation. For each condition, >150 cells were counted on each of at least six coverslips. Bars represent relative percent BrdUrd incorporation, and error bars represent the standard deviation. (B) PC3 cells were transfected and analyzed as in A for BrdUrd incorporation (∗∗, P < 0.01; ∗∗∗, P < 0.001 compared with vector; +, P > 0.05 compared with vector).