Androgen Receptor regulation of Vitamin D receptor in response of castration-resistant prostate cancer cells to 1α-Hydroxyvitamin D5 - a calcitriol analog

Abstract

Calcitriol (1,25(OH)(2)D3) is cytostatic for prostate cancer (CaP), but had limited therapeutic utility due to hypercalcemia-related toxicities, leading to the development of low-calcemic calcitriol analogs. We show that one analog, 1-α-Hydroxyvitamin-D5 (1α(OH)D5), induced apoptosis in castration-sensitive LNCaP prostate cancer cells, but unlike calcitriol, did not increase androgen receptor (AR) transcriptional activity. LNCaP-AI, a castrate-resistant (CRCaP) LNCaP subline, was resistant to 1α(OH)D5 in the presence of androgens; however, androgen withdrawal (AWD), although ineffective by itself, sensitized LNCaP-AI cells to 1α(OH)D5. Investigation of the mechanism revealed that the vitamin D receptor (VDR), which mediates the effects of 1α(OH)D5, is downregulated in LNCaP-AI cells compared to LNCaP in the presence of androgens, whereas AWD restored VDR expression. Since LNCaP-AI cells expressed higher AR compared to LNCaP and AWD decreased AR, this indicated an inverse relationship between VDR and AR. Further, AR stimulation (by increased androgen) suppressed VDR, while AR downregulation (by ARsiRNA) stimulated VDR levels and sensitized LNCaP-AI cells to 1α(OH)D5 similar to AWD. Another cell line, pRNS-1-1, although isolated from a normal prostate, had lost AR expression in culture and adapted to androgen-independent growth. These cells expressed the VDR and were sensitive to 1α(OH)D5, but restoration of AR expression suppressed VDR levels and induced resistance to 1α(OH)D5 treatment. Taken together, these results demonstrate negative regulation of VDR by AR in CRCaP cells. This effect is likely mediated by prohibitin (PHB), which was inhibited by AR transcriptional activity and stimulated VDR in CRCaP, but not castrate-sensitive cells. Therefore, in castration sensitive cells, although the AR negatively regulates PHB, this does not affect VDR expression, whereas in CRCaP cells, negative regulation of PHB by the AR results in concomitant negative regulation of the VDR by the AR. These data demonstrate a novel mechanism by which 1α(OH)D5 prolong the effectiveness of AWD in CaP cells.

Figure 1.

1α(OH)D5 inhibits cell growth and survival but does not induce AR levels or transcriptional activity in androgen-dependent LNCaP prostate cancer cells. LNCaP cells cultured in medium with androgens (FBS) and exposed to either calcitriol (D3) or 1α(OH)D5 (D5). (A) Cell number increase was estimated by MTT assay and indicated comparable cytostatic effects of 100 nM D3 and 1 to 2 µM D5. Data represent mean ± standard deviation (SD) of 3 independent readings. (B) Flow cytometric analysis to compare effects of calcitriol versus 1α(OH)D5 on cell proliferation and apoptosis. LNCaP cells were treated with vehicle or calcitriol or different concentrations of 1α(OH)D5 for 48 hours. Change in S-phase is recorded as percentage change over vehicle-treated cells and change in apoptosis as fold change over vehicle-treated cells. (C) AR levels assessed by immunoblotting in LNCaP cells showing AR induction by D3 but not by D5. β-actin was assessed as loading control. Numbers under each lane represent fold change in band intensity normalized to that of the control band. These experiments were repeated at least 3 times with similar results. (D) LNCaP cells left untransfected or transfected with hPSA-luc and β-gal were treated as shown for 72 hours. AR transcriptional activity was determined by luciferase assay and shows increased activity in D3- but not D5-treated cells. Data of hPSA-gal/β-gal shown as mean ± SD of 3 independent readings.

Figure 2.

1α(OH)D5 inhibits growth of LNCaP-AI cells, a castration-resistant subline of LNCaP, in charcoal stripped serum (CSS) but not in complete medium containing FBS. LNCaP-AI cells were cultured in (A) complete medium containing FBS (upper panel) or phenol red–free medium containing CSS (lower panel) for the periods indicated in the presence of vehicle (ethanol) or 100 nM versus 1 µM 1α(OH)D5 (D5). Growth rates estimated by MTT assay indicate greater effect of D5 in CSS compared to FBS. Data represent mean ± standard deviation (SD) (n = 3). (B) Flow cytometry to determine the effect of D3 and increasing doses of D5 on LNCaP-AI cell apoptosis. Cells were cultured in medium containing FBS or CSS for 5 days, and the cells were trypsinized and stained with propidium iodide and annexin V prior to flow cytometry. Data represent fold change over number of cells undergoing apoptosis in cells treated with ethanol (EtOH control). (C) AR transcriptional activity with calcitriol versus 1α(OH)D5 in FBS versus CSS as determined by luciferase assay in hPSA-luc and β-gal transfected cells. Note the increased AR activity in the presence of D3, which was not apparent in the presence of D5. Data represent mean ± SD of 3 independent readings.

Figure 3.

The androgen receptor (AR) negatively regulates the levels of the vitamin D receptor (VDR). (A) 1α(OH)D5-induced growth inhibition is mediated by the VDR. (Upper) LNCaP-AI cells cultured in CSS medium treated with control siRNA were growth inhibited by 48-hour treatment with 1 to 2 µM 1α(OH)D5, as determined by MTT assay (note that the higher inhibition rates observed in previous figures were achieved after longer periods of treatment), but (lower) the same cell line subjected to VDR siRNA for the same time period showed no statistically significant response to 1α(OH)D5. Data represent mean ± standard deviation (SD) (n = 3). (B) Negative correlation between the AR and the VDR. (Left) LNCaP-AI cells express higher levels of AR and lower levels of VDR compared to LNCaP, (right) but when LNCaP-AI cells were cultured for >24 hours in CSS, AR levels declined while VDR levels increased. (C) Prolonged treatment with DHT inhibited VDR levels. LNCaP-AI cells were treated with increasing doses of DHT. AR expression increased with increasing concentration of DHT (upper panel). In contrast, VDR levels decreased after 4 days but not 2 days of treatment (middle panel). Numbers under each lane represent fold change in band intensity normalized to that of control.

Figure 4.

Downregulation of the AR by AR siRNA sensitizes LNCaP-AI cells to 1α(OH)D5. (A) An AR siRNA, which downregulated AR expression, but not a pool of control (nonspecific) siRNA duplexes, stimulated VDR levels. The AR siRNA, but not a control siRNA, downregulated AR expression, especially that induced by vitamin D3 and D5 treatment (upper panel), and induced VDR expression (middle panel). Results were normalized to levels of actin, which acted as loading control (lower panel). Numbers under each band represent fold change in band intensity normalized to actin with respect to that of the vehicle-treated control cells. (B) LNCaP-AI cells were transfected with control siRNA or AR-specific siRNA in the presence of D5, and changes in cell numbers in the presence of control or AR siRNA were estimated by MTT assay (upper). In the presence of the control siRNA, the effect of D5 on the growth of these cells was not significant, (lower) but AR downregulation with AR siRNA sensitized these cells to D5, which induced a 41.43% decrease in cell growth rates (P = 0.0092) versus 14.93% decrease (P > 0.5) with control siRNA (mean ± standard deviation [SD], n = 3).

Figure 5.

Expression of AR in AR-null pRNS-1-1 cells derived from a normal prostate desensitized these cells to 1α(OH)D5. (A) pRNS-1-1 cells stably transfected with vector only responded to 1α(OH)D5. (B) pRNS-1-1 cells stably transfected with wild-type AR (wtAR) demonstrated decreased response to 1α(OH)D5. (C) pRNS-1-1 cells stably transfected with a mutant AR (T877A) responded even less to 1α(OH)D5. (D) Expression of wild-type or mutant AR in pRNS-1-1 cells inhibited VDR and prohibitin expression. (Upper panel) Parental pRNS-1-1 cells were transfected with vector only or wild-type or mutant AR. (Second panel) Overexpression of AR in pRNS-1-1 cells, but not the vector, suppressed the expression of VDR, (third panel) as well as that of prohibitin. (Bottom panel) Loading control as detected by the expression of α-tubulin.

Figure 6.

Prohibitin (PHB) mediates the effects of 1α(OH)D5 in androgen-insensitive, but not androgen-sensitive, prostate cancer cells. (A) LNCaP (left) and LNCaP-AI cells (right) were subjected to control (nonspecific) siRNA or PHB siRNA and the effect of these treatments on PHB levels (upper panel), VDR levels (second panel), AR levels (third panel), and tubulin (fourth panel) determined by Western blotting. Note that PHB downregulation stimulated AR levels in both LNCaP and LNCaP-AI, as reported by others, but affected VDR expression only in LNCaP-AI. (B) Downregulation of AR expression in LNCaP-AI cells increases PHB levels. LNCaP-AI cells were subjected to control or AR siRNA, and PHB levels were determined by Western blotting. Tubulin levels were determined as loading control. (C) The growth-inhibitory effect of 1α(OH)D5 in LNCaP-AI cells in CSS medium requires an active PHB. Androgen-independent LNCaP-AI cells treated with control siRNA cultured in CSS medium respond to 1α(OH)D5 as determined by MTT assay (upper), whereas those treated with PHB siRNA duplexes do not (lower). (D) Scheme depicting PHB mediation of the effects of AR on VDR in androgen-insensitive cells. Red lines depict effects seen only in androgen-independent prostate cancer.