Regulation of Androgen Receptor Transcriptional Activity by Rapamycin

SUPPLEMENTAL DATA

Regulation of androgen receptor transcriptional activity by rapamycin

Yu Wang1,2, Margarita Mikhailova3, Swagata Bose1, Chong-Xian Pan1,2, Ralph W. deVere White1 and Paramita M. Ghosh1,2

1University of California Davis, School of Medicine, Sacramento, CA; 2VA Northern California Health Care System, Mather, CA and 3University of Texas Health Science Center at San Antonio, TX

PSA expression and AR transcriptional activity on the PSA promoter are adequate read-outs of AR transcriptional activity on other AR target genes involved in cell cycle progression. The AR is a transcription factor and promotes the transcription of many genes, including PSA, by binding to the androgen response element (ARE) in their promoter region. The AR has also been shown to regulate proliferation and survival, but the exact targets of AR in the cell cycle and survival machinery, other than p21Cip1/Waf1(Lu et al., 1999), are not known. Previous studies from our laboratory showed that increasing stimulation with dihydrotestosterone (DHT) stimulated AR transcriptional activity on a PSA promoter region (Wang et al., 2007).Here we show that low levels of AR stimulation (represented by 3-4 fold increase in PSA) stimulated proliferation in prostate cancer cells, whereas high levels of stimulation (>15-fold) were growth inhibitory (Figure S1).Growth inhibition at high concentrations of DHT is likely caused by increased expression of p21, but the AR target in the cell cycle, which stimulates cell growth at low DHT concentrations,has not been identified. Another cdk inhibitor, p27, which is not an AR target gene, and whose expression was not affected by DHT, has been used as a loading control. Based on these data, it appears that at low levels of stimulation, PSA expression, or AR transcriptional activity measurements on a PSA promoter,are adequate readouts of the effect of the AR on proliferation.

FIGURE S1. Low levels of PSA expression is an indicator of AR transcriptional activity on cell cycle proteins resulting in increased proliferation. LNCaP and C4-2 cells were cultured in medium containing charcoal stripped serum (CSS) together with increasing levels of DHT as indicated. Cells were collected after 48 hours and analyzed by Western blotting for protein levels and by flow cytometry for proliferation rates. Flow cytometric data was statistically analyzed to determine the percentage of cells in S-phase. For each experiment, the S-phase data was divided by that for control cells, for which the S-phase was normalized to 100. This was done to realistically compare changes in S-phase in the different cell lines under the various treatment conditions. In the experiment described here, the percentage S-phase in the control cells was 8.04% for LNCaP cells and 17.32% for C4-2. PSA western blots were scanned and quantitated, then normalized to the band intensity for the control band.

C4-2 cells are androgen-sensitive but androgen-independent. Although the sublines of LNCaP cells arising from culture in androgen deprived conditions (C4-2 and LNAI) are still androgen sensitive and are growth stimulated upon an increase in AR transcriptional activity, they are also androgen-independent, because upon withdrawal of androgens, they do not undergo growth arrest. This is illustrated in Figure S2, which shows that treatment with bicalutamide, which inhibited PSA expression in both LNCaP and C4-2 cells, did not inhibit proliferation rates in C4-2 cells.

Figure S2. Effect of the anti-androgen bicalutamide on proliferation and survival of androgen-dependent LNCaP and androgen-independent C4-2 cells. (A) Immunoblots demonstrating the effect of 10 μM bicalutamide on PSA expression in LNCaP and C4-2 cells. (B) Flow cytometric analysis of LNCaP and C4-2 cells treated with increasing concentration of bicalutamide. The S-phase was calculated as described elsewhere (Ghosh et al., 2005), and normalized by calculating percent change from control (vehicle treated) cells, which was taken as 100. This was done in order to adequately compare proliferation rates in LNCaP and C4-2 cells, since these cells grew at vastly different rates (for vehicle treated LNCaP cells, %S-phase = 25.51, C4-2: 33.96). (C)Effect of androgen removal and bicalutamide on proliferation and survival of LNCaP cells. LNCaP cells were cultured in FBS or CSS-containing media in vehicle or 10 μM bicalutamide for 48 hours prior to flow cytometric analysis.

Androgens are necessary for cell survival in androgen-sensitive prostate cancer cells. Figure S2C illustrates that AR transcriptional activity is needed for both cell proliferation and survival. Inhibition of AR transcriptional activity by culture in CSS-containing medium reduced proliferation rates and also induced apoptosis (albeit at low levels) in LNCaP cells. These results indicate that stimulation of AR transcriptional activity would stimulate both cell proliferation and survival.

SUPPLEMENTAL REFERENCES

Ghosh PM, Malik SN, Bedolla RG, Wang Y, Mikhailova M, Prihoda TJ, et al. (2005). Signal transduction pathways in androgen-dependent and -independent prostate cancer cell proliferation (2005). Endocr Relat Cancer, 12, 119-34.

Lu S, Liu M, Epner DE, Tsai SY and Tsai MJ (1999). Androgen regulation of the cyclin-dependent kinase inhibitor p21 gene through an androgen response element in the proximal promoter. Mol Endocrinol, 13, 376-84.

Wang Y, Kreisberg JI, Bedolla RG, Mikhailova M, deVere White RW and Ghosh PM. (2007) A 90 kDa fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells. Oncogene, 26, 6061-70.