A promoting role of androgen receptor in androgen-sensitive and -insensitive prostate cancer cells

Abstract

Although the vital role of the androgen receptor (AR) has been well demonstrated in primary prostate cancers, its role in the androgen-insensitive prostate cancers still remains unclear. Here, we used a small hairpin RNA approach to directly assess AR activity in prostate cancer cells. Reduction of AR expression in the two androgen-sensitive prostate cancer cell lines, LNCaP and LAPC4, significantly decreased AR-mediated transcription and cell growth. Intriguingly, in two androgen-insensitive prostate cell lines, LNCaP-C42B4 and CWR22Rv1, knockdown of AR expression showed a more pronounced effect on AR-induced transcription and cell growth than androgen depletion. Using cDNA microarrays, we also compared the transcriptional profiles induced by either androgen depletion or AR knockdown. Although a significant number of transcripts appear to be regulated by both androgen depletion and AR knockdown, we observed a subset of transcripts affected only by androgen depletion but not by AR knockdown, and vice versa. Finally, we demonstrated a direct role for AR in promoting tumor formation and growth in a xenograft model. Taken together, our results elucidate an important role for the AR in androgen-insensitive prostate cancer cells, and suggest that AR can be used as a therapeutic target for androgen-insensitive prostate cancers.

Figure 1.

Down-regulation of endogenous AR expression by AR shRNA in prostate cancer cells. (A) LNCaP cells were infected with either the GFP adenovirus or the different AR shRNA adenovirus at an MOI of 40. Whole-cell lysates were prepared after 48 h of viral infection, and then analyzed by western blotting. Specific antibodies used to detect protein expression are labeled in the figure. (B) Identical experiments performed in LAPC4 cells. (C) LNCaP cells were infected with either the GFP adenovirus or AR shRNA3 adenovirus at an MOI of 40. Cells were fixed and immunostained 72 h after viral infection. Representative confocal laser scanning microscopy images of cells are shown. (D) Identical experiments performed in LAPC4 cells.

Figure 2.

Down-regulation of AR expression reduces AR transactivation in prostate cancer cells. (A) LNCaP cells infected with either the GFP adenovirus (control) or AR shRNA3 adenovirus at an MOI of 40 for 6 h were transiently transfected with PSA7 kb-Luc (PSA-Luc), reporter, MMTV-Luc or ARE2-Luc reporter and pcDNA3-β-gal in T-medium with 5% CS-FBS for 24 h, and then incubated in different amounts of DHT for another 24 h. Luciferase and β-gal activities were measured and reported as RLU. (B) Transient transfection experiments carried out in LAPC4 cells infected with the AR shRNA3 or control adenoviruses at an MOI of 40. (C) LNCaP cells infected with control or AR shRNA3 adenovirus in the presence or absence of 10 nM DHT. Total RNA was isolated and analyzed by northern blot using radiolabeled probes for PSA, KLK2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) LAPC4 cells were infected with control adenovirus or AR shRNA3 adenovirus in the presence or absence of 10 nM DHT. Total RNA was isolated, reverse transcribed and analyzed by PCR.

Figure 3.

Identification of genes down-regulated by androgen deprivation and AR knockdown using cDNA microarray. (A) Treatment scheme for microarray experiment. (B) The number of genes with at least 1.5-fold decrease in expression in response to androgen deprivation, AR knockdown or both. (C) Fold change distribution of genes that showed decreased expression (>1.5 fold) in response to androgen deprivation or AR knockdown. Brightness of green in the figures below the graphs corresponds to the degree of decreased expression (fold change). (D) Genes whose expression decreased after AR knockdown (1.5-fold) but were not affected by androgen deprivation.

Figure 4.

Down-regulation of AR expression inhibits the growth of androgen-sensitive prostate cancer cells. (A) LNCaP cells were seeded into 96-well plates in media with or without DHT after 3 h adenovirus infection at an MOI of 10. Cell growth was measured every other day by MTS assay. The data represent the mean ± SD of three independent experiments. (B) Identical experiments performed in LAPC4 cells. (C) LNCaP cells were seeded into 24-well plates at 400 cells/well after 3 h adenovirus infection at an MOI of 10. Cells were cultured with the media in the presence or absence of DHT for 14 days and colonies were fixed and stained with crystal violet. (D) Similar experiments performed in LAPC4 cells.

Figure 5.

Down-regulation of AR expression affects AR-mediated transcription and cell growth in androgen-insensitive prostate cancer cells. (A) LNCaP, LNCaP C4-2B4 and CWR22-Rv1 cells were infected with control or AR shRNA3 lentivirus, and incubated in medium with 10 μg/ml blastcidin for selection. Whole-cell lysates were prepared at Day 7, and analyzed by western blotting with AR and tubulin antibodies. (B) LNCaP and LNCaP C4-2B4 cells were infected with either control or AR shRNA3 adenovirus at an MOI of 20 for 6 h, and transfected with PSA7 kb-luc reporter (PSA-Luc) and pcDNA3-β-gal. Cells were incubated in T-medium with 5% CS-FBS for 24 h, and then induced with different amounts of DHT for another 24 h. Luciferase and β-gal activities were measured and reported as RLU. (C) LNCaP C4-2B4 and CWR22-Rv1 cells were infected with control or AR shRNA3 lentivirus and incubated with medium containing 10 μg/ml of blastcidin for 7 days. Cell growth was measured by the MTS assay. The data represent the mean ± SD of three independent experiments. (D) CWR22-Rv1 and LNCaP C4-2B4 cells were infected with the control or AR shRNA3 lentivirus, and cultured with 10 μg/ml blastcidin for 7 days. Cells resistant to the selection were re-plated into 12-well plates and cultured for 14 days. Colonies were fixed and stained with crystal violet.

Figure 6.

Reduction of AR expression inhibits tumor xenograft formation in athymic mice. (A) LAPC4 cells were transduced with the AR shRNA or GFP lentiviruses at a MOI of 3 for 24 h. Cells were harvested, resuspended in PBS and mixed with an equal volume of Matrigel ECM. Here, 100 μl of cell suspension (1 × 10⁷ cells/ml) was injected subcutaneously in opposite lateral flanks of 6–8-week-old athymic male mice. Mice were monitored twice weekly. Tumors were measured in two dimensions with calipers, and tumor volume (mm³) was calculated with the formula V = (length × width²)/2. ‘Asterisk’ indicates a significant difference (P < 0.05) between the two groups of animals. (B) The tumor specimens isolated from xenograft animals at Day 15 were fixed in 10% neutral-buffered formalin and embedded in paraffin, and then analyzed by immunohistochemistry using anti-AR antibodies.