MicroRNA let-7c suppresses androgen receptor expression and activity via regulation of Myc expression in prostate cancer cells

Abstract

Castration-resistant prostate cancer continues to rely on androgen receptor (AR) expression. AR plays a central role in the development of prostate cancer and progression to castration resistance during and after androgen deprivation therapy. Here, we identified miR-let-7c as a key regulator of expression of AR. miR-let-7c suppresses AR expression and activity in human prostate cancer cells by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation of human prostate cancer cells. Down-regulation of Let-7c in prostate cancer specimens is inversely correlated with AR expression, whereas the expression of Lin28 (a repressor of let-7) is correlated positively with AR expression. Our study demonstrates that the miRNA let-7c plays an important role in the regulation of androgen signaling in prostate cancer by down-regulating AR expression. These results suggest that reconstitution of miR-let-7c may aid in targeting enhanced and hypersensitive AR in advanced prostate cancer.

FIGURE 1.

Let-7c suppresses AR expression. Relative expression levels of let-7c (A) and AR (B) were determined in LNCaP, C4-2B, LNCaP-s17, and LN-IL6+ cells by qRT-PCR. Results are presented as relative fold change compared to expression levels in LNCaP cells. C, LNCaP cells were transfected with let-7c antisense oligonucleotides, and AR mRNA levels were analyzed by qRT-PCR. Results are presented as relative fold change compared to expression levels in LNCaP cells transfected with control oligonucleotides. Data points represent mean ± S.D. of triplicate samples from two independent experiments. Error bars denote mean ± S.D. * denotes p ≤ 0.05. AR expression was increased when let-7c expression was down-regulated. D, Western blot analysis showing the increase in AR expression in LNCaP cells transfected with let-7c antisense. Actin is shown as loading control. E, LN-IL6+ cells were transfected with let-7c, and AR mRNA levels were analyzed by qRT-PCR. AR expression was reduced when let-7c was overexpressed. Con, control. * denotes p ≤ 0.05. F, Western blot analysis showing the down-regulation of AR expression in LN-IL6+ cells transfected with let-7c. G and H, AR levels were analyzed in LNCaP and C4-2B cells stably expressing let-7c by Western blotting. AR protein levels were reduced when let-7c was expressed. I, protein levels of AR were analyzed in LNCaP cells stably expressing Lin28 by Western blotting. AR expression was enhanced in cells expressing Lin28.

FIGURE 2.

Let-7c inhibits AR activity and transcription of AR target genes. A, LNCaP cells were cotransfected with let-7c and pGL3-PSA6.0-Luc reporter. Data points represent mean ± S.D. of triplicate samples from two independent experiments. Error bars denote mean ± S.D. * denotes p ≤ 0.05. Transactivation of the PSA promoter by AR was reduced with let-7c overexpression. B, LNCaP cells were transfected with control or let-7c or let-7c antisense, and levels of PSA and NKX3.1 mRNAs were analyzed by qRT-PCR. Results are presented as relative fold change compared with expression levels in LNCaP cells transfected with control oligonucleotides. Down-regulation of let-7c enhanced the levels of PSA and NKX3.1 mRNAs, whereas overexpression of let-7c reduced their expression. Con, control. C, PSA secretion in LNCaP cells was enhanced when let-7c expression was down-regulated. * denotes p ≤ 0.05. D, LNCaP cells stably expressing control, let-7c, or Lin28 were transfected with pGL3-PSA6.0-Luc reporter, and luciferase activities were assayed. Transactivation of the reporter by AR was reduced in cells expressing let-7c, whereas it was increased in cells expressing Lin28. RLU, Relative Luciferase Units. E, secretion of PSA by LNCaP cells stably expressing let-7c or Lin28 was analyzed by ELISA. PSA secretion was reduced in cells expressing let-7c and increased in cells expressing Lin28. Recruitment of AR to AREs in PSA (F) and NKX3.1 (G) promoters was analyzed by ChIP assays in LNCaP cells transfected with let-7c or Lin28. Recruitment of AR to the AREs was reduced in cells overexpressing let-7c and was enhanced in cells expressing Lin28. IP, immunoprecipitation.

FIGURE 3.

Let-7c regulates AR expression at the level of transcription. LNCaP cells were transfected with a control (Con) or let-7c plasmid in the presence or absence of actinomycin D, and AR mRNA levels were analyzed by Northern blotting (A) or qRT-PCR (B). Half-life of AR mRNA was not altered in the presence of let-7c. C, LNCaP cells were cotransfected with control or let-7c or with let-7c antisense oligos along with the pGL4-AR-Prom-Luc reporter. Data points represent the mean ± S.D. of triplicate samples from two independent experiments. AR promoter activity was repressed in let-7c-overexpressing cells and was enhanced when let-7c was down-regulated. RLU, Relative Luciferase Units.

FIGURE 4.

Down-regulation of AR by let-7c is mediated by down-regulation of Myc. Expression levels of AR and c-Myc were analyzed by Western blotting in LNCaP cells transfected with let-7c (A) or let-7c antisense oligos (B). Protein levels of AR and c-Myc were reduced in let-7c-overexpressing cells and were increased when let-7c was down-regulated. Con, control. C, LNCaP cells were transfected with plasmids expressing Myc or shRNA against Myc along with the pGL4-AR prom-Luc reporter. Luciferase assays showed that down-regulation of Myc reduced transactivation of the AR promoter, whereas overexpression of Myc enhanced reporter activity. RLU, Relative Luciferase Units. D and E, mRNA levels of AR, PSA, and NKX3.1 were measured by qRT-PCR in LNCaP cells transfected with Myc or shRNA against Myc. Expression levels of all three genes were decreased when Myc was down-regulated and were enhanced when Myc was overexpressed. F, LNCaP cells were transfected with let-7c and Myc alone or together along with the pGL4-AR prom-Luc reporter. Overexpression of Myc could overcome the repression of AR promoter activity by let-7c. G, recruitment of Myc to the Myc binding site in the AR promoter was analyzed by ChIP assays. Overexpression of let-7c reduced and expression of Lin28 enhanced the recruitment of Myc to the AR promoter. IP, immunoprecipitation.

FIGURE 5.

Let-7c reduces proliferation of PCa cells. A, cell growth of LNCaP cells stably expressing Lin28 was determined by cell counting up to 96 h. Expression of Lin28 enhanced the growth rate of LNCaP cells. The inset (Western blot analysis) shows the increase in expression of AR with increased expression of Lin28. B, cell growth of C4-2B cells stably expressing let-7c was analyzed up to 72 h. Expression of let-7c decreased the growth rate of C4-2B cells. The inset (Western blot analysis) shows the reduction in AR protein levels with expression of let-7c. C, relative expression levels of let-7c, AR, Lin28, and Myc were analyzed by qRT-PCR in PCa xenografts injected intratumorally with let-7c expressing lentiviruses. Enhanced expression of let-7c suppressed expression of AR, Lin28, and Myc. Data points represent mean ± S.D. of triplicate samples from two independent experiments. Error bars denote mean ± S.D. p ≤ 0.05.

FIGURE 6.

Expression levels of let-7c and AR are negatively correlated in human PCa. A, relative expression levels of let-7c and AR were measured by qRT-PCR in total RNAs extracted from 22 human prostate cancer samples. Levels of let-7c and AR were correlated negatively with each other. p = 0.015 by two-tailed Student's t test. B, plot showing the negative correlation between levels of let-7c and AR mRNAs in the above samples. The correlation coefficient (R) was found to be −0.5135. C and D, plots showing negative correlation between relative expression levels of let-7c and Lin28 and let-7c and Myc, respectively, in the above samples. r = −0.1765 (Lin28) and r = −0.3354 (Myc). E, representative Western blot analysis showing protein levels of Lin28 and AR in extracts from normal, benign (B), and adjacent tumor (T) containing human prostate samples. Actin is shown as a loading control. The levels of Lin28 and AR are positively correlated with each other.

FIGURE 7.

Expression levels of AR, Lin28 and Myc are correlated with each other in human PCa. A, comparison of AR, Lin28, and Myc expression in the dataset GDS1439 (23). Benign, n = 6; primary prostate cancer, n = 7; and metastatic prostate cancer, n = 6. B, comparison of AR, Lin28, and Myc levels in GDS2547 (24). Normal prostate tissue, n = 18; normal tissue adjacent to tumor, n = 16; primary prostate cancer, n = 65; and metastatic prostate cancer, n = 25. Data are expressed as mean ±S.E. (in percentages) of the maximum single channel count determined in each data set. C, gene expression analysis using the Oncomine database showing the relative expression levels of AR, Lin28, and Myc in three datasets comparing normal prostate tissue and prostate cancer. Wallace_prostate: normal, n = 20; cancer, n = 69. Yu_prostate: normal, n = 23; cancer, n = 64. Vanaja_prostate: normal, n = 8; cancer, n = 32. Data are presented as mean ± S.E. of normalized expression units according to Oncomine output.