The role of the androgen-signaling pathway in survival of the prostate epithelial and prostate cancer cells has been appreciated for some time, but the mechanisms underlying this process have not been addressed fully. In this study, we found that androgens up-regulated c-FLIP gene expression through multiple AREs within the c-FLIP gene. ChIP analysis indicated that AR directly bound to the promoter region of the c-FLIP gene in response to androgen treatment, suggesting that it directly targeted the c-FLIP gene. The ectopic expression of c-FLIP promoted androgen-independent growth of LNCaP tumors in nude mice. Our data shed light on a transcription-mediated mechanism for the effects of AR on cell survival and apoptosis in the prostate gland.
The identification and characterization of androgen-responsive genes have advanced our understanding of androgen action and prostate cancer in a variety of ways. One of the best-characterized androgen-responsive genes, PSA, is widely used as a diagnostic marker for prostate cancer. Other androgen-responsive genes such as hKLK2 (25
) and PSCA (26
) have been proposed as potential biomarkers for prostate cancer or for prostate cancer progression. The promoters of the androgen-responsive genes have been successfully used to generate mouse transgenic models for prostate cancer (27
). Our results show androgen directly activated c-FLIP gene expression. Supporting this view was the discovery of multiple AREs in the c-FLIP promoter that complexed with AR-DBD and activated the c-FLIP promoter reporter plasmids in the presence of androgens. The c-FLIP AREs are similar to those found in the androgen-responsive enhancers in PSA (28
), pro-basin (19
), kKH2 (29
), and PSCA (26
) genes. These enhancers contain multiple low-affinity AREs that activate transcription in response to androgens. The pivotal role of c-FLIP in the control of Fas/FasL-mediated apoptosis suggests that it may be involved in androgen-mediated prostate cell survival.
Although the mechanisms underlying androgen functions in survival of the prostate gland as well as in promoting prostate tumor cell growth have not been clearly defined, the effects of androgen on both pro-and antiapoptotic gene expression have been demonstrated (15
). Our present data provide additional information about how androgen can determine survival and apoptosis in the prostate gland. Specifically, in the presence of androgens, AR in the nucleus acts as a transcriptional activator for expression of the c-FLIP gene the function of which is to inhibit apoptosis. If androgens are depleted by chemical or surgical castration, however, this protective function is abolished, resulting in apoptosis in the prostate gland.
Whereas prostate cancer begins as an androgen-dependent tumor that regresses in response to androgen ablation, tumors eventually reappear and progress in an androgen-independent manner (4
). Altered gene expression patterns in prostate cancer cells may contribute to the androgen-independent growth of prostate cancer cells (30
). Consistent with this hypothesis, ectopic expression of some genes promotes androgen-independent growth in human prostate cancer cells (32
). c-FLIP expression is increased in human melanoma, and overexpression of c-FLIP contributed to tumor escape from the Fas/FasL-dependent apoptosis (34
). Consistent with these observations, the c-FLIP gene was overexpressed in most prostate cancer specimens we surveyed. Loss or down-regulation of c-FLIP expression could sensitize various cells to the Fas/FasL-mediated apoptosis (35
). Our finding that LNCaP tumors expressing high c-FLIP grew more quickly in castrated nude mice supports the relevance of the increased c-FLIP expression to prostate cancer. Moreover, androgen-independent growth correlated with the inhibition of apoptosis in the LNCaP tumors expressing high levels of c-FLIP. Whether dysfunction of the AR-c-FLIP signaling pathway might contribute to the survival of the androgen-independent prostate tumors in prostate cancer patients is currently being examined in our laboratory.