Evaluation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy in antagonizes therapeutic

Purpose— To characterize the importance of cellular Fas-associated death domain (FADD)-like interleukin 1 β -converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase 8 (FLICE)-promoted apoptosis, in modulating the response of prostate cancer (CaP) cells to androgen receptor (AR)-targeted therapy. Experimental Design— c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacological interventions. Results— c-FLIP expression was increased in high-grade prostatic intra-epithelial neoplasia (HGPIN) and CaP tissue relative to normal prostate epithelium (P<0.001). Maximal c-FLIP expression was detected in castrate-resistant CaP (CRPC) (P<0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also down-regulated c-FLIP expression, induced caspase-8 and caspase-3/7 mediated apoptosis and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of cFLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance. Conclusion— c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of CaP cells. A combination of HDACi with androgen-deprivation therapy (ADT) may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP however may be relevant to enhance the response of existing and novel therapeutics in CRPC. compensatory cytokine/growth factor-induced signaling, important the transition CRPC. The of to characterize the expression of c-FLIP in prostatectomy tissue and determine whether expression of c-FLIP modulates the sensitivity of androgen-dependent CaP cells to bicalutamide. In addition to direct targeting of c-FLIP using we also demonstrate that two histone deacetylase inhibitors (HDACi), droxinostat and SAHA (Vorinostat®) can repress c-FLIP expression in androgen-dependent cells and that these agents can potentiate the apoptosis induced by bicalutamide in non-castrate models. Conversely, we show that SAHA is ineffective in cell-based models of castrate-resistant suggesting that applicable during may Moreover, we show that c-FLIP plays an important role in the viability of castrate-resistant prostate cancer cells. Geometric were from the FL2 fluorescence signal, normalized against background AR signaling is the principal pathway driving prostate cancer (CaP) growth. Inhibition of androgen-driven signaling is the predominant therapeutic strategy for CaP, reinforced by the recent development of two clinically effective agents, abiraterone-acetate and MDV3100. Here we show that c-FLIP, an AR-regulated anti-apoptotic protein constitutes a novel mode of resistance to AR-targeted therapeutics. Down-regulation of c-FLIP in response to siRNA-targeting or histone deacetylase inhibitors (HDACi) induced apoptosis co-incident with down-regulation of c-FLIP expression and potentiated the response to the AR-targeted drug bicalutamide in non-castrate-resistant cells. In contrast, the elevated c-FLIP expression detected in castrate-resistant cells antagonized HDACi and bicalutamide response. Accordingly, HDACi may be useful in prolonging the therapeutic response to AR-targeted drugs in non-castrate conditions. Moreover, the increase in c-FLIP expression determined in castrate-resistant disease may assist in predicting patient response to existing and emerging androgen deprivation strategies and alternatively, may itself be a rationale therapeutic target to improve treatment of CRPC.