FLICE-Like Inhibitory Protein Blocks Transforming Growth Factor β1–Induced Caspase Activation and Apoptosis in Prostate Epithelial Cells

Abstract

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawalinduced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor B1 (TGFB1), mimicking androgen withdrawal -induced apoptosis. FLIP levels decline with TGFB1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFB1-induced apoptosis. Small interfering RNA -mediated knockdown of FLIP recapitulates and enhances TGFB1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFB1-induced apoptosis. TGFB1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFB-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFB1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFB stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis. (Mol Cancer Res 2008;6(2):231 -42)

FLIP-ping out: Death receptor signaling in the prostate

“…3). Interestingly, we observed that insulin 108 (likely acting via the IGF-1 receptor 123 ) can block the effect of TGFβ on FLIP expression, consistent with the antagonistic roles of TGFβ and IGF-1 in AWIA.…”

“…116 We found that TGFβ decreased both FLIP mRNA and protein, similar to the effects of androgen withdrawal. 108 NRP-152 cells overexpressing FLIP were resistant to TGFβ induced apoptosis, while siRNA directed against FLIP induced apoptosis, even in the absence of TGFβ. 108 These data indicate that FLIP can directly regulate prostate epithelial cell apoptosis in vitro.…”

“…108 NRP-152 cells overexpressing FLIP were resistant to TGFβ induced apoptosis, while siRNA directed against FLIP induced apoptosis, even in the absence of TGFβ. 108 These data indicate that FLIP can directly regulate prostate epithelial cell apoptosis in vitro. Since both androgen withdrawal and TGFβ addition reduce FLIP levels, and since androgen withdrawal induces TGFβ production ( Fig.…”

“…However, the demonstration that LNCaP xenografts overexpressing FLIP are resistant to castration induced growth inhibition 115 provides some in vivo support for FLIP downregulation in AWIA. We have also examined FLIP regulation in NRP-152 cells, 108 a prostate epithelial cell line that apoptoses in response to TGFβ, and therefore models the paracrine effects of TGFβ on prostate epithelial cells in AWIA. 116 We found that TGFβ decreased both FLIP mRNA and protein, similar to the effects of androgen withdrawal.…”

“…120 In some tumors, high level Hedgehog signaling via the Gli2 transcription factor leads to high FLIP levels. 121 IGF-1, insulin, and other growth factors can specifically upregulate FLIP levels, both by activating NFκB as well as via activation of the PI3K/AKT pathway 97,108 while ERK/MEK regulation of FLIP levels may occur in T cells but is otherwise not frequently observed. 122 At present, there is no evidence that TGFβ signaling directly regulates FLIP transcription via Smads, although we have identified a series of Smad binding elements by sequence scanning of the promoter (Fig.…”