FOXO3a mediates the androgen-dependent regulation of FLIP and contributes to TRAIL-induced apoptosis of LNCaP cells

Abstract

Androgen-withdrawal-induced apoptosis (AWIA) is deregulated in androgen refractory prostate cancer. Androgens have been shown to positively regulate expression of the antiapoptotic FADD-like interleukin-1b-converting enzyme (FLICE)-like inhibitory protein (FLIP), and reduced FLIP expression precedes apoptosis after androgen withdrawal. Here, we show that FLIP protein expression is downregulated in castrated rats, while in LNCaP cells, androgens regulate FLIP in a manner that is dependent on phosphoinositol-3-kinase (PI3K) and Akt signaling. Specifically, treatment of LNCaP cells with LY294002, or expression of either PTEN or a nonphosphorylatable form of FOXO3a (FOXO3aTM), downregulates FLIP protein and mRNA. Conversely, treatment with androgens in the absence of PI3/Akt signaling, or following expression of FOXO3aTM, leads to increased FLIP expression. A FOXO3a binding site was identified in the FLIP promoter and shown necessary for the combined effects of androgens and FOXO3a on FLIP transcription. FOXO3a binds the androgen receptor, suggesting that the transcriptional synergy depends on an interaction between these proteins. Finally, LNCaP cells are sensitized to TRAIL-induced apoptosis by PTEN or LY294002, and rescued by androgens. FOXO3aTM also sensitizes cells to androgen-inhibited TRAIL apoptosis. Androgen rescue was diminished when either FOXO3a or FLIP was reduced by siRNA. These data support a role for FOXO3a in AWIA.

Nodal enhances the activity of FoxO3a and its synergistic interaction with Smads to regulate cyclin G2 transcription in ovarian cancer cells

“…Specifically, FoxO3a has been reported to activate genes that induce cell cycle arrest, such as p27 (Dijkers et al, 2000), p21 (Seoane et al, 2004), p15 and p19 (Katayama et al, 2008), and to suppress the expression of cyclin D2 (Fernandez de Mattos et al, 2004). FoxO3a is also involved in apoptosis by activating proapoptotic genes Bim (Sunters et al, 2003), Puma (You et al, 2006), TRAIL (Cornforth et al, 2008) and Fas ligand (Brunet et al, 1999). Inhibition of FoxO3a expression promotes cell transformation, tumor progression and angiogenesis, indicating that FoxO3a has tumor-suppressive roles (Ramaswamy et al, 2002).…”

FLIP-ping out: Death receptor signaling in the prostate

“…Alternatively, androgens might act independently of TGFβ to directly regulate FLIP. Support for the latter possibility comes from studies of androgen regulation of FLIP transcription in our lab 113 and by Gao et al, 114 (detailed below).…”

“…3) and demonstrated that this element mediates the transcription of a FLIP promoter reporter in a FOXO3a-and androgen-dependent manner. 113 We have also observed that AR co-immunoprecipitates with FOXO3aTM, again in an androgen dependent manner, indicating that these proteins can interact and function as a complex. 113 The AR contains an amino-terminal LxxLL-related FQNLF motif which mediates an interaction with the carboxyl terminal AF2 domain (Fig.…”

Androgen Action During Prostate Carcinogenesis

“…This discrepancy might be explained by the involvement of an AKT-regulated transcription factor, FOXO3a. Androgen induction of c-FLIP requires the presence of FOXO3a, which binds to the AR and potentially to the Forkhead-binding site within the c-FLIP promoter (102). Expression of FOXO3a TM, a constitutively active form of FOXO3a, rescues the androgen induction of c-FLIP in LNCaP cells, supporting a critical role of FOXO3a in androgen-induced c-FLIP upregulation (102).…”

“…LNCaP xenografts overexpressing c-FLIP are also resistant to castrationinduced growth inhibition, suggesting a role of c-FLIP in the development of CRPC (99). It has also been observed that both c-FLIP mRNA and protein levels are reduced during progression to CRPC in animal models (101,102). C-FLIP is directly regulated by androgens via a cluster of four AREs within a 156-bp region downstream from the transcription start site (99).…”