Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3␤. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.Akt, also named protein kinase B (PKB) 1 or RAC kinase, is a family of phosphatidylinositol 3-OH-kinase-regulated serine/ threonine kinase (1-3). Three isoforms of Akt have been identified: Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥, all of which are activated by growth factors in a phosphatidylinositol 3-OHkinase-dependent manner (4 -6). Accumulated evidence shows that Akt and its downstream targets constitute a major cell survival pathway. Akt promotes cell survival and suppresses apoptotic death in a number of cell types induced by a variety of stimuli, including growth factor withdrawal, cell cycle discordance, and loss of cell adhesion (7). Several downstream targets containing the Akt phosphorylation consensus sequence (R-X-R-X-X-S/T) have been identified which shed light on the mechanisms by which Akt promotes cell survival and blocks apoptosis. The first anti-apoptotic Akt target identified was the pro-apoptotic protein BAD. BAD is a pro-death member of the Bcl-2 family that initiates apoptosis by binding to Bcl-x L on the outer mitochondrial membrane, causing the release of cytochrome c into the cytosol. Akt phosphorylates BAD on Ser 136 , promoting the association of BAD with 14-3-3 proteins in the cytosol and inactivating its proapoptotic function (7). The execution of cellular apoptosis also involves changes in the transcriptional program (7). Akt decreases the transcription of a subset of death genes by phosphorylation of the Forkhead family of transcription factors, which causes their nuclear exclusion and inactivation (7). Akt also phosphorylates and activates the cyclic AMP-response element-binding pr...

Akt is a determinant of cisplatin [cis-diammine-dichloroplatinum (CDDP)] resistance in ovarian cancer cells, and this may be related to the regulation of p53. Precisely how Akt facilitates CDDP resistance and interacts with p53 is unclear. Apoptotic stimuli induce second mitochondria-derived activator of caspase (Smac) release from mitochondria into the cytosol, where it attenuates inhibitor of apoptosis proteinmediated caspase inhibition. Whereas Smac release is regulated by p53 via the transactivation of proapoptotic Bcl-2 family members, it is unclear whether p53 also facilitates Smac release via its direct mitochondrial activity. Here we show that CDDP induces mitochondrial p53 accumulation, the mitochondrial release of Smac, cytochrome c, and HTR/Omi, and apoptosis in chemosensitive but not in resistant ovarian cancer cells. Smac release was p53 dependent and was required for CDDP-induced apoptosis. Mitochondrial p53 directly induced Smac release. Akt attenuated mitochondrial p53 accumulation and Smac/cytochrome c/Omi release and conferred resistance. Inhibition of Akt facilitated Smac release and sensitized chemoresistant cells to CDDP in a p53-dependent manner. These results suggest that Akt confers resistance, in part, by modulating the direction action of p53 on the caspase-dependent mitochondrial death pathway. Understanding the precise etiology of chemoresistance may improve treatment for ovarian cancer.

Resistance to cisplatin-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Wild-type TP53 status is often, but not always, associated with cisplatin sensitivity, suggesting that additional factors may be involved. Overexpression/ activation of the phosphatidylinositol-3-kinase/Akt pathway is commonly observed in ovarian cancer, and Akt activation is a determinant of chemoresistance in ovarian cancer cells, an effect that may be due, in part, to its inhibitory actions on p53-dependent apoptosis. To that end, we examined the role and regulation of p53 in chemosensitive ovarian cancer cells, as well as in their chemoresistant counterparts, and investigated if and how Akt influences this pathway. Cisplatin induced apoptosis in chemosensitive, but not chemoresistant cells, and this was inhibited by downregulation of p53. Cisplatin upregulated PUMA in a p53-dependent manner, and the presence of PUMA was necessary, but not sufficient for cisplatin-induced apoptosis. p53 was phosphorylated on numerous N-terminal residues, including Ser15, Ser20, in response to cisplatin in chemosensitive, but not chemoresistant cells. Furthermore, activation of Akt inhibited the cisplatininduced upregulation of PUMA, and suppressed cisplatin-induced p53 phosphorylation, while inhibition of Akt increased total and phospho-p53 contents and sensitized p53 wild-type, chemoresistant cells to cisplatin-induced apoptosis. Finally, mutation of Ser15 and/or Ser20, but not of Ser37, to alanine significantly attenuated the ability of p53 to facilitate CDDP-induced apoptosis, and this was independent of PUMA expression. These results support the hypothesis that p53 is a determinant of CDDP sensitivity, and suggest that Akt contributes to chemoresistance, in part, by attenuating p53-mediated PUMA upregulation and phosphorylation of p53, which are essential, but independent determinants of sensitivity to CDDP-induced apoptosis. ' 2007 Wiley-Liss, Inc.Key words: ovarian cancer; apoptosis; p53; Akt; cisplatin Resistance to cisplatin (CDDP: cis-diaminedichloroplatinum)-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Chemoresistance is a multifactorial phenomenon, the molecular mechanisms of which are poorly understood. While alterations in DNA platination do not appear to play a significant role in chemoresistance, 1 induction of apoptosis is a key effect of CDDP chemotherapy, 2 and alterations in the apoptotic capacity are frequently observed and are important determinants of chemosensitivity. 3-5 Indeed, we and others have shown that key regulators of the apoptotic response to CDDP, including X-linked inhibitor of apoptosis protein (XIAP), FLIP, and the MAP kinases (p38, JNK, ERK), are dysregulated in chemoresistant ovarian cancer cells, 1,3,4,[6][7][8] relative to their sensitive counterparts.The normal function of the p53 tumor suppressor is associated with chemosensitivity and improved clinical outcome in ovarian cancer. 3,9-11 Indeed, our previous data suggest that p53 is a determina...