Inhibition of protein kinase C (PKC) activity in transformed cells and tumor cells containing activated p21RAS results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21 RAS oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21RAS . Dominant-negative mutants of PKC, short interfering RNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKC␦ isozyme demonstrated that PKC␦ plays a critical role in p21 RAS -mediated apoptosis. An activating p21 RAS mutation, or activation of the phosphatidylinositol 3-kinase (PI3K) Ras effector pathway, increased the levels of PKC␦ protein and activity in cells, whereas inhibition of p21 RAS activity decreased the expression of the PKC␦ protein. Activation of the Akt survival pathway by oncogenic Ras required PKC␦ activity. Akt activity was dramatically decreased after PKC␦ suppression in cells containing activated p21RAS . Conversely, constitutively activated Akt rescued cells from apoptosis induced by PKC␦ inhibition. Collectively, these findings demonstrate that p21 RAS , through its downstream effector PI3K, induces PKC␦ expression and that this increase in PKC␦ activity, acting through Akt, is required for cell survival. The p21 RAS effector molecule responsible for the initiation of the apoptotic signal after suppression of PKC␦ activity was also determined to be PI3K. PI3K (p110 CAAX , where AA is aliphatic amino acid) was sufficient for induction of apoptosis after PKC␦ inhibition. Thus, the same p21 RAS effector, PI3K, is responsible for delivering both a pro-apoptotic signal and a survival signal, the latter being mediated by PKC␦ and Akt. Selective suppression of PKC␦ activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21 RAS .The ras oncogene family is among the most commonly mutated group of genes in human cancer. Its protein products code for three closely related p21 RAS proteins, including H-Ras, K-Ras, and N-Ras. p21RAS proteins are localized in the inner plasma membrane, bind GDP and GTP, and possess an intrinsic GTPase activity. p21RAS proteins function as plasma membrane-bound guanine nucleotide-binding proteins and act as molecular switches, thereby regulating signal transduction pathways for hormones, growth factors, and cytokine receptors (1). Several downstream effector proteins of p21 RAS have been identified that bind preferentially to p21 RAS in the GTP-bound state, including Raf, phosphatidylinositol 3-kinase (PI3K), 2 and a family of GDP-GTP exchange factors for the Ral small GTPases (Ral-GDS). Raf proteins, which are proto-oncogeneencoded serine/threonine kinases, activate the MEK-ERK signaling pathway. PI3K activation results in the activation of the anti-apoptotic serine/threonine kinase Akt, among other molecules. Other p21 RAS targets include the GTPase-activating proteins, p120 GAP and neurofibromin (2). p21 RAS proteins were shown to influence proliferation, d...

SIRT1, an NAD-dependent histone/protein deacetylase, has classically been thought of as a nuclear protein. In this study, we demonstrate that SIRT1 is mainly localized in the nucleus of normal cells, but is predominantly localized in the cytoplasm of the cancer / transformed cells we tested. We found this predominant cytoplasmic localization of SIRT1 is regulated by elevated mitotic activity and PI3K/IGF-1R signaling in cancer cells. We show that aberrant cytoplasmic localization of SIRT1 is due to increased protein stability and is regulated by PI3K/IGF-1R signaling. In addition, we determined that SIRT1 is required for PI3K-mediated cancer cell growth. Our study represents the first identification that aberrant cytoplasm localization is one of the specific alternations to SIRT1 that occur in cancer cells, and PI3K/IGF-1R signaling plays an important role in the regulation of cytoplasmic SIRT1 stability. Our findings suggest that the over-expressed cytoplasmic SIRT1 in cancer cells may greatly contribute to its cancer-specific function by working downstream of the PI3K/IGF-1R signaling pathway.

Androgen antagonists or androgen deprivation is a primary therapeutic modality for the treatment of prostate cancer. Invariably, however, the disease becomes progressive and unresponsive to androgen ablation therapy (hormone refractory). The molecular mechanisms by which the androgen antagonists inhibit prostate cancer proliferation are not fully defined. In this report, we demonstrate that sirtuin 1 (SIRT1), a nicotinamide adenosine dinucleotide-dependent histone deacetylase (HDAC) linked to the regulation of longevity, is required for androgen antagonist-mediated transcriptional repression and growth suppression. Androgen antagonist-bound androgen receptor (AR) recruits SIRT1 and nuclear receptor corepressor to AR-responsive promoters and deacetylates histone H3 locally at the prostate-specific antigen promoter. Furthermore, SIRT1 down-regulation by small interfering RNA or by pharmacological means increased the sensitivity of androgen-responsive genes to androgen stimulation, enhanced the sensitivity of prostate cancer cell proliferative responses to androgens, and decreased the sensitivity of prostate cancer cells to androgen antagonists. In this study, we demonstrate the ligand-dependent recruitment of a class III HDAC into a corepressor transcriptional complex and a necessary functional role for a class III HDAC as a transcriptional corepressor in AR antagonist-induced transcriptional repression. Collectively, these findings identify SIRT1 as a corepressor of AR and elucidate a new molecular pathway relevant to prostate cancer growth and approaches to therapy.