Regulation of survivin by PI3K/Akt/p70S6K1 pathway

Abstract

PI3K activation is commonly observed in many human cancer cells. Survivin expression is elevated in cancer cells, and induced by some growth factors through PI3K activation. However, it is not clear whether PI3K activation is sufficient to induce survivin expression. To investigate the role of PI3K pathway in the regulation of survivin, we expressed an active form of PI3K, v-P3k in chicken embryonic fibroblast cells (CEF), and found that overexpression of PI3K-induced survivin mRNA expression. Forced expression of wild-type but not mutant tumor suppressor PTEN in CEF decreased survivin mRNA levels. PI3K regulates survivin expression through Akt activation. To further investigate downstream target of PI3K and Akt in regulating the expression of survivin mRNA, we found that PI3K and Akt-induced p70S6K1 activation and that overexpression of p70S6K1 alone was sufficient to induce survivin expression. The treatment of CEF cells by rapamycin decreased the survivin mRNA expression. This result demonstrated that p70S6K1 is an important target downstream of PI3K and Akt in regulating suvivin mRNA expression. The knockdown of survivin mRNA expression by its specific siRNA induced apoptosis of cancer cells when the cells were treated with LY294002 or taxol. Taken together, these results demonstrated that PI3K/Akt/p70S6K1 pathway is essential for regulating survivin mRNA expression.

Introduction

Apoptosis is an important process for cell and tissue homeostasis. Apoptotic effector molecules and disordered apoptosis are involved in various diseases. The inhibitor of apoptosis protein (IAP) inhibits apoptosis by inactivating several caspases [1], [2]. Survivin is a newly described member of the IAP family [3]. Survivin is constitutively expressed in most cancers, including carcinomas of the lung, colon, pancreas, prostate, breast, stomach, ovarian and in most hematopoietic malignancies, and is an unfavorable prognostic marker [4]. Survivin can inhibit apoptosis by blocking a common step downstream of mitochondrial cytochrome c release by inhibiting terminal effectors caspase-3 and caspase-7, and by interfering with caspase-9 activity and processing [5], [6]. Overexpression of survivin can lead to resistance to apoptotic stimuli including chemotherapy.

Survivin is expressed primarily in fetal, but not adult tissues. The expression of survivin is cell cycle dependent. It is selectively expressed at the G2/M phase of the cell cycle in a cell cycle-regulated manner, and localized with caspase-3 to mitotic spindle microtubules [7]. Hematopoietic and vascular remodeling cytokines, STAT3-dependent signaling and phosphotidylinositol-3-kinase (PI3K) activity affect survivin expression through non-cell cycle dependent mechanisms [8]. PI3K/Akt signaling pathway has been implicated to play an important role in the upregulation of survivin in both vascular endothelial cells and tumor cells. For example, in endothelial cells, angiopoietin-1 inhibits cell apoptosis via the Akt/survivin pathway [9]. VEGF binding to VEGF-R2 activates the PI3K pathway and induces the expression of survivin in endothelial cells [10]. In cancer cells, survivin protein level is upregulated by coexpression of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) through PI3K/Akt signaling pathway in breast cancer cells [11]. Hematopoietic cytokine GM-CSF exerts the anti-apoptotic and mitogenic effects partially by increasing survivin levels through the activation of PI3K, while PI3K inhibitor LY294002 inhibits the effect of GM-CSF in acute myeloid leukemia cells by decreasing survivin expression at mRNA and protein levels [12]. Exposure of human neuroblastoma cells to exogenous VEGF results in an increased expression of survivin protein and phosphorylated Akt, and inhibition of PI3K abrogates those effects [13]. Inhibition of PI3K pathway also downregulates survivin expression, and enhances TRAIL-mediated apoptosis in neuroblastomas [14]. Geranylgeranyltransferase I inhibitors (GGTIs) induce apoptosis in both cisplatin-sensitive and -resistant human ovarian epithelial cells by inhibition of PI3K/Akt and survivin pathways [15]. These results suggest that PI3K/Akt signaling pathway is involved in regulating survivin expression in response to cytokines, growth factors, and chemotherapeutic drugs. However, it is unknown whether PI3K is sufficient to induce survivin expression.

Epithelial ovarian cancer is the fourth biggest cause of cancer-related death in women. Taxol is one of the first-line chemotherapeutic drugs for ovarian cancer, especially for advanced ovarian cancer [16]. Mitotic deregulation by survivin in ErbB2-overexpressing breast cancer cells and ovarian cancer cells is proposed to contribute to taxol resistance [17], [18]. A recent study has shown that survivin counteracts the therapeutic effect of microtubule de-stabilizers by stabilizing tubulin polymers, which may contribute to taxol resistance through microtubule-targeting [19]. Our previous studies have demonstrated that PI3K/Akt/p70S6K1 is an important signaling pathway in regulating ovarian tumorigenesis and angiogenesis [20], [21], [22]. Since high levels of survivin protein are detected in advanced ovarian carcinomas [23], we hypothesize that PI3K is sufficient to induce surviving expression. In this study, we will use chicken embryo fibroblast (CEF) cells as a model to test: (1) whether PI3K activation alone induced endogenous survivin expression, (2) whether inhibition of PI3K signaling by PTEN and LY294002 decreased survivin expression; and (3) what are the downstream molecules of PI3K that regulate survivin expression. These results will allow us to identify the role of PI3K in mediating survivin expression, and to understand the mechanism of PI3K in regulating survivin expression.