Regulation of p53-dependent apoptosis: Implications for cell fate determination and cisplatin resistance in human ovarian cancer cells in vitro

Abstract

Programmed cell death (apoptosis), is a normal physiological process which involves a distinct, well-characterized signaling cascade. However, dysregulation of the apoptotic machinery is commonly observed in many pathophysiological conditions. In particular, human cancer is commonly associated with a reduced capacity of cells to undergo apoptosis in response to cell stress, leading to an abnormal accumulation of damaged cells. Human ovarian cancer is the most lethal gynecological malignancy, a statistic that is due, in part, to the phenomenon a chemoresistance, by which tumour cells evade the cytotoxic effects of chemotherapeutic agents used to eradicate the disease. Cisplatin (CDDP) and paclitaxel are first-line chemotherapeutics for human ovarian cancer, but resistance to these agents is common, and significantly attenuates positive clinical outcomes. Recent evidence suggests that aberrant regulation of the apoptotic cascade may be a causative factor for chemoresistance. To that end, gene products implicated in the regulation of apoptosis, including the P13K/Akt and Inhibitor of Apoptosis Protein family are frequently over-expressed and/or dysregulated in chemoresistant cells. Activation of the TP53 tumor suppressor gene product, p53, is critical for DNA-damage-induced apoptosis in many cell types, and mutation of this gene is the most frequently observed abnormality in all of human cancer. p53 mutations are commonly, but not always, associated with poor prognosis and chemoresistance. Since the aberrant regulation of p53 may have critical implications for the clinical management of human ovarian cancer, it is of paramount importance to understand the molecular and cellular mechanisms by which p53 is regulated in these cells, and if and how dysregulated p53 activation may play a role in the etiology of chemoresistance. Cultured human ovarian cancer cells were used to establish the in vitro regulation of p53 and to assess the requirement for p53-dependent apoptosis for CDDP-induced apoptosis. We observed that CDDP induced apoptosis in chemosensitive ovarian cancer cells, but not in their chemoresistant variants. In addition, we demonstrated that over-expression of X-Linked Inhibitor of Apoptosis Protein (XIAP) induces chemoresistance in ovarian cancer cells, while down-regulation of XIAP sensitizes chemoresistant ovarian cancer cells to CDDP-induced apoptosis. Attenuation of Akt signalling using a dominant-negative Akt attenuated XIAP-mediated chemoresistance, suggesting a functional link between XIAP and Akt with respect to the regulation of chemosensitivity. Akt activation, a frequently observed event in human ovarian cancer, inhibited CDDP-induced apoptosis in chemosensitive cells, while dominant-negative Akt up-regulated p53 and sensitized chemoresistant cells to CDDP in a p53-dependent manner, suggesting a functional link between Akt activation and p53-mediated apoptosis. Akt-mediated chemoresistance was also associated with ablated CDDP-induced down-regulation of the anti-apoptotic protein XIAP. We further demonstrated that CDDP induced the up-regulation of the p53-responsive gene product PUMA, and induced apoptosis in a PUMA-dependent manner, although PUMA expression was not sufficient to confer a chemosensitive phenotype. CDDP also induced phosphorylation of p53 on numerous N-terminal residues in chemosensitive, but not chemoresistant cells. Activation of Akt inhibits the CDDP-induced phosphorylation of p53, while inhibition of Akt function induces p53 phosphorylation. Phosphorylation of Ser15 and Ser20, but not Ser37, was required for p53-dependent apoptosis but not for p53-dependent up-regulation of PUMA. Moreover, we showed that basal p53 levels are maintained, in part, by the constitutive activation of the soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway, and inhibition of this pathway depletes basal cGMP levels, up-regulates p53 content, stability, and phosphorylation, and induces apoptosis in a partially p53-dependent manner. These results suggest that: (a) p53 is a critical determinant of cell fate in human ovarian cancer cells, and is regulated by diverse signaling cascades including the Akt and sGC/cGMP pathways, (b) CDDP-induced apoptosis proceeds via a p53-dependent mechanism involving PUMA and p53 phosphorylation, (c) Akt confers resistance to ovarian cancer cells, in part, by suppressing the CDDP-induced activation and phosphorylation of p53. Akt also contributes to chemoresistance by attenuating the CDDP-induced down-regulation of XIAP. The current study significantly extends our understanding of how the p53 pathway is regulated, and how dysregulation of p53 activation may have profound effects upon the sensitivity of ovarian cancer cells to stress-induced apoptosis. This, in turn, may have important effects upon our understanding of the pathophysiology of chemoresistance in human ovarian cancer; a phenomenon that significantly attenuates successful treatment outcomes for this disease.