Abstract 1645: The phosphorylation status of BAD pathway influence to chemo-sensitivity

Abstract

Abstract Objective: Patients with advanced or recurrent endometrial cancer have a poor prognosis in part due to lack of efficacy of currently available chemotherapies and incomplete understanding of the biology that underlies chemo-response. In the current study we used genomic expression data to identify molecular signaling pathways associated with endometrial cancer cell resistance to cisplatin, doxorubicin chemotherapy and further characterized the role of the BAD apoptosis pathway as a therapeutic target and determinant of chemo-response. Methods: Eight endometrial cell lines were subject to genome-wide expression analysis using Affymetrix HG-U133Plus GeneChips and in parallel, ranked by relative sensitivity to cisplatin and doxorubicin using MTS cell proliferation assays. Gene array data from sensitive versus resistant cell lines was compared by student's t-test and Pearson's correlation to identify those genes associated with drug resistance and further evaluated by GeneGo Metacore software for representation of molecular signaling pathways. Furthermore, the role of the BAD pathway in chemo-resistance was evaluated by selective inhibition of BAD pathway members, PKA, PP2C, AKT1, AKT2 and AKT3 by siRNA or pharmacologic agents in the presence and absence of cisplatin. Cell death and growth arrest in the presence of cisplatin were evaluated by Western blot for PARP cleavage as well as nuclear condensation and fragmentation and MTS assays. Results: Student's t-test and Pearson's correlation identified the expression of 325 and 1136 genes to be associated with cisplatin (p<0.01) and doxorubicin (p<0.01) resistance, respectively. Pathway analysis of genes associated with both cisplatin and doxorubicin resistance indicated significant representation of the BAD apoptosis pathway (p<0.002). Selective depletion of the phosphatase, PP2C by siRNA resulted in increased phosphorylation of BAD, serine 118 and increased resistance to cisplatin induced cell growth arrest and cell death. In contrast, depletion of the kinase, PKA resulted in decreased phosphorylation of BAD, serine 118 and increased cell death in the presence of cisplatin. In addition, depletion of AKT1 using siRNA as wells as treatment of cells with the AKT inhibitor, triciribine, increased cisplatin-induced growth arrest and cell death, whereas depletion of AKT2 or AKT3 did not affect cisplatin sensitivity. Conclusions: The phosphorylation status BAD may influence endometrial cancer cell sensitivity to chemotherapy and represents a compelling target for future therapeutic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1645.