Abstract 3225: SHetA2 targets mortalin-p53 interaction in differential induction of apoptosis in ovarian cancer over normal cells

Introduction: Identical TP53 mutations observed in fallopian tube pre-neoplastic lesions and high grade serous ovarian cancer (HGSOC) from the same patient suggest that these mutations drive ovarian carcinogenesis. While elevated p53 protein expression caused by these mutations increase cell survival, persistently high levels can cause apoptosis. Mortalin (HSPA9) is a chaperone protein which plays a major role in protecting cancer cells from p53-mediated apoptosis by binding to p53 and preventing it from translocating to the nucleus where it transactivates apoptosis-inducing genes and to the mitochondria where it inhibits Bcl-2 and induces Bax pore formation. SHetA2 is an anti-cancer drug that binds mortalin and induces apoptosis in cancer, but not in normal cells. We hypothesized that release of p53 from mortalin contributes to the mechanism of SHetA2-induced apoptosis in cancer, but not in normal cells. Methods: SHetA2 effects on mortalin and p53 interaction and subcellular localization were evaluated by co-immunoprecipitation in cell extracts and fluorescent cytochemistry of human ovarian cancer cell lines and tissues and human fallopian tube secretory epithelial cells (hFTSECs) cultures and tissues. Effects of SHetA2 and manipulations of mortalin and p53 expression, function and localization in ovarian cancer cells were measured using the MTS dye to detect viable cells, Flow cytometric analysis of Annexin-V/PI cell staining to detect apoptosis and a PCR array to measure p53 regulated gene expression. Results: Mortalin bound to p53 in protein extracts of cultures and tissues from ovarian cancer, but not from hFTSECs. SHetA2 caused dose-responsive apoptosis in association with nuclear and mitochondrial accumulation of p53 in ovarian cancer cell lines harboring wild type or S215R, L130V TP53 mutants, but not in hFTSECs. Mortalin overexpression or p53 knockdown reduced SHetA2 cytotoxicity in ovarian cancer cells. Mortalin knockdown, p53 overexpression, and chemical inhibitors of p53 transcription (Pifithrin-α) and p53 mitochondrial localization (Pifithrin-µ) were lethal to ovarian cancer cells at effective concentrations and therefore ineffective tools for probing the SHetA2 apoptosis mechanism. In the A2780 cell line containing wild type p53, SHetA2 caused ≥2 fold regulation of p53 signaling genes (p21, FOXO3, IGF1R, MDM4, SESN2, SIAH1, DR5, CAPS2, CCNE1, CDC25A, E2F1, PCNA and TP63). Our analysis of p53 mutations using The Cancer Genome Atlas (TCGA) data revealed that majority of HGSOC exhibited p53 mutations spanning from exon 3-9, with the majority being missense mutations in the DNA binding domain associated with increased protein stability and gain of function. Conclusion: Release of wild type and mutant p53 proteins from mortalin contributes to the mechanism of SHetA2-induced apoptosis in cancer cells and does not occur in healthy cells. Funded by R01CA196200. Citation Format: Satish Kumar Ramraj, Richard Pelikan, Vishal Chandra, Doris Benbrook. SHetA2 targets mortalin-p53 interaction in differential induction of apoptosis in ovarian cancer over normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3225. doi:10.1158/1538-7445.AM2017-3225