Amplicon-Dependent CCNE1 Expression Is Critical for Clonogenic Survival after Cisplatin Treatment and Is Correlated with 20q11 Gain in Ovarian Cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers

Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model

BACKGROUND:Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. METHODOLOGY/PRINCIPAL FINDING:Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c-VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. SIGNIFICANCE:Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis

Overexpression of Fatty Acid Synthase in Middle Eastern Epithelial Ovarian Carcinoma Activates AKT and Its Inhibition Potentiates Cisplatin-Induced Apoptosis

Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of fatty acids, has been shown to be deregulated in several cancers, including epithelial ovarian carcinoma (EOC). In this study, we investigated the function of the FASN signaling pathway in a large series of Middle Eastern EOC patient samples, a panel of cell lines and nude mouse model. Using immunohistochemistry, we detected overexpression of FASN in 75.5% (114/151) of the tumor samples. Overexpression of FASN was associated significantly with tumor proliferative marker Ki-67 (P = 0.0009), activated AKT (P = 0.0117) and XIAP (P = 0.0046). Treatment of EOC cell lines with C-75, a selective inhibitor of FASN, caused inhibition of EOC cell viability via induction of apoptosis. Inhibition of FASN by C-75 led apoptosis via the mitochondrial pathway. FASN inhibition caused downregulation of activated AKT and its downstream targets. In addition, inhibition by FASN siRNA caused downregulation of FASN and activation of caspases, suggesting the role of FASN in C-75 mediated apoptosis. Furthermore, treatment of EOC cells with subtoxic doses of C-75 augmented the effect of cisplatin-mediated induction of apoptosis. Finally, treatment of EOC cell line xenografts with a combination of C-75 and cisplatin resulted in growth inhibition of tumors in nude mice through downregulation of FASN and activation of caspases. Altogether, our results show overexpression of FASN in Middle Eastern EOC, suggesting that FASN may be a potential therapeutic target in a subset of EOC, alone or in combination with other conventional chemotherapeutic agents