Abstract C208: Chemosensitization of ovarian cancer after topotecan modulation of HIF and p53.

Abstract

Abstract Background: Topotecan [TPT] is used to treat ovarian cancer (OC) and targets topoisomerase I (Topo I). OC grows under hypoxic conditions and inevitably fails to respond to chemotherapy. Hypoxia inducible factor-1α (HIF) is an adverse prognostic factor in OC associated with drug resistance. Based on reports that topotecan downregulates HIF1α, and that HIF binds directly to p53, we determined whether TPT-mediated modulation of HIF1α would lead to increased p53 function and chemosensitivity. Methods: Three ovarian cancer cell lines were studied: CAOV3, CAOV4 and TOV112D. Direct p53:HIF binding was determined using monoclonal co-immunoprecipitation (Co-IP) of nuclear extracts from cells grown under hypoxic conditions (1% O2). The effects of TPT on ovarian cancer cell line HIF-1α and p53 levels was determined by ELISA and western blot, as well as on HIF-related downstream reporter gene products VEGF (ELISA), ABCB1 and ABCB5 (Flow Cytometry) and p53-related BAX protein levels (ELISA) and thrombospondin-1 (Thbs1) transcript levels (RT-PCR). The association between TPT effects and expression levels of Topo I (western blot) between cell lines and after Topo I knock-down by siRNA was examined. The effects of sub-lethal doses of TPT on cisplatin dose response were examined under normoxic and hypoxic conditions (XTT assay). RNA immunoprecipitation (RIP) using monoclonal and polyclonal Topo I antibodies was used to obtain HIF1α mRNA interrogated by RT-PCR using three independent HIF-specific primers. Results: Co-IP of CAOV4 and TOV112D nuclear extracts with p53-specific antibodies yielded both HIF1 α and p53 bands on western blots. TPT treated CAOV3, TOV112D, and CAOV4 cells grown under hypoxia showed a dose dependent drop in HIF-1α levels with increasing p53 levels. Hypoxia-related upregulation of vascular endothelial growth factor (VEGF), ABCB1 (p-glycoproteins) and ABCB5 was blocked in CAOV3 and TOV112D and partially blocked in CAOV4 cells after pretreatment with 25 nM TPT, a clinically achievable concentration. With respect to p53 function, increased BAX protein and Thbs1 transcript levels were seen after TPT. Cell lines grown in hypoxia compared to normoxia were more sensitive to TPT. TPT IC50 values were inversely associated with Topo I expression (r= 0.99, p<0.01). Hypoxia related resistance to cisplatin in both CAOV3 and TOV112D cells was reversed after pre-treatment with 25 nM TPT. Topo I silencing RNA (siRNA) blocked TPT-mediated inhibition of HIF-1α stabilization under hypoxic conditions. HIF-1α protein stabilization under hypoxia was unaffected by Topo I siRNA. RIP specific for Topo I yielded a HIF-specific RT-PCR product in both CAOV3 and TOV112D cells. Taken together, these data indicate that topotecan modulates HIF and p53 expression levels and reverses hypoxia related chemoresistance to cisplatin. Topotecan may act by targeting Topo 1 resident on HIF-1α mRNA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C208.