Cell Fate Decisions HIF Inactivation of p53 in Ovarian Cancer Can Be Reversed by Topotecan, Restoring Cisplatin and Paclitaxel Sensitivity Basmina Parmakhtiar 1 , Robert A. Burger 2 , Jai-Hyun Kim 3 , and John P. Fruehauf 1,3 Abstract Ovarian cancer growth under hypoxic conditions results in hypoxia-inducible factor-1a (HIF1a) stabilization. HIF1a is an adverse prognostic factor that may contribute to worse outcomes via its capacity to bind to p53, potentially blocking p53-mediated apoptosis. We determined whether HIF1a-p53 binding occurred in hypoxic ovarian cancer cell lines, and if this blocked p53 transcriptional activity. Topotecan (TPT), used in the treatment of ovarian cancer, inhibits HIF1a trans- lation via a topoisomerase-1 (TOPO1)–dependent mecha- nism. We examined if TPT knockdown of HIF1a restored p53 transcriptional function. TPT effects on HIF1a and p53- related transcriptional targets were assessed by PCR. Associa- tions between TPT effects and TOPO1 expression levels were examined by Western blots and knockdown by siRNA. RNA- binding protein immunoprecipitation was used to assess if TOPO1 was resident on HIF1a mRNA. We determined if sublethal doses of TPT, used to knockdown HIF1a, reversed hypoxia-related cisplatin and paclitaxel resistance (XTT assay). Flow cytometry was used to assess HIF1a-mediated upregula- tion of ABCB1 and ABCB5 efflux pump expression. We found that HIF1a binding to, and inhibition of, p53 transcriptional activity in hypoxic ovarian cancer cells was associated with drug resistance. TPT-mediated downregulation of HIF1a in hypoxic cells required TOPO1 resident on HIF1a mRNA, restored p53 transcriptional activity, downregulated ABCB1/ ABCB5 cell surface expression, and reversed hypoxia-related cisplatin and paclitaxel resistance. Implications: TPT-mediated reduction of HIF1a accumula- tion in hypoxic ovarian cancer cell lines restores p53 tumor- suppressor function, offering a novel approach to reverse chemoresistance. Further clinical investigation is warranted. Introduction Ovarian cancer is the second most common and the most lethal gynecologic malignancy (1). Primary treatment after cytoreduc- tion consists of platinum/taxane-based chemotherapy, with response rates of up to 70% (2). Acquired resistance to platins and taxanes occurs for most patients, rendering them less respon- sive to further therapy (3, 4). Although newer agents have emerged, including bevacizumab and PARP inhibitors, the out- look for advanced-stage ovarian cancer remains challenging (5, 6). Chemoresistance is associated with adaptive responses to tumor hypoxia, including the transition to a cancer stem cell pheno- type (4, 7–10). Cancer survival pathways in hypoxia are driven primarily by the HIF complex, comprised of hypoxia-inducible factor-1a (HIF1a) and HIF1b (8, 11). Under normoxic conditions, cytosolic HIF1a is rapidly hydroxylated by prolyl hydroxylase (PHD), promoting Von Hippel–Lindau binding and HIF1a degradation (12). Under hypoxic conditions, mitochondria efflux hydrogen peroxide, inhibiting PHD-mediated HIF1a hydroxylation, resulting in HIF1a accumulation and formation of the functional HIF1a: HIF1b (HIF) transcription factor (13, 14). HIF binds to hypoxia response elements (HRE), activating cassettes that support angio- genesis and acquisition of a drug-resistant stem-cell pheno- type (4, 10, 14, 15). These include VEGF and multidrug resistance (MDR) genes ABCB1, ABCG2, and ABCB5 (9, 10, 15–17). The p53 tumor-suppressor protein modulates cell-cycle pro- gression, apoptosis, and DNA repair, and has been implicated in ovarian cancer treatment response (18, 19). P53 mutations are found in >60% of epithelial ovarian cancer (20). P53 gene mutations alter protein function, with some downstream com- ponents conserved and/or altered (21–24). Direct binding interactions between HIF's oxygen-dependent domain and the DNA-binding domain of tetrameric p53 have been reported (25). Hypoxic conditions are associated with p53 accumulation and decreased downstream transcription (26–28). We speculated that HIF1a accumulation in hypoxia could pro- mote its binding to p53, interfering with p53's capacity to trigger apoptosis after chemotherapy (18, 19, 23). Topotecan (TPT), a topoisomerase-1 (TOPO1) inhibitor used in second-line treatment of ovarian cancer, prevents hypoxia- related HIF1a accumulation in a TOPO1-dependent manner, leading to decreased VEGF expression (29, 30). Metronomic TPT downregulates HIF in vivo (31). We determined if HIF:p53 com- plex formation occurred in ovarian cancer cell lines under hypoxic 1 Department of Biological Chemistry, University of California Irvine, Irvine, California. 2 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania. 3 Chao Family Comprehensive Cancer Center, University of California Irvine, Orange, California. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: John P. Fruehauf, UCI Health, 101 The City Drive South, Bldg. 56 Heme Onc, Orange, CA 92868. Phone: 714-456-7670; Fax: 714-456- 7668; E-mail: jfruehau@uci.edu Mol Cancer Res 2019;17:1675–86 doi: 10.1158/1541-7786.MCR-18-1109 Ó2019 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org 1675 on November 6, 2019. © 2019 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst May 14, 2019; DOI: 10.1158/1541-7786.MCR-18-1109