Supplemental Figure 1. Effect of TPT on VEGF levels in ovarian cancer cell Lines. Supplemental Figure 2. P53 knockdown reverses topotecan effects on downstream levels of p53 target transcripts BAX, CDKN1A, GADD45, and Thbs1. Supplemental Figure 3. TPT dose response for CAOV3, TOV112D, and CAOV4 cells treated with topotecan for 72 hours +/- hypoxia and tested for proliferation inhibition using the XTT assay. Supplemental Figure 4. Confirmation that free TOPO1 bound to HIF-1α mRNA in solution. Supplemental Table 1. OC cell line expression levels of TOPO1 determined by western blotting as the ratio of TOPO1 to actin band density read on a Kodak 2000MM photoimager.
ARTICLE ABSTRACT
Ovarian cancer growth under hypoxic conditions results in hypoxia-inducible factor-1α (HIF1α) stabilization. HIF1α 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 HIF1α-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 HIF1α translation via a topoisomerase-1 (TOPO1)–dependent mechanism. We examined if TPT knockdown of HIF1α restored p53 transcriptional function. TPT effects on HIF1α and p53-related transcriptional targets were assessed by PCR. Associations 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 HIF1α mRNA. We determined if sublethal doses of TPT, used to knockdown HIF1α, reversed hypoxia-related cisplatin and paclitaxel resistance (XTT assay). Flow cytometry was used to assess HIF1α-mediated upregulation of ABCB1 and ABCB5 efflux pump expression. We found that HIF1α binding to, and inhibition of, p53 transcriptional activity in hypoxic ovarian cancer cells was associated with drug resistance. TPT-mediated downregulation of HIF1α in hypoxic cells required TOPO1 resident on HIF1α mRNA, restored p53 transcriptional activity, downregulated ABCB1/ABCB5 cell surface expression, and reversed hypoxia-related cisplatin and paclitaxel resistance.
TPT-mediated reduction of HIF1α accumulation in hypoxic ovarian cancer cell lines restores p53 tumor-suppressor function, offering a novel approach to reverse chemoresistance. Further clinical investigation is warranted.
