Homologous Recombination Is the Principal Pathway for the Repair of DNA Damage Induced by Tirapazamine in Mammalian Cells

Self Cite

Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcome and few validated drug targets. Two prevalent features of TNBC, tumor hypoxia and derangement of homologous recombination (HR) repair, are potentially exploitable for therapy. This study investigated whether hypoxia-activated prodrugs (HAP) of DNA-damaging cytotoxins may inhibit growth of TNBC by simultaneously addressing these two targets. We measured in vitro activity of HAP of DNA breakers (tirapazamine, SN30000) and alkylators (TH-302, PR-104, SN30548) in TNBC cell lines and isogenic models, and related this to measures of HR repair and expression of prodrug-activating enzymes. Antitumor activity of HAP was examined in isogenic BRCA2-knockout xenograft models and compared with platinum chemotherapy. All five HAP selectively inhibited growth of TNBC cell lines under hypoxia. Sensitivity to HAP was not strongly associated with BRCA1 genotype. However, HAP sensitivity was enhanced by suppression of HR (assessed by radiation-induced RAD51 focus formation) when BRCA1 and PALB2 were knocked down in a common (MDA-MB-231) background. Furthermore, knockout of BRCA2 markedly sensitized DLD-1 cells to the clinical nitrogen mustard prodrugs TH-302 and PR-104 and significantly augmented sterilization of clonogens by these agents in xenografts, both as monotherapy and in combination with radiotherapy, but had less effect on activity of the benzotriazine di-N-oxide SN30000. PR-104 monotherapy was more effective than cisplatin at inhibiting growth of BRCA2-knockout tumors at equitoxic doses. This study demonstrates the potential for HAP of nitrogen mustards to simultaneously exploit hypoxia and HR defects in tumors, with translational implications for TNBC and other HR-deficient malignancies. Mol Cancer Ther; 13(11);

Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 89%

Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in Triple-Negative Breast Cancer

Hunter

Hsu

et al. 2014

Self Cite

“…Interestingly, using one of the same pairs of lines (AA8 and irs1SF), Evans and colleagues observed a 4Â enhanced potency of tirapazamine in the irs1SF compared with the wild-type whereas we observed a 50Â enhancement with TH-302. This difference is consistent with the activity of TH-302 being more specific to the HDR mechanism of action as tirapazamine was shown to also yield lesion repair by BER (13). The findings that TH-302 activity is enhanced in the context of HDR repair pathways is of particular note because of the recent finding that hypoxia results in decreased expression of homologous recombination proteins, including XRCC-3, (the protein mutated in the irs1SF line), BRCA1, as well as other HDR-related genes (42)(43)(44).…”

supporting

confidence: 80%

“…DNA repair mutant Chinese hamster ovary (CHO) cell lines were provided by Martin Brown (Stanford University, Stanford, CA; ref. 13). The UWB1.289 pair was from Elizabeth Swisher (University of Washington, Seattle, WA), the VC-8 pair was from Graeme Smith (KuDOS), and the FANCA pair was from Sara Rockwell (Yale University, New Haven, CT).…”

Section: Cell Linesmentioning

confidence: 99%

Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302

Meng

Evans

Bhupathi

et al. 2012

Self Cite

172

183

TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxiaselective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of gH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell-based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect.

“…The hypoxic selectivity of tirapazamine is due to the rapid reoxidation of the initial radical to the parent prodrug by O 2 . Thus, tirapazamine functions as a hypoxia-activated agent (20,21), exhibiting the capability of potentiating the antitumor efficacy of many anticancer drugs (22)(23)(24)(25). The putative mechanism may be attributed to its suppression of the accumulation of HIF-1a protein.…”

Section: Introductionmentioning

confidence: 99%

Tirapazamine Sensitizes Hepatocellular Carcinoma Cells to Topoisomerase I Inhibitors via Cooperative Modulation of Hypoxia-Inducible Factor-1α

Cai

Liu

Zhu

et al. 2014

Topoisomerase I inhibitors are a class of anticancer drugs with a broad spectrum of clinical activity. However, they have limited efficacy in hepatocellular cancer. Here, we present in vitro and in vivo evidence that the extremely high level of hypoxia-inducible factor-1a (HIF-1a) in hepatocellular carcinoma is intimately correlated with resistance to topoisomerase I inhibitors. In a previous study conducted by our group, we found that tirapazamine could downregulate HIF-1a expression by decreasing HIF-1a protein synthesis. Therefore, we hypothesized that combining tirapazamine with topoisomerase I inhibitors may overcome the chemoresistance. In this study, we investigated that in combination with tirapazamine, topoisomerase I inhibitors exhibited synergistic cytotoxicity and induced significant apoptosis in several hepatocellular carcinoma cell lines. The enhanced apoptosis induced by tirapazamine plus SN-38 (the active metabolite of irinotecan) was accompanied by increased mitochondrial depolarization and caspase pathway activation. The combination treatment dramatically inhibited the accumulation of HIF-1a protein, decreased the HIF-1a transcriptional activation, and impaired the phosphorylation of proteins involved in the homologous recombination repair pathway, ultimately resulting in the synergism of these two drugs. Moreover, the increased anticancer efficacy of tirapazamine combined with irinotecan was further validated in a human liver cancer Bel-7402 xenograft mouse model. Taken together, our data show for the first time that HIF-1a is strongly correlated with resistance to topoisomerase I inhibitors in hepatocellular carcinoma. These results suggest that HIF-1a is a promising target and provide a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and tirapazamine in hepatocellular cancers. Mol Cancer Ther; 13(3); 630-42. Ó2013 AACR.