Abstract 453: Identification of human oxidoreductases involved in the hypoxia-dependent activation of bioreductive prodrugs
Authors: Guise Christopher P. , Tipparaju Smitha R. , Abbattista Maria R. , Dachs Gabi U. , Su Jiechuang , Foehrenbacher Annika B. , Wilson William R. , Patterson Adam V. 
Published: February 2011

Abstract
Hypoxia occurs in most human tumors and is associated with resistance to conventional therapies and poor patient outcomes. Eliminating hypoxic tumor cells therefore remains an important therapeutic goal. A number of bioreductive prodrugs are undergoing clinical evaluation as hypoxia selective cytotoxins, including PR-104, TH-302, tirapazamine (TPZ) and porfiromycin (POR). These prodrugs are activated in tumor cells by one-electron oxidoreductases (1eR) in a process that is reversed by molecular oxygen. Relatively few 1eR have been studied, with the notable exception of cytochrome P450 reductase (CYPOR), known to activate many prodrug substrates including the dinitrobenzamide mustard prodrug PR-104A (Guise et al., Biochem Pharmacol 2007;74:810). We now describe the identification of three additional PR-104A 1eR enzymes. Expression of twelve candidate 1eR in the HCT116 colorectal cancer cell line was demonstrated by induction of an occult V5-tag. Significant increases in hypoxic PR-104A metabolism were observed in cells expressing CYPOR, methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1) and inducible nitric oxide synthase (NOS2A). Hypoxic PR-104A metabolism was enhanced in the order CYPOR (3420 ± 250.5) &gt; MTRR (1514.8 ± 240.1) &gt; NOS2A (1018.1 ± 204.9) &gt; NDOR1 (819.1 ± 9.8) as compared to HCT116 WT (411.5 ± 54.3 pmol metabolites/106 cells/hr). We further characterized the role of these 1eR in the cytotoxicity of PR-104A, TPZ, POR and TH-302, and complemented this analysis by determining the extent to which this catalytic activity can be detected by the hypoxia imaging agents pimonidazole (PIMO) and pentafluoro-etanidazole (EF5). Binding of the imaging agents was differentially enhanced by the expression of these 1eR in the order CYPOR &gt;NDOR1 &gt;MTRR &gt;NOS2A. These data suggest it may be possible to utilise hypoxia PET probes as biomarkers for non-invasive detection of hypoxic cell metabolic capacity.
A specific mAb was identified for each of the four 1eR and validated by immunoblot against matched V5-tag induced lysates. Next, mAb validation was extended to paraffin-embedded formalin-fixed (PEFF) cell plugs of each clonal cell population. The CYPOR mAb (1:50 dilution, Santa Cruz Biotech, sc-25263) was found to be of excellent specificity for IHC detection of CYPOR, and this validation was extended using PEFF tissue sections of parental (WT), CYPOR-overexpressing and CYPOR knockout HCT116 tumor xenografts. In order to translate these findings, tissue microarrays of various human tumors were evaluated for CYPOR expression. We identified several tumor types, including lung, breast, prostate and pancreas, where a proportion of cases displayed elevated levels of CYPOR protein in tumor epithelium. Thus CYPOR may be a suitable biomarker to identify diseases or individuals that may benefit from treatment with hypoxic cytotoxins.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 453.