Abstract 1390: shRNA mediated suppression of thymidylate synthase results in an increased sensitivity to ionizing radiation similar to 5-fluoro-2′-deoxyuridine

Abstract

Abstract The fluoropyrimidines (FPs), 5-fluorouracil (5-FU) and 5-fluoro-2’-deoxyuridine (FdUrd), are widely used as radiosensitizers in the treatment of gastrointestinal cancer. The FPs exert their cytotoxic effects through activation to 5-fluoro-2’-deoxyuridine 5’-monophosphate (FdUMP), an inhibitor of thymidylate synthase (TS), resulting in depletion of dTTP and inhibition of DNA synthesis. Incorporation of fluorodeoxyuridine 5’-triphosphate (FdUTP) and dUTP into DNA can also elicit cytotoxicity. Radiosensitization by FdUrd correlates with dTTP depletion and S-phase arrest, but not with cytotoxicity. We hypothesized that depletion of dTTP leads to incorrect nucleotide incorporation into DNA which, if not repaired, augments cell death following irradiation. This hypothesis was supported by our findings that mismatch repair (MMR)-deficient HCT116 were better radiosensitized by FdUrd compared to MMR-proficient HCT 116 cells. Furthermore, FdUrd produced nucleotide misincorporations in DNA, measured directly as pSP189 plasmid mutations in HCT116 and SW620 cells, but only at radiosensitizing concentrations. We wished to determine whether suppression of TS protein would be as effective as FPs as a radiosensitizing strategy. To selectively inhibit TS we used each of two shRNAs, TS1 and TS2, both of which produced a ≥ 90% decrease in TS expression. Suppression of TS expression in HCT116 and HT29 cells elicited an increase in sensitivity to ionizing radiation (IR) that was similar to the increase observed with FdUrd (IC50) (Radiation Enhancement Ratio (RER) = HCT116: 1.4 ± 0.08 (TS1), 1.5 ± 0.04 (TS2) vs. 1.5 ± 0.08 (FdUrd); HT29: 1.4 ± 0.06 (TS1), 1.4 ± 0.04 (TS2) vs. 1.6 ± 0.3 (FdUrd)). Additionally, a similar increase in pSP189 plasmid mutations was observed following suppression of TS, and FdUrd (IC50) in both cell lines. S-phase accumulation following TS suppression was slightly attenuated compared to drug (HCT116: 70% (TS1), 60% (TS2) vs. 75% (FdUrd); HT29: 55% (TS1), 75% (TS2) vs. 85% (FdUrd)). FdUrd and TS suppression produced a similar initial depletion of dTTP (HCT116: 65% (TS1), 70% (TS2) vs. 40% (FdUrd); HT29: 75% (TS1), 50% (TS2) vs. 50% (FdUrd)), however dTTP levels rebounded with FdUrd, but remained low with TS suppression. dATP levels increased profoundly with FdUrd, but decreased slightly with TS suppression. Despite these differences in dNTPs, TS suppression produced less cytotoxicity than FdUrd (IC50), but both produced a similar increase in sensitivity to IR. These results support our hypothesis that the mechanism of radiosensitzation by FdUrd is the decrease in dTTP, and not the incorporation of the drug into DNA or its cytotoxicity. Therefore, clinical treatment with FdUrd and IR could be titrated to maximize DNA mismatches in tumors rather than cytotoxicity, or TS could be targeted directly by shRNA methods to decrease normal tissue toxicity. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 1390.