282 Downregulation of BRCA2 and Thymidylate Synthase (TS) Sensitizes Human Tumour Cells to Chemotherapy: Induction of ‘Complementary Lethality’ by Targeting DNA Repair

Abstract

The capacity of tumour cells to sustain high mutation rates leads to phenotypic heterogeneity that drives cancer advancement, malignancy, and recurrence. Although the inherent genomic instability present in most human cancer cells can drive both tumour initiation and malignant progression, it is also a liability: genome instability in the absence of sufficient DNA repair can lead to cancer cell death. Thus, suppression of tumour cell DNA repair machinery may be a strategy to turn the apparent strengths of mutation and heterogeneity into a weakness by promoting an increase in deleterious mutations from which tumour cells cannot recover. A promising target is BRCA2, which is involved in homologous recombination (HR) repair and has been implicated in hereditary breast, ovarian, and other cancers − a phenomenon termed ‘BRCAness’. Clinical data reveals that although individuals with BRCA2 mutations are at a higher risk of cancer, they also respond more favourably to conventional chemotherapy. It is hypothesized that in the absence of functional BRCA2 protein, tumour cells are unable to efficiently repair the DNA damage induced by anticancer agents and thus succumb more readily to their effects. We report that siRNA knockdown of BRCA2 in human A549 non-small cell lung cancer cells inhibited growth and also induced sensitization to cisplatin, melphalan and (to a lesser extent) the thymidylate synthase (TS) targeting agent 5-FUdR. Interestingly, BRCA2 downregulation did not potentiate the toxicity of pemetrexed which also targets the TS pathway. BRCA2 siRNA treatment was combined with siRNA targeting TS, previously shown by our group to sensitize Hela cells to 5-FU, 5-FUdR and pemetrexed. The aim was to promote sensitization to alkylating agents and TS targeting drugs in the same cell and thus induce a state of combined ‘complementary lethality’ whereby each siRNA potentiates the effect of a drug with a mechanism of action complementary to the function of the targeted protein. In this context, knockdown of both TS and BRCA2 followed by treatment with an alkylating drug (cisplatin or melphalan) plus 5-FUdR was more effective at inhibiting tumour cell growth than would be predicted by knockdown of TS and BRCA2 followed by treatment with any single drug (5-FUdr, cisplatin, or melphalan). These data suggest that (1) targeting BRCA2 function is a viable strategy to increase the effectiveness of common chemotherapeutics, and (2) it is possible to use antisense-mediated targeting of both TS and a DNA repair protein to sensitize the same cell to two different drugs, and thus potentiate overall therapeutic effectiveness. This research is supported by a grant from the Canadian Institutes of Health Research (CIHR) to J.K and MDV. MR is a scholar of the CIHR Strategic Training Program in Cancer Research and Technology Transfer (CaRTT) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Unit (TBCRU) scholarship program.