Combining Small Interfering RNAs Targeting Thymidylate Synthase and Thymidine Kinase 1 or 2 Sensitizes Human Tumor Cells to 5-Fluorodeoxyuridine and Pemetrexed

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting IS protein are a mainstay in cancer treatment, but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumor milieu, and may modulate resistance to TS-targeting drugs. Combined down-regulation of these enzymes is an attractive strategy to enhance cancer therapy. We have shown previously that antisense-targeting IS enhanced tumor cell sensitivity to TS-targeting drugs in vitro and in vivo. Because both TS and TKs contribute to increased cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of IS small interfering RNA (siRNA) to sensitize tumor cells to TS-targeting anticancer drugs. We assessed the effects of targeting TK1 or TK2 with siRNA alone and in combination with siRNA targeting IS and/or TS-protein targeting drugs on tumor cell proliferation. Down-regulation of TK with siRNA enhanced the capacity of TS siRNA to sensitize tumor cells to traditional TS protein-targeting drugs [5-fluorodeoxyuridine (5FUdR) and pemetrexed]. The sensitization was greater than that observed in response to any siRNA used alone and was specific to drugs targeting TS. Up-regulation of TK1 in response to combined 5FUdR and IS siRNA suggests that TK knockdown may be therapeutically useful in combination with these agents. TKs may be useful targets for cancer therapy when combined with molecules targeting IS mRNA and TS protein.