Abstract C147: siRNA targeting TS and TK as an anticancer therapy

Abstract

Abstract Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis, repair and proliferation. Cytosolic thymidine kinase 1 (TK1) and mitochondrial thymidine kinase 2 (TK2) represent “scavenger” pathways for alternative production of essential dTMP by phosphorylation of intracellular thymidine (Thd). Both TS and TKs are upregulated in multiple human tumors, supporting a role for both in malignancy. Drugs targeting TS-protein are a mainstay in cancer treatment but toxicity to normal tissues and tumor cell drug resistance limit their effectiveness. We hypothesize that, when TS-protein is inhibited, the capacity of TKs to generate dTMP may modulate resistance to anti-TS drugs. Antisense molecules, including oligonucleotides (ODNs) and siRNAs, can selectively target specific mRNAs (including TS and TK mRNAs) encoding proteins mediating drug resistance. We have previously reported that antisense ODNs targeting TS inhibit human tumor cell growth in vitro and in immunocompromised host mice, and antisense TS ODNs and siRNAs enhance the growth inhibitory effects of TS-targeting drugs. Antisense TS, therefore, is an attractive candidate for development as an anticancer drug, both alone and in combination with drugs targeting TS protein (raltitrexed, pemetrexed, 5FUdR, and others). The ability of antisense TS drugs to inhibit tumor cell growth and response to other TS-targeting drugs does not correlate with the degree of antisense downregulation of TS mRNA, suggesting that factors other than TS attenuate antitumor effects. We used siRNAs targeting TK1 or TK2 to knock down TK expression in attempt to reduce human tumor cell growth when targeting TKs alone, in combination with siRNAs targeting TS, and in combination with TS siRNA and the anti-TS protein drug 5FUdR. We report that TK and TS levels are highly variable among a panel including human cervical (HeLa) and breast carcinoma (MCF7), colorectal cancer (HT-29) and human mesothelioma cell lines. Furthermore, 24 hrs post-transfection, siRNAs targeting TK1 or TK2 reduce targeted TK-mRNA by greater than 85% at concentration of 5 nM in human cervical carcinoma (HeLa) and human breast carcinoma (MCF7) cells. At 96 hrs post-transfection, siRNA targeting TK1 or TK2 do not independently reduce human tumor cell growth in vitro. Furthermore, simultaneous siRNA targeting of TS and TK effectively, independently, and non-antagonistically reduce both TS and TK mRNA. Antisense targeting TK1 or TK2 alone, unlike siRNA targeting TS alone, does not enhance the capacity of 5FUdR to inhibit human tumor cell growth in vitro. We suggest, however, that combined antisense targeting of TS and TK1/TK2 (particularly under conditions where extracellular thymidine levels are high) will be more effective than either used alone to reduce tumor cell growth and sensitize them to the effects of antitumor TS-targeting chemotherapeutics. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C147.