We have demonstrated for the first time that shRNA mediated TS suppression produced excellent radiosensitization that was at least as effective as that observed with FdUrd. Furthermore, these studies demonstrate that TS inhibition alone is less cytotoxic compared to FdUrd. FdUrd and other FPs target TS at the same concentrations at which they can be incorporated into nucleic acids. As a result, prior studies correlating the depletion of dTTP with radiosensitization by FPs were hampered by the concomitant presence of FP nucleotides (4
) making it difficult to separate the contribution of each pathway to radiosensitization. The shRNA mediated suppression of TS eliminates the possibility of fraudulent nucleotide incorporation into nucleic acids. Therefore, these results provide evidence that TS inhibition alone is sufficient for radiosensitization and also establish definitively that cytotoxicity with FdUrd requires mechanisms in addition to TS inhibition, such as DNA incorporation of FP nucleotides.
We recently provided evidence for a novel mechanism of radiosensitization with FdUrd in which dTTP depletion, as a result of TS inhibition, produced mismatches in DNA that augmented cell death following irradiation (5
). However, the contribution of FdUTP in producing mismatches could not be evaluated. The present study revealed a similar increase in both the extent and type of nucleotide misincorporation events following either FdUrd or shRNA suppression of TS under radiosensitizing conditions, despite the differences in cytotoxicity. Furthermore, the elevation of dATP characteristic of FdUrd treatment did not occur with TS shRNA, thus eliminating this imbalance as a source of mismatches. These results prove that the nucleotide misincorporation events are a direct result of the dNTP effects mediated by TS suppression, and cannot be explained by FdUTP incorporation into DNA. Furthermore, these results amplify and support our earlier findings, demonstrating that radiosensitization occurs as a result of mismatches in DNA due to TS inhibition (5
Acute elevation in TS protein has been observed following exposure to FPs or analogs in preclinical and clinical studies (17
), as shown here (). This increase in TS expression is clinically undesirable because high TS expression has been associated with poorer overall survival in colorectal cancer, while low TS expression is prognostic for better outcome in patients treated with 5-FU (18
). Thus, use of small molecule TS inhibitors can actually antagonize therapy by promoting overexpression of TS. Previous studies evaluated suppression of TS with siRNA or ODNs on tumor cell proliferation, but not on radiosensitization (6
). Only modest effects were observed on cell growth when ODNs or siRNAs were used as single agents, with little to no additional effect when administered with a FP (19
). Importantly, the combination of a FP or antifolate and TS siRNA or ODN still allowed overexpression of TS (7
), rendering this combination clinically undesirable. These findings provide strong rationale for developing new approaches that inhibit TS without causing TS overexpression. The shRNA approach used in our studies provided a more profound (≥ 90% decrease) and prolonged silencing of TS protein (≥8 days). Although cytotoxicity was modest, the excellent radiosensitization suggests that combining TS shRNA with concurrent irradiation has potential to impact tumor growth in vivo
while also eliminating negative clinical effects associated with elevation of TS.
Despite lengthy, nearly complete suppression of TS protein observed with TS shRNA, this approach produced less cytotoxicity than FdUrd. Previous studies have implicated FdUTP incorporation into DNA as an important contributor to cytotoxicity (3
). Products of drug metabolism, such as FdUTP and its incorporation into DNA, are absent following shRNA suppression of TS, which likely accounts for its lower cytotoxicity (). ATR-mediated phosphorylation of Chk1 is a common response to DNA damaging drugs such as FPs and plays a role in the initiation of the S-phase checkpoint. Both FdUrd and TS shRNA induced S-phase arrest and ATR dependent phosphorylation of Chk1 (data not shown). Therefore, while FdUTP and/or its incorporation into DNA may elicit additional effects that contribute to an increase in cytotoxicity, TS suppression alone is sufficient to activate the Chk1 damage response.
The TS shRNA strategy allowed us to examine the effects of TS suppression without the confounding variables of FP metabolism and its associated effects. TS shRNA produced less cytotoxicity than FdUrd, but was equally effective at radiosensitizing tumor cells. This work marks the first demonstration of a shRNA strategy targeting TS to produce radiosensitization. Furthermore, this study has advanced the understanding of the lesion associated with radiosensitization by FdUrd. That compromised TS expression induced both mismatches and radiosensitization similar to FdUrd demonstrates a causal and sufficient role for the depletion of dTTP and consequent misincorporation of nucleotides into DNA in the underlying mechanism of action of FdUrd mediated radiosensitization. TS suppression may be particularly valuable as a radiosensitizing approach in vivo
because concurrent irradiation with FPs is limited by normal tissue toxicity due to, at least in part, the toxic effects of the FPs and their catabolites (20
). Furthermore, use of TS shRNA with radiotherapy may help to eliminate the negative prognostic role imparted by the increase in TS expression observed with traditional drug therapies and warrants further investigation.