The deacetylase inhibitor SAHA prevents thymidylate synthase (TS) inhibitor-induced upregulation of TS and is synergistic with TS inhibitors in A431 cells and in α-folate receptor transfected A431-FBP cells

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HDAC inhibitors such as trichostatin A (TSA) have been reported to lower TS levels in tumor cells and to synergise with 5-fluorouracil, a TS inhibitor. This has been ascribed to either inhibition of TS transcription or, particularly at low concentrations, acetylation of HSP90 and increased proteasomal degradation of TS (Lee et al, MCT, 2006). This sensitisation may be particularly important as TS inhibitors lead to an acute upregulation of TS ascribed to either release of the translational autoregulation by TS protein, or to an increase in TS protein half-life. We have therefore investigated the individual effects of plevitrexed, a folate-based TS inhibitor, and SAHA, a class II HDAC inhibitor, on TS protein expression in A431 epidermoid tumor cells and its α-folate receptor (α-FR) transfected isogenic partner cell line, A431-FR. These were chosen because plevitrexed, although predominantly transported via the reduced-folate carrier (RFC) can also be transported via the tumour-associated α-FR. The IC50 for plevitrexed are 90nM and 23nM for A431 and A431-FR cells respectively as determined by a 72h MTT assay. Plevitrexed (90nM) induced TS upregulation in both cell lines at ~24h. An equitoxic concentration in A431-FBP cells (20nM) induced an increase in TS at 24h but was more marked at 48h. SAHA, at the IC50 concentration of 1µM, reduced TS levels at ~24h in both cell lines. Furthermore, SAHA prevented plevitrexed-mediated TS upregulation in both cell lines when it was added at the same time as plevitrexed, and reduced the level of the upregulated protein when added 24h after plevitrexed. The combination of plevitexed and SAHA was synergistic in A431 cells but antagonistic in A431-FR cells (combination index (CI) at IC50 of 0.46 ± 0.077 and 1.9 ± 0.64 respectively) as determined by median-effect analysis when the drugs were added at a fixed ratio of the IC50. In light of the delayed induction of TS observed in A431-FR cells we pre-exposed these cells to plevitrexed for 24h and obtained synergy (CI = 0.66 ± 0.11). This schedule was also synergistic in A431 cells (CI = 0.56 ± 0.081). In addition, synergy was observed in this schedule when SAHA was combined with 5-fluorodeoxyuridine in A431 cells and with BGC 945, a novel first generation α-FR-targeted TS inhibitor in preclinical development, in A431-FR cells. The molecular mechanisms underlying the synergistic interaction between SAHA and the TS inhibitors may be more complicated than simply reduction in TS levels. Indeed, SAHA at the IC50 concentrations in these cell lines gives an HSP90 inhibition molecular signature, i.e. increased HSP70 and decreased CRAF and phosphorylated-AKT. The clinical potential for the combination of HDAC inhibitors with pyrimidine- or folate-based TS inhibitors should be investigated further. Funded by Cancer Research UK