Background:Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in non-small cell lung cancer (NSCLC). We have now investigated the effect of TS overexpression on pemetrexed sensitivity in NSCLC cells.Methods:We established NSCLC cell lines that stably overexpress TS and examined the effects of such overexpression on the cytotoxicity of pemetrexed both in vitro and in xenograft models. We further examined the relation between TS expression in tumour specimens from NSCLC patients and the tumour response to pemetrexed by immunohistochemical analysis.Results:The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells. The inhibition of DNA synthesis and induction of apoptosis by pemetrexed were also greatly attenuated by forced expression of TS. Furthermore, tumours formed by TS-overexpressing NSCLC cells in nude mice were resistant to the growth-inhibitory effect of pemetrexed observed with control tumours. Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.Conclusion:A high level of TS expression confers a reduced sensitivity to pemetrexed. TS expression is thus a potential predictive marker for response to pemetrexed-based chemotherapy in NSCLC patients.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the therapeutic response to EGFR tyrosine kinase inhibitors (TKI) in patients with advanced non-small cell lung cancer (NSCLC). The response rate to these drugs remains low, however, in NSCLC patients with wild-type EGFR alleles. Combination therapies with EGFR-TKIs and cytotoxic agents are considered a therapeutic option for patients with NSCLC expressing wild-type EGFR. We investigated the antiproliferative effect of the combination of the oral fluorouracil S-1 and the EGFR-TKI gefitinib in NSCLC cells of differing EGFR status. The combination of 5-fluorouracil and gefitinib showed a synergistic antiproliferative effect in vitro in all NSCLC cell lines tested. Combination chemotherapy with S-1 and gefitinib in vivo also had a synergistic antitumor effect on NSCLC xenografts regardless of the absence or presence of EGFR mutations. Gefitinib inhibited the expression of the transcription factor E2F-1, resulting in the down-regulation of thymidylate synthase at the mRNA and protein levels. These observations suggest that gefitinib-induced down-regulation of thymidylate synthase is responsible, at least in part, for the synergistic antitumor effect of combined treatment with S-1 and gefitinib and provide a basis for clinical evaluation of combination chemotherapy with S-1 and EGFR-TKIs in patients with solid tumors. [Mol Cancer Ther 2008;7(3):599 -606]

Background:Thymidylate synthase (TS), a key enzyme in the de novo synthesis of thymidine, is an important chemotherapeutic target for malignant tumours including lung cancer. Although inhibition of TS has an antiproliferative effect in cancer cells, the precise mechanism of this effect has remained unclear.Methods:We examined the effects of TS inhibition with an RNA interference-based approach. The effect of TS depletion on the growth of lung cancer cells was examined using colorimetric assay and flow cytometry.Results:Measurement of the enzymatic activity of TS in 30 human lung cancer cell lines revealed that such activity differs among tumour histotypes. Almost complete elimination of TS activity by RNA interference resulted in inhibition of cell proliferation in all tested cell lines, suggestive of a pivotal role for TS in cell proliferation independent of the original level of enzyme activity. The antiproliferative effect of TS depletion was accompanied by arrest of cells in S phase of the cell cycle and the induction of caspase-dependent apoptosis as well as by changes in the expression levels of cyclin E and c-Myc. Moreover, TS depletion induced downregulation of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP), and it seemed to activate the mitochondrial pathway of apoptosis.Conclusion:Our data provide insight into the biological relevance of TS as well as a basis for clinical development of TS-targeted therapy for lung cancer.

Aberrant DNA methylation is a commonly observed epigenetic change in lung cancer. Folate has been suggested to play a role in the homeostasis of DNA methylation and has also been implicated in cancer chemotherapy. We investigated a possible role for folate in DNA methylation by measuring folate concentrations in tumors and adjacent normal tissues from 72 non-small cell lung cancer (NSCLC) patients. These were compared to DNA methylation levels and to clinicopathological features. Folate concentrations were determined as the sum of 5,10-methylenetetrahydrofolate and tetrahydrofolate. The MethyLight assay was used to quantitate methylation in promoter regions of P16(CDKN2A), APC, CDH13, RARB, RASSF1, RUNX3, and MYOD1. Methylation of LINE-1 repeats was used as a surrogate for global methylation. Folate levels in tumors correlated positively with LINE-1, CDH13, and RUNX3 methylation. Folate concentrations and methylation of LINE-1, RASSF1, and RUNX3 were significantly higher in adenocarcinoma compared to squamous cell carcinoma (SCC). Two sets of array-based data retrieved from the Gene Expression Omnibus consistently showed that expression of FOLR1, a folate transport enzyme, and GGH, an enzyme that prevents folate retention, were higher and lower, respectively, in adenocarcinomas compared to SCC. This was independently validated by quantitative RT-PCR in 26 adenocarcinomas and 13 SCC. Our results suggest that folate metabolism plays a role in aberrant DNA methylation in NSCLC. The histological subtype differences in folate concentration and DNA methylation observed here were associated with distinct expression patterns for folate metabolizing enzymes. These findings may have clinical applications for histology-directed chemotherapy with fluoropyrimidine and anti-folates in NSCLC. (Cancer Sci 2009; 100: 2325-2330 L ung cancer is a leading cause of cancer death worldwide. Genetic and epigenetic aberrations accumulate throughout lung carcinogenesis. Global hypomethylation of genomic DNA and hypermethylation of gene promoter regions occur simultaneously in a wide variety of malignancies including lung cancer.(1,2) Little is known however about the mechanism leading to these epigenetic alterations in human primary lung cancer.Epidemiological studies have demonstrated that dietary folate supplementation can prevent the development of lung cancer. Folate is an important precursor of one-carbon units required for DNA methylation. Therefore, folate metabolism has been suggested to influence epigenetic alterations in lung cancer and this could provide a mechanism to explain the prevention of lung cancer by folate supplementation. High folate might contribute to the maintenance of global methylation through an adequate supply of one-carbon units for the methylation machinery, thereby stabilizing the genome. Although the status of dietary folate intake has been analyzed in relation to DNA methylation, (4) an association between folate concentration and global methylation in human lung tissue has so far not been reported.In ad...

Purpose: Most non^small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (EGFR-TKI) such as gefitinib and erlotinib, but they almost invariably develop resistance to these drugs. A secondary mutation in EGFR (T790M) and amplification of the MET protooncogene have been identified as mechanisms of such acquired resistance to EGFR-TKIs. We have now investigated whether addition of the oral fluoropyrimidine derivative S-1to gefitinib might overcome gefitinib resistance in NSCLC cell lines. Experimental Design: The effects of gefitinib on EGFR signaling and on the expression both of thymidylate synthase and of the transcription factor E2F-1 in gefitinib-resistant NSCLC cells were examined by immunoblot analysis. The effects of S-1 (or 5-fluorouracil) and gefitinib on the growth of NSCLC cells were examined in vitro as well as in nude mice. Results: Gefitinib induced down-regulation of thymidylate synthase and E2F-1 in gefitinibresistant NSCLC cells with MET amplification but not in those harboring theT790M mutation of EGFR. The combination of 5-fluorouracil and gefitinib synergistically inhibited the proliferation of cells with MET amplification, but not that of those with the T790M mutation of EGFR, in vitro. Similarly, the combination of S-1and gefitinib synergistically inhibited the growth only of NSCLC xenografts with MET amplification. Conclusions: Our results suggest that the addition of S-1to EGFR-TKIs is a promising strategy to overcome EGFR-TKI resistance in NSCLC with MET amplification.