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...

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Although postoperative adjuvant chemotherapy (PAC) with uracil -tegafur significantly improves the prognosis of patients with stage I lung adenocarcinoma, subset analysis has revealed that only 11.5% of patients with stage IB derive actual benefit from such therapy. Therefore, it is extremely important to identify patients for whom adjuvant chemotherapy will be beneficial. We performed comprehensive protein analysis of 24 surgically resected specimens of stage I adenocarcinoma using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by bioinformatical investigations to identify protein molecules. Furthermore, we carried out immunohistochemical studies of 90 adenocarcinoma specimens to validate the results of LC-MS/MS. We detected two kinds of protein molecules (myosin IIA and vimentin) by LC-MS/MS. We confirmed their immunohistochemical expression and distribution, and evaluated the relationship between the expression of these proteins and prognosis after adjuvant chemotherapy. Patients with no expression of either myosin IIA or vimentin showed a significantly better outcome regardless of PAC using uracil -tegafur. However, we were unable to select responders to uracil -tegafur using these proteins. Cases of adenocarcinoma lacking expression of either myosin IIA or vimentin show a good outcome without PAC, and therefore do not require such treatment.

Lower TS expression in adenocarcinoma of the lung was confirmed in a large-scale study.

The expression level of FPGS, GGH and ABCC1 in CRC tissues could predict the reduced folate level after LV administration, and these factors may determine the efficacy of LV treatment.

Due to hypoxic conditions, colorectal cancer (CRC) tissues have a specific metabolism compared with normal mucosa, and this study aimed to investigate blood plasma metabolite differences between CRC patients and healthy subjects. Non-targeted methods have previously been used to characterize CRC disease diagnosis and identify small molecules in biological samples, but here we used a metabolomic approach based on liquid chromatography-mass spectrometry (LC-MS) of blood plasma from CRC patients (n=10) and healthy subjects (n=10). The analysis detected 130 identified compounds including free amino acids, fatty acids and nucleotides. Of these, many free amino acids were decreased in CRC patients while nucleotide-related and stress-related metabolites were increased in CRC patients compared with healthy subjects. Multivariate analysis clearly separated CRC patients and healthy subjects. These results demonstrate that plasma metabolite would reflects tumoral specific metabolism and metabolomic profiling of blood plasma using LC-MS is a valuable tool for characterizing CRC patients based on plasma metabolite diagnostic markers.

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Abstract. It has been reported that the expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil (5-FU)-based therapy in cancer patients. We investigated the differences in the mRNA and protein expression of these enzymes in various tumor tissues. A total of 17,613 specimens of head and neck, gastric, colorectal, breast, lung and pancreatic cancer were collected from multiple facilities in Japan, and the mRNA and protein expression levels of the above enzymes were examined in 4,830 and 12,783 of these specimens, respectively. The mRNA levels were analyzed using RT-PCR in laser-captured microdissected formalinfixed paraffin-embedded specimens, while the protein levels were analyzed by enzyme-linked immunosorbent assays. The median values of the relative TS, DPD and OPRT mRNA levels were 2.06, 0.803 and 1.17, respectively, while the median protein levels were 22.1, 134.8 and 3.81 ng enzyme/mg protein, respectively. The carcinomas were classified into two sets of four groups each using the overall median levels of TS and DPD or TS and OPRT as cutoff values. Approximately 60% of the gastric cancers exhibited elevated mRNA and protein expression levels of DPD, while >65% of the colorectal cancers showed low levels of DPD expression. Overall, 75% of the head and neck cancers exhibited high expression levels of DPD. Among the lung and pancreatic cancers, 50-74% showed low TS/high DPD expression. In conclusion, the mRNA expression and protein levels of TS, DPD and OPRT differed according to the type of cancer. The results of this large-scale population analysis are expected to be useful as reference data for predicting the relationship between the respective enzyme levels and the efficacy of 5-FU-based chemotherapy.