Different histological types of non-small cell lung cancer have distinct folate and DNA methylation levels

金沢大学がん研究所分子標的がん医療研究開発センター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. © 2009 Japanese Cancer Association

Folic Acid-Metabolizing Enzymes Regulate the Antitumor Effect of 5-Fluoro-2′-Deoxyuridine in Colorectal Cancer Cell Lines

<div><p>In colorectal cancer chemotherapy, the current standard of care includes combination therapy with 5-fluorouracil (5-FU) and leucovorin (LV). However, the factors that determine the LV-mediated enhancement of 5-FU antitumor activity are not fully understood. Therefore, we investigated the roles of thymidine synthase (TYMS), folate receptor 1 (FOLR1), dihydrofolate reductase (DHFR), phosphoribosylglycinamide formyltransferase (GART), methylenetetrahydrofolate dehydrogenase (MTHFD1), and methylenetetrahydrofolate reductase (MTHFR) in LV-mediated enhancement of 5-fluoro-2′-deoxyuridine (FdUrd) cytotoxicity <i>in vitro</i> as a model of 5-FU antitumor activity. These genes were downregulated in DLD-1 and HCT116 human colorectal cancer cells by using small-interfering RNA. Reduced expression of <i>TYMS</i> mRNA significantly increased FdUrd cytotoxicity by 100- and 8.3-fold in DLD-1 and HCT116 cells, respectively. In contrast, reducing the expression of <i>FOLR1</i>, <i>DHFR</i>, <i>GART</i>, <i>MTHFD1</i>, and <i>MTHFR</i> decreased FdUrd cytotoxicity by 2.13- to 12.91-fold in DLD-1 cells and by 3.52- to 10.36-fold in HCT116 cells. These results demonstrate that folate metabolism is important for the efficacy of FdUrd. Overall, the results indicate that it is important to clarify the relationship between folate metabolism-related molecules and 5-FU treatment in order to improve predictions of the effectiveness of 5-FU and LV combination therapy.</p></div

Effect of TYMS silencing on the increased FdUrd cytotoxicity induced by leucovorin (LV).

<p>DLD-1 and HCT116 cells were treated with control or TYMS siRNA, and then with 0.03 μM FdUrd plus increasing concentrations of LV for 72 h. LV-enhanced cytotoxicity was prevented by silencing of TYMS. All data are expressed as the mean ± SD (n = 3). ***<i>p</i> < 0.001, statistically significant when compared to a control using Dunnett’s multiple <i>t</i>-test.</p

Effect of folate receptor 1 (<i>FOLR1</i>) silencing on FdUrd cytotoxicity.

<p>At 48 h after treatment of DLD-1 and HCT116 cells with control or FOLR1 siRNA, cells were treated with FdUrd for 72 h and cell viability was determined. Silencing of <i>FOLR1</i> reduced the efficacy of FdUrd at 0.03–30 μM in DLD-1 cells and at 0.03–3 μM in HCT116 cells. All data are expressed as the mean ± SD. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005, statistically significant when compared to a control using Student’s <i>t</i>-test.</p

Effect of phosphoribosylglycinamide formyltransferase (<i>GART</i>) silencing on FdUrd cytotoxicity.

<p>At 48 h after treatment of DLD-1 and HCT116 cells with control or GART siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of <i>GART</i> reduced the efficacy of FdUrd at 0.03–10 μM in DLD-1 cells and at 0.1–3 and 30 μM in HCT116 cells. All data are expressed as the mean ± SD. *<i>p</i> < 0.05, ***<i>p</i> < 0.005, statistically significant when compared to a control using Student’s <i>t</i>-test.</p

Effect of TYMS silencing on the cytotoxic effect of FdUrd in DLD-1 and HCT116 cells.

<p>At 48 h after treatment with control or TYMS siRNA, cells were treated with FdUrd for 72 h. The siRNA significantly enhanced FdUrd toxicity at low concentrations in both cell lines. All data are expressed as the mean ± standard deviation (SD). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005, statistically significant when compared to a control using Student’s <i>t</i>-test.</p

5-Chloro-2,4-dihydroxypyridine, CDHP, prevents lung metastasis of basal-like breast cancer cells by reducing nascent adhesion formation

A drug for metastasis prevention is necessary. The orally administered anticancer drug S‐1 contributes to cancer therapy. In a mouse xenograft model of metastatic breast cancer from our previous study, the administration of S‐1 inhibited lung metastasis. However, the mechanism of inhibition remains elusive. S‐1 contains 5‐chloro‐2, 4‐dihydroxypyridine (CDHP), which does not have the antigrowth activity, but prevents the degradation of 5‐fluorouracil, an anticancer reagent. In this study, we found that CDHP treatment shrinks cell morphology in metastatic basal‐like breast cancer cell lines. Wound healing assays showed reduced cell migration in CDHP‐treated cells. At the molecular level, CDHP treatment reduced the number of nascent adhesions, whereas the number of mature focal adhesions was not changed. These findings indicate that CDHP impairs focal adhesion formation, which results in a reduction in cell migration. For the in vivo metastasis assay, we used a highly lung‐metastatic cell line. We xenografted them into immunodeficient mice, and administered CDHP. To determine whether CDHP prevents metastasis, we measured the weights of harvested lungs. The results showed that the lung weights of the CDHP‐treated animals were not significantly different compared to the no‐tumor controls, whereas the vehicle group showed a number of metastatic foci and an increase in lung weight. These observations indicate that CDHP administration prevents metastasis. This study reveals a novel effect of CDHP for lung metastasis prevention. Our findings may facilitate the establishment of future metastasis prevention therapies

Effect of <i>MTHFR</i> silencing on FdUrd cytotoxicity.

<p>At 48 h after treatment of DLD-1 and HCT116 cells with control or MTHFR siRNA, cells were treated with FdUrd for 72 h, and cell viability was determined. Silencing of <i>MTHFR</i> reduced the efficacy of FdUrd at 0.3 μM in HCT116 cells. All data are expressed as the mean ± SD. *<i>p</i> < 0.05, statistically significant when compared to a control using Student’s <i>t</i>-test.</p