Background

The MFSD2A gene maps within a linkage disequilibrium block containing the MYCL1-EcoRI polymorphism associated with prognosis and survival in lung cancer patients. Survival discrepancies between Asians and Caucasians point to ethnic differences in allelic frequencies of the functional genetic variations.

Results

Analysis of three single-nucleotide polymorphisms (SNPs) mapping in the MFSD2A 5'-regulatory region using a luciferase reporter system showed that SNP rs12072037, in linkage disequilibrium with the MYCL1-EcoRI polymorphism and polymorphic in Asians but not in Caucasians, modulated transcriptional activity of the MFSD2A promoter in cell lines expressing AHR and ARNT transcription factors, which potentially bind to the SNP site.

Conclusion

SNP rs12072037 modulates MFSD2A promoter activity and thus might affect MFSD2A levels in normal lung and in lung tumors, representing a candidate ethnically specific genetic factor underlying the association between the MYCL1 locus and lung cancer patients' survival.

In DNA methylation microarray analysis, quantitative assessment of intermediate methylation levels in samples with various global methylation levels is still difficult. Here, specifically for methylated DNA immunoprecipitation-CpG island (CGI) microarray analysis, we developed a new output value. The signal log ratio reflected the global methylation levels, but had only moderate linear correlation (r = 0.72) with the fraction of DNA molecules immunoprecipitated. By multiplying the signal log ratio using a coefficient obtained from the probability value that took account of signals in neighbouring probes, its linearity was markedly improved (r = 0.94). The new output value, Me value, reflected the global methylation level, had a strong correlation also with the fraction of methylated CpG sites obtained by bisulphite sequencing (r = 0.88), and had an accuracy of 71.8 and 83.8% in detecting completely methylated and unmethylated CGIs. Analysis of gastric cancer cell lines using the Me value showed that methylation of CGIs in promoters and gene bodies was associated with low and high, respectively, gene expression. The degree of demethylation of promoter CGIs after 5-aza-2'-deoxycytidine treatment had no association with that of induction of gene expression. The Me value was considered to be useful for analysis of intermediate methylation levels of CGIs.

Cancers arise as a consequence of multiple genetic and epigenetic alterations. Many genes aberrantly methylated in cancers have been identified in recent years, and their use in cancer diagnosis and therapy is currently under investigation. During our genome-wide screening for a novel tumor-suppressor gene in gastric cancers, we found that only a small amount of aberrant methylation was present, even in non-cancerous gastric mucosae. A subsequent large-scale analysis of the gastric mucosae of healthy individuals and gastric cancer patients using quantitative methylation-specific PCR (qMSP) revealed that Helicobacter pylori infection potently induced aberrant DNA methylation in non-cancerous gastric mucosae and that these high methylation levels can decrease following cessation of the H. pylori infection. Helicobacter pylori infection induced the methylation of specific genes among 48 genes that can be methylated in gastric cancer cell lines. Most importantly, the methylation levels in the gastric mucosae of individuals without H. pylori infection correlated with their risk of gastric cancer. These findings show that a field for cancerization is formed by H. pylori infection and that this field can be measured using DNA methylation as a marker. The concept of an “epigenetic field for cancerization” has been also demonstrated for colon and breast cancers, and it is possibly present for other cancers and other diseases. Applied knowledge of epigenetic changes in human diseases has now started to make an impact on the prevention, diagnostics, and therapeutics of these diseases.

RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor β1 (TGF-β1). It is known that TGF-β1 induces cell growth arrest by activating CDKN1A (p21WAF1/Cip1), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-β-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-β-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.