BackgroundDNA methylation regulates together with other epigenetic mechanisms the transcriptional activity of genes and is involved in the pathogenesis of malignant diseases including lung cancer. In non-small cell lung cancer (NSCLC) various tumor suppressor genes are already known to be tumor-specifically methylated. However, from the vast majority of a large number of genes which were identified to be tumor-specifically methylated, tumor-specific methylation was unknown so far. Thus, the major aim of this study was to investigate in detail the mechanism(s) responsible for transcriptional regulation of the genes SPAG6 and L1TD1 in NSCLCs.MethodsWe analysed publically available RNA-sequencing data and performed gene expression analyses by RT-PCR. DNA methylation analyses were done by methylation-sensitive high-resolution melt analyses and bisulfite genomic sequencing. We additionally investigated protein expression using immunohistochemistry. Cell culture experiments included tumor cell growth, proliferation, viability as well as colony formation assays. Moreover, we performed xenograft experiments using immunodeficient mice.ResultsWe observed frequent downregulation of SPAG6 and L1TD1 mRNA expression in primary tumor (TU) samples compared to corresponding non-malignant lung tissue (NL) samples of NSCLC patients. We furthermore observed re-expression of both genes after treatment with epigenetically active drugs in most NSCLC cell lines with downregulated SPAG6 and L1TD1 mRNA expression. Frequent tumor-specific DNA methylation of SPAG6 and L1TD1 was detected when we analysed TU and corresponding NL samples of NSCLC patients. ROC curve analyses demonstrated that methylation of both genes is able to distinguish between TU and NL samples of these patients. Immunohistochemistry revealed a close association between SPAG6/L1TD1 methylation and downregulated protein expression of these genes. Moreover, by performing functional assays we observed reduced cell growth, proliferation and viability of pCMV6-L1TD1 transfected NSCLC cells. In addition, reduced volumes of tumors derived from pCMV6-L1TD1 compared to pCMV6-ENTRY transfected NCI-H1975 cells were seen in a xenograft tumor model.ConclusionsOverall, our results demonstrate that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs and that DNA methylation is involved in the transcriptional regulation of these genes. Moreover, in vitro as well as in vivo experiments revealed tumor-cell growth suppressing properties of L1TD1 in NSCLC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0568-5) contains supplementary material, which is available to authorized users.

Purpose: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on miRNA silencing in non-small cell lung cancers (NSCLC).Experimental Design: We conducted microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2 0 -deoxycytidine (Aza-dC)and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. miRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding nonmalignant lung tissue samples of 101 patients with stage I-III NSCLC.Results: By comparing microarray data of untreated and drug-treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analyzed. Moreover, miR-9-3 and miR-193a were found to be tumor specifically methylated in patients with NSCLC. We observed a shorter disease-free survival of patients with miR-9-3 methylated lung squamous cell carcinoma (LSCC) than patients with miR-9-3 unmethylated LSCC by multivariate analysis [HR ¼ 3.8; 95% confidence interval (CI), 1.3-11.2, P ¼ 0.017] and a shorter overall survival of patients with miR-9-3 methylated LSCC than patients with miR-9-3 unmethylated LSCC by univariate analysis (P ¼ 0.013).Conclusions: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in patients with LSCC.

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumorspecifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs.

In our study, we investigated the role of ZNF677 in non-small cell lung cancers (NSCLC). By comparing ZNF677 expression in primary tumor (TU) and in the majority of cases also of corresponding non-malignant lung tissue (NL) samples from > 1,000 NSCLC patients, we found tumor-specific downregulation of ZNF677 expression (adjusted p-values < 0.001). We identified methylation as main mechanism for ZNF677 downregulation in NSCLC cells and we observed tumor-specific ZNF677 methylation in NSCLC patients (p < 0.0001). In the majority of TUs, ZNF677 methylation was associated with loss of ZNF677 expression. Moreover, ZNF677 overexpression in NSCLC cells was associated with reduced cell proliferation and cell migration. ZNF677 was identified to regulate expression of many genes mainly involved in growth hormone regulation and interferon signalling. Finally, patients with ZNF677 methylated TUs had a shorter overall survival compared to patients with ZNF677 not methylated TUs (p = 0.013). Overall, our results demonstrate that ZNF677 is trancriptionally regulated by methylation in NSCLCs, suggest that ZNF677 has tumor cell growth suppressing properties in NSCLCs and that ZNF677 methylation might serve as prognostic parameter in these patients.