SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

Corinna Altenberger, Gerwin Heller, Barbara Ziegler, Erwin Tomasich, Maximilian Marhold, Thais Topakian, Leonhard Müllauer, Petra Heffeter, György Lang, Adelheid End-Pfützenreuter, Balazs Döme, Britt-Madeleine Arns, Walter Klepetko, Christoph C. Zielinski, Sabine Zöchbauer-Müller

DNA 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. We 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. We 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. Overall, 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.