Abstract 2772: SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

DNA methylation is one of the major epigenetic mechanisms regulating transcriptional activity and is involved in the pathogenesis of non-small cell lung cancers (NSCLC). In a recent study we identified a large number of tumor-specifically methylated genes in NSCLCs (Heller et al., Carcinogenesis 2013). For further detailed analyses we selected the genes SPAG6 (Sperm Associated Antigen 6) and L1TD1 (LINE-1 Type Transposase Domain Containing 1). By analysing publically available IlluminaHiSeq RNA-seq data 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. In addition, we investigated SPAG6 and L1TD1 mRNA expression in 5 NSCLC cell lines and found SPAG6 as well as L1TD1 mRNA expression frequently downregulated in all of these cell lines compared to normal human bronchial epithelial cells (NHBECs). Subsequently, we treated cells of NSCLC cell lines which did not express SPAG6 or L1TD1 with the epigenetically active drugs 5-aza-2′-deoxycytidine and Trichostatin A and observed re-expression of both genes suggesting that transcriptional regulation of SPAG6 and L1TD1 is mediated by DNA methylation in NSCLCs. Bisulfite genomic sequencing of parts of the 5′ region of SPAG6 and L1TD1 revealed that the vast majority of CpG sites indeed are methylated in NSCLC cells in contrast to NHBECs. Moreover, we analysed SPAG6 and L1TD1 methylation in TU and NL samples of 147 stage I-III NSCLC patients using the gene-specific approach methylation-sensitive high resolution melt analysis (MS-HRM). Differences in SPAG6 as well as in L1TD1 methylation between TU and NL samples were statistically significant for both genes and confirmed that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs. Additionally, we investigated SPAG6 and L1TD1 protein expression in TU and NL samples of 35 NSCLC patients by immunohistochemistry. In the vast majority of SPAG6 or L1TD1 methylated TU samples, protein expression of these genes was downregulated in tumor cells. Moreover, we performed cell proliferation, cell viability and colony formation assays in vitro and observed that ectopic expression of L1TD1 but not of SPAG6 reduced tumor cell proliferation, viability and the ability of NSCLC cells to form colonies. To further investigate the role of L1TD1 in the pathogenesis of NSCLCs, in vivo studies are being carried out. Overall, our results demonstrate that DNA methylation is the major mechanism for frequent downregulation of SPAG6 and L1TD1 expression in NSCLCs. Citation Format: Corinna Altenberger, Gerwin Heller, Barbara Ziegler, Erwin Tomasich, Maximilian Marhold, Leonhard Mullauer, Gyorgy Lang, Adelheid End-Pfutzenreuter, Balazs Dome, Britt-Madeleine Arns, Walter Klepetko, Christoph C. Zielinski, Sabine Zochbauer-Muller. SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2772.