Epigenetic abnormalities, especially DNA methylation, are involved in the pathogenesis of non-small cell lung cancers (NSCLC). By using a genome-wide approach, we recently identified ∼ 500 tumor-specifically methylated genes in a large number of NSCLC patients and from most of them tumor-specific methylation in NSCLCs was unknown so far.

One of these genes is the Sperm Associated Antigen 6 (SPAG6) gene. By analysing publically available IlluminaHiSeq RNA-seq data from “The Cancer Genome Atlas” database, we observed that SPAG6 mRNA expression is frequently lost in tumor samples compared to corresponding non-malignant lung tissue samples of NSCLC patients (http://cancergenome.nih.gov/). To further investigate the mechanism of SPAG6 downregulation, we studied SPAG6 expression in 5 NSCLC cell lines. We observed loss of SPAG6 expression in all of these cell lines compared to normal human bronchial epithelial cells (NHBECs). Subsequently, we treated cells of NSCLC cell lines which do not express SPAG6 with the epigenetically active drugs 5-aza-2´-deoxycytidine and Trichostatin A and observed SPAG6 reexpression. These results suggest that transcriptional regulation of SPAG6 is epigenetically regulated. Bisulfite genomic sequencing of parts of the 5´ region of SPAG6 revealed that the vast majority of CpG sites indeed are methylated in cells of NSCLC cell lines which do not express SPAG6 while no methylation was found in NHBECs. Moroever, we analysed SPAG6 methylation in tumor and corresponding non-malignant tissue samples of 147 stage I-III NSCLC patients using methylation-sensitive high-resolution melting (MS-HRM) assays. Differences in SPAG6 methylation between tumor and corresponding non-malignant lung tissue samples were statistically significant (p < 0.0001) demonstrating that SPAG6 is tumor-specifically methylated. Furthermore, ROC curve analyses of methylation results revealed that methylation of SPAG6 is able to distinguish tumor samples from corresponding non-malignant lung tissue samples of NSCLC patients. We additionally investigated SPAG6 protein expression in tumor and corresponding non-malignant tissue samples of 35 NSCLC patients by immunohistochemistry. In the vast majority of tumor samples which were found to be SPAG6 methylated, SPAG6 protein expression was lost in tumor cells while SPAG6 protein expression was observed in bronchial and bronchiolar epithelial cells of non-malignant lung tissue samples. Comparison of SPAG6 methylation results with clinico-pathological data of NSCLC patients suggests a potential negative prognostic relevance of SPAG6 methylation for NSCLC patients. Currently, we determine the biological function of SPAG6 in NSCLC cell lines. Overall, our results demonstrate that DNA methylation is the major mechanism for frequent loss of SPAG6 expression in NSCLCs.

Citation Format: Corinna Altenberger, Gerwin Heller, Bianca Schmid, Barbara Ziegler, Leonhard Müllauer, György Lang, Adelheid End-Pfützenreuter, Balazs Döme, Britt-Madeleine Arns, Kwun M. Fong, Casey M. Wright, Ian A. Yang, Walter Klepetko, Christoph C. Zielinski, Sabine Zöchbauer-Müller. Transcriptional regulation of SPAG6 by DNA methylation in NSCLCs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2014-397