Altenberger Corinna, Heller Gerwin, Ziegler Barbara, Tomasich Erwin, Marhold Maximilian, Topakian Thais, Müllauer Leonhard, Heffeter Petra, Lang György, End-Pfützenreuter Adelheid, Döme Balazs, Arns Britt-Madeleine, Klepetko Walter, Zielinski Christoph C, Zöchbauer-Müller Sabine
Department of Medicine I, Clinical Division of Oncology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.
Mol Cancer. 2017 Jan 5;16(1):1. doi: 10.1186/s12943-016-0568-5.
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.
DNA甲基化与其他表观遗传机制共同调节基因的转录活性，并参与包括肺癌在内的恶性疾病的发病机制。在非小细胞肺癌（NSCLC）中，各种抑癌基因已被证实存在肿瘤特异性甲基化。然而，在大量已被鉴定为肿瘤特异性甲基化的基因中，绝大多数基因的肿瘤特异性甲基化情况至今仍不清楚。因此，本研究的主要目的是详细探究NSCLC中SPAG6和L1TD1基因转录调控的机制。
我们分析了公开的RNA测序数据，并通过RT-PCR进行基因表达分析。DNA甲基化分析采用甲基化敏感的高分辨率熔解分析和亚硫酸氢盐基因组测序。我们还使用免疫组织化学研究了蛋白质表达。细胞培养实验包括肿瘤细胞生长、增殖、活力以及集落形成测定。此外，我们使用免疫缺陷小鼠进行了异种移植实验。
与NSCLC患者相应的非恶性肺组织（NL）样本相比，我们观察到原发性肿瘤（TU）样本中SPAG6和L1TD1 mRNA表达频繁下调。此外，在用表观遗传活性药物处理后，大多数SPAG6和L1TD1 mRNA表达下调的NSCLC细胞系中，这两个基因重新表达。当我们分析NSCLC患者的TU和相应NL样本时，检测到SPAG6和L1TD1频繁出现肿瘤特异性DNA甲基化。ROC曲线分析表明，这两个基因的甲基化能够区分这些患者的TU和NL样本。免疫组织化学显示SPAG6/L1TD1甲基化与这些基因的蛋白表达下调密切相关。此外，通过进行功能测定，我们观察到转染pCMV6-L1TD1的NSCLC细胞的生长、增殖和活力降低。另外，在异种移植肿瘤模型中，与转染pCMV6-ENTRY的NCI-H1975细胞相比，源自pCMV6-L1TD1的肿瘤体积减小。
总体而言，我们的结果表明SPAG6和L1TD1在NSCLC中存在肿瘤特异性甲基化，并且DNA甲基化参与了这些基因的转录调控。此外，体外和体内实验均显示L1TD1在NSCLC细胞中具有抑制肿瘤细胞生长的特性。
