The online version of this article (doi:10.1186/s12943-016-0568-5) contains supplementary material, which is available to authorized users.
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.
Additional file 1: Table S1. Description of NSCLC cell lines used in this study. Information about histology, origin and disease stage of donors was obtained from ATCC catalogue ( https://www.lgcstandards-atcc.org). EGFR, KRAS and TP53 mutational status and MET amplification according to supplementary references (1–3). *activating EGFR mutation in exon 19 (E746-E749 del), **activating EGFR mutation in exon 21 (L858R). N/A, not available; wt, wildtype; mut, mutated. Table S2. Clinico-pathological characteristics of 983 NSCLC patients. Overview of gender, histology, stage of disease and ethnicity of NSCLC patients obtained from TCGA database and used for mutation and copy number changes analyses of SPAG6 and L1TD1 is shown. ADC, adenocarcinoma; SCC, squamous cell carcinoma. Clinical data based on Caleydo software version 16/04/14. Table S3. Primer sequences. Summary of oligonucleotide sequences used for mRNA expression, MS-HRM, BGS analyses and construction of pCMV6-GFP expression vector. Y, random integration of C or T in fwd primer; R, random integration of G or A in rev primer. Table S4. Methylation of SPAG6 and L1TD1 in tumor cells of other tumor types. *Morphology, histology and origin of cell lines according to ATCC catalogue ( https://www.lgcstandards-atcc.org). Percentage of methylation was calculated as described previously (4). (DOCX 33 kb)
Additional file 2: Figure S1. SPAG6 and L1TD1 mRNA expression in different datasets of TCGA database. SPAG6 and L1TD1 mRNA expression was analysed using IlluminaHiSeq RNAseq data from TCGA database. Datasets LUAD and LUSC (lung), BRCA (breast), COADREAD (colorectal), HNSC (head and neck), KIRC (kidney), LIHC (liver) and PRAD (prostate) were analysed. Normalized log2 mRNA expression values are shown. Each dot represents a single sample. (TIF 176 kb)12943_2016_568_MOESM2_ESM.tif
Additional file 3: Figure S2. Impact of SPAG6 and L1TD1 mRNA expression on OS of NSCLC patients. (A) A shorter OS of squamous cell carcinoma patients with low SPAG6 mRNA expression (N = 155) compared to high SPAG6 mRNA expression (N = 267) was observed. (B) Adenocarcinoma patients with low L1TD1 mRNA expression (N = 138) showed a shorter OS compared to adenocarcinoma patients with high L1TD1 mRNA expression (N = 350). Gene expression microarray datasets (Affymetrix IDs 210032_s_at and 219955_at) were analysed and Kaplan-Meier plots were generated using all datasets and default settings of KM plotter. The cut-off values for “low” and “high” SPAG6 and L1TD1 mRNA expression were automatically defined by KM plotter software (Version 2013). (TIF 50 kb)12943_2016_568_MOESM3_ESM.tif
Additional file 4: Figure S3. SPAG6 and L1TD1 SNVs and deletions in NSCLC patients. TCGA LUAD and LUSC datasets were analysed with Caleydo software (version April 2014). Mutation of TP53 was used to demonstrate reliability of TCGA data analysis. ADC, adenocarcinoma patients; SCC, squamous carcinoma patients. (TIF 90 kb)12943_2016_568_MOESM4_ESM.tif
Additional file 5: Figure S4. L1TD1 mRNA expression in xenograft tumors. Expression of L1TD1 in 4 xenografts derived from pCMV6-L1TD1 transfected NCI-H1975 cells was confirmed by RT-PCR. GAPDH was used as housekeeping gene to normalize mRNA expression of L1TD1. (TIF 17 kb)12943_2016_568_MOESM5_ESM.tif
Kulis M, Esteller M. DNA methylation and cancer. Adv Genet. 2010;70:27–56. PubMed
Heller G, Altenberger C, Schmid B, Marhold M, Tomasich E, Ziegler B, et al. DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers. Oncotarget. 2015;6:394–408. PubMed
Zöchbauer-Müller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res. 2001;61:249–55. PubMed
Yang B, Wang L, Luo X, Chen L, Yang Z, Liu L. SPAG6 silencing inhibits the growth of the malignant myeloid cell lines SKM-1 and K562 via activating p53 and caspase activation-dependent apoptosis. Int J Oncol. 2015;46:649–56. PubMed
Silina K, Zayakin P, Kalnina Z, Ivanova L, Meistere I, Endzelins E, et al. Sperm-associated antigens as targets for cancer immunotherapy: expression pattern and humoral immune response in cancer patients. J Immunother. 2011;34:28–44.
Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013;6:l1. CrossRef
Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489:519–25.
Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 511:543–50.
Toyooka S, Toyooka KO, Maruyama R, Virmani AK, Girard L, Miyajima K, et al. DNA methylation profiles of lung tumors. Mol Cancer Therapeutics. 2001;1:61–7.
Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res. 2001;61:3225–9. PubMed
Toyooka S, Toyooka KO, Miyajima K, Reddy JL, Toyota M, Sathyanarayana UG, et al. Epigenetic down-regulation of death-associated protein kinase in lung cancers. Clin Cancer Res. 2003;9:3034–41. PubMed
- SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers
Christoph C. Zielinski
- BioMed Central
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