title: SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers.
abstract: 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.
authors.affiliation: 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.
authors.forename: Corinna
authors.identifier: 0000-0002-5836-5809
authors.initials: C
authors.lastname: Altenberger
authors.affiliation: 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.
authors.forename: Gerwin
authors.initials: G
authors.lastname: Heller
authors.affiliation: 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.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ziegler
authors.affiliation: 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.
authors.forename: Erwin
authors.initials: E
authors.lastname: Tomasich
authors.affiliation: 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.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Marhold
authors.affiliation: 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.
authors.forename: Thais
authors.initials: T
authors.lastname: Topakian
authors.affiliation: Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.|Department of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Leonhard
authors.initials: L
authors.lastname: Müllauer
authors.affiliation: Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.|Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Heffeter
authors.affiliation: Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.|Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.|Department of Thoracic Surgery, Semmelweis University and National Institute of Oncology, Budapest, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Lang
authors.affiliation: Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.|Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
authors.forename: Adelheid
authors.initials: A
authors.lastname: End-Pfützenreuter
authors.affiliation: Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.|Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.|Department of Thoracic Oncology and Tumor Biology, National Koranyi Institute of Pulmonology, Budapest, Hungary.|Department of Thoracic Surgery, Semmelweis University and National Institute of Oncology, Budapest, Hungary.
authors.forename: Balazs
authors.initials: B
authors.lastname: Döme
authors.affiliation: Department of Respiratory and Critical Care Medicine and Ludwig Boltzmann Institute for COPD, Otto-Wagner Hospital, Vienna, Austria.
authors.forename: Britt-Madeleine
authors.initials: BM
authors.lastname: Arns
authors.affiliation: Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.|Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Klepetko
authors.affiliation: 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.
authors.forename: Christoph C
authors.initials: CC
authors.lastname: Zielinski
authors.affiliation: Department of Medicine I, Clinical Division of Oncology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. sabine.zoechbauer-mueller@meduniwien.ac.at.|Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria. sabine.zoechbauer-mueller@meduniwien.ac.at.
authors.forename: Sabine
authors.initials: S
authors.lastname: Zöchbauer-Müller
mesh_terms: D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D019175:DNA Methylation; D030541:Databases, Genetic; D004195:Disease Models, Animal; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D064593:Heterografts; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008868:Microtubule Proteins / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D047368:Tumor Burden; D014410:Tumor Stem Cell Assay
chemical_list: C562346:L1TD1 protein, human; D008868:Microtubule Proteins; D011506:Proteins; D012333:RNA, Messenger; C400106:SPAG6 protein, human
keywords: DNA methylation; Epigenetics; L1TD1; Non-small cell lung cancer; SPAG6