title: Cell Free DNA of Tumor Origin Induces a 'Metastatic' Expression Profile in HT-29 Cancer Cell Line.
abstract: BACKGROUND
  Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.
  
  
  OBJECTIVE
  To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.
  
  
  METHODS
  DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin), DNA methyltransferase 3a (DNMT3a) and NFκB (for treated HDFα cells).
  
  
  RESULTS
  Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05) mRNA level alteration in 118 genes (logFc≥1, p≤0.05), including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A), metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1), tumor biomarker (CEACAM5), metabolic genes (i.e. INSIG1, LIPG), messenger molecule genes (i.e. DAPP, CREB3L2). Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05), including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β), STING pathway (ADAR, IRF7, CXCL10, CASP1) and the FGF2 gene.
  
  
  CONCLUSIONS
  DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling pathway in normal fibroblasts.
authors.affiliation: Hungarian Academy of Sciences, Molecular Medicine Research Unit, Budapest, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Fűri
authors.affiliation: 2nd Dept. of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kalmár
authors.affiliation: Hungarian Academy of Sciences, Molecular Medicine Research Unit, Budapest, Hungary.
authors.forename: Barnabás
authors.initials: B
authors.lastname: Wichmann
authors.affiliation: Hungarian Academy of Sciences, Molecular Medicine Research Unit, Budapest, Hungary.
authors.forename: Sándor
authors.initials: S
authors.lastname: Spisák
authors.affiliation: 2nd Dept. of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Andrea
authors.initials: A
authors.lastname: Schöller
authors.affiliation: 2nd Dept. of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Barbara
authors.initials: B
authors.lastname: Barták
authors.affiliation: Hungarian Academy of Sciences, Molecular Medicine Research Unit, Budapest, Hungary.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Tulassay
authors.affiliation: Hungarian Academy of Sciences, Molecular Medicine Research Unit, Budapest, Hungary.
authors.forename: Béla
authors.initials: B
authors.lastname: Molnár
mesh_terms: D004247:DNA / Q000235:genetics*; D000090205:DNA Methyltransferase 3A; D004847:Epithelial Cells / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D052138:Genes, Neoplasm; D019073:HT29 Cells; D006801:Humans; D009362:Neoplasm Metastasis / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*
chemical_list: C000717967:DNMT3A protein, human; D004247:DNA; D000090205:DNA Methyltransferase 3A