Cell Free DNA of Tumor Origin Induces a ‘Metastatic’ Expression Profile in HT-29 Cancer Cell Line

Abstract

BackgroundEpithelial 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.AimsTo 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.Materials and MethodsDNA 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).ResultsAdministration 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.ConclusionsDNA 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.

Tumor microenvironment complexity and therapeutic implications at a glance

“…In the context of cancer, they are involved in horizontal gene transfer and oncogenic transformation of normal cells as well as in the metastasis development [197]. Although there is no clear mechanistic evidence for their oncogenic potential, several proposals include (ii) overexpression of several pro-metastatic genes through the tolllike receptor 9 (TLR9)/ myeloid differentiation primary response 88 (MYD88) independent pathway [198]; (ii) transposable elements [199] and finally the cellular uptake of exosomes [200,201]. Another consequence of cell-free contacts involving cfDNA is the increased chemo-, radioresistance of cancer cells, as radiotherapy produces oxidized DNA which triggers reactive oxygen species and induces DNA damage response pathways [202].…”

First landscape of binding to chromosomes for a domesticated mariner transposase in the human genome: diversity of genomic targets of SETMAR isoforms in two colorectal cell lines

“…However, HSMAR1 is still capable of mediating 5' end cleavage and DNA integration [8,19]. This raises the interesting possibility that SETMAR isoforms might catalyse integration of extracellular DNA, because circulating free DNA is often released from cell death or infection and can be passively transferred into cells [21,22]. Given the limited number of possible integration sites, mostly located outside of genes, SETMAR would then represent a nuclear mechanism protecting cells against the genotoxic effects of integration of circulating cell free DNA.…”

“…However, the precise molecular mechanism is unknown and whether the choice of integration sites is governed by the binding properties of SETMAR is still an open question. Also, it is not clear whether (and how) this mechanism is connected to metastatic processes, by mediating integration of the free DNA released by tumor cells into the chromosomal DNA of healthy cells [21,22].…”

Сell-free D N A in follicular fluid of women with different parameters of ovarian function

“…Среди различных биомаркеров патологических процессов свободная внеклеточная ДНК (свДНК) в последнее время привлекает большое внимание [1]. Несвязанная с клетками ДНК выявляется в различных биологических жидкостях организма [2][3][4], ее концентрация возрастает при целом ряде заболеваний (онкологические, аутоиммунные, посттравматический синдром) [5][6][7][8][9], включая акушерско-гинекологическую патологию [10], что обусловливает интерес к исследованию диагностической и прогностической значимости свДНК [11][12][13][14].…”