Hypomethylation of LINE-1 retrotransposon in breast cancer cells exposed to pesticides. Role of TGF-β1 pathway

MIRET NOELIA V; ALTAMIRANO, GABRIELA; ZAPPIA C. DANIEL; AYELEN GÓMEZ; CAROLINA A PONTILLO; ZARATE LORENA; LASAGNA MARIANELA; CLAUDIA COCCA; FEDERICO MONCZOR; KASS, LAURA; ANDREA RANDI

Mar Del Plata

Congreso; LXIV Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2019

SAIC

Long interspersed nuclear element 1 (LINE-1) is the most common non-LTR retrotransposon in thehuman genome and comprises 17% of the genome. Reactivation of LINE-1 has been linked to breast cancer progression and metastasis. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-ß1 (TGFß1) pathway in human liver cancer cells. We have observed that environmental doses of two weak AhR ligands: the organochlorine pesticide hexachlorobenzene (HCB, 0.005 µM) and the organophosphate chlorpyrifos (CPF, 0.5 µM) activate the TGF-ß1 signaling and enhance LINE-1 mRNA expression in the human breast cancer cell line MDA-MB-231. Our aim was to investigate the role of the TGF-ß1 signaling in the mechanism of action and if the pesticides induce epigenetic changes. To study the involvement of the TGF-ß1 signaling on LINE-1 expression, cells were exposed to HCB or CPF in the presence of the TGF-ß1receptor I inhibitor (SB431542, 2 µM). qPCR results showed that the enhancement exerted by the pesticides was prevented by the treatment with the inhibitor (p?0.05). Considering that LINE-1expression is regulated by methylation of its internal promoter, MDA-MB-231 cells were exposed to HCB or CPF and the methylation status of the LINE-1 5´-UTR was determined. Six sites (+38, +103, +167, +234, +306, +373) were analyzed by a combination of single digestions with methylation-sensitive restriction enzymes and qPCR. We found that the methylation was reduced by HCB at the sites +38, +103 and +167 (p?0.05) and by CPF at the sites +38 and +103 (p?0.05). In conclusion,HCB and CPF induce the demethylation of LINE-1 and enhance mRNA expression through TGF-ß1 pathway. Our findings support previous investigations showing that methylation of the first sitesin 5´-UTR are essential for the LINE-1 transcriptional regulation. Finally, environmental doses of pesticides promote epigenetic changes that could increase human breast cancer risk.