Characterization of Acyl-CoA Sinthetase 4 (ACSL4) promoter in human breast cancer cells

DATTILO MELINA; BENZO YANINA; LOPEZ PAULA; NATALIA MORDUCHOWICZ; CASTILLO FERNANDA; MALOBERTI PAULA

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2016

ACSL4 expression in human breast cancer cells correlates with tumor aggressiveness but its regulation remains unknown. Our objective is to characterize the transcriptional regulation of ACSL4 in breast cancer cells according to their level of aggressiveness (MDA-MB-231 and MCF-7). A 1.8 kb fragment of promoter was cloned upstream Nluc gene, a luminiscent reporter. Deletions were made from both 5´ and 3? ends. Results show there is at least one element in 5´ region that downregulates the promoter activity in both cell lines. Unidirectional deletion of 3´ end shows a positive regulatory 43 bp region only in MDA-MB-231 as responsible of the higher expression of ACSL4. Bioinformatic identification of possible transcription factors involved in this regulation showed consensus sites for RORa in the 5´ region and E2F consensus sites in 3? region of the promoter among others sites like CREB, SP1 and ESRR. Mutations were constructed for consensus sites potentially involved. RORa mutation increased activity in both cell lines suggesting this transcription factor is involved at least in part in the negative regulation of promoter activity in the 5´ end in these breast cancer cells. For the 3´ region, E2F mutants sites were obtained within the region related to the differential promoter activity. A transcriptional activity decrease was observed only in MDA-MB-231. Thus E2F is enhancing at least in part the promoter activity in the 3´ end only in MDA-MB-231. Overexpression of ERa in MDA-MB-231 reduced promoter activity, indicating that ERa is a negative regulator of ACSL4 expression. In regard the epigenetic mechanisms, a histone deacetylases inhibitor increases transcription in both cell lines and a cytosine methylation inhibitor increases transcription only in MDA-MB-231. Therefore, with these results we could observe different transcription factors and epigenetic mechanisms that could regulate differentially ACSL4 promoter in both breast cancer cell lines.