GPAT2, a gene involved in piRNA biogenesis, is epigenetically regulated and promotes tumoral phenotype of MDA-MB-231 cell line

GARCIA FABIANI, M.B.; MONTANARO, M.A.; LACUNZA, E.; QUIROGA, I.Y.; ABBA, M.; COLEMAN, R.A.; GONZALEZ BARO, M.R.; PELLON-MAISON, M.

Smithfield, RI

Congreso; Gordon Research Conference "Epigenetics Mechanisms and Implications"; 2013

Gordon Research Conference

The first step in de novo glycerolipid synthesis is catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2, located in the outer mitochondrial membrane, is highly expressed in germ cell line in testis, and it was recently found that is essential for piRNA biogenesis. GPAT2 was also proposed as a ?cancer-testis gene?, which encode a heterogeneous group of proteins (CT antigens) expressed almost exclusively in normal testis and certain cancer cells. The aim of this work was to evaluate the contribution of GPAT2 expression to tumoral phenotype and to understand the mechanism of GPAT2 overexpression in cancerous cells. By in silico analysisn, we selected MDA-MB-231 as a model of  tumorigenic cell line that express GPAT2. A stable GPAT2 KO MDA-MB-231 cell line, with only 5% of GPAT2 expression had 50% lower cell growing rate on MTT growing curve assays, a reduction of 90 % in the number of colonies in soft agar colony assay, and a reduction in the percentage of closure in wound healing assay (60 % at 8 hours). Additionally, apoptotic cell to total cell ratio was determined by TUNEL assay. At 30 min of staurosporin treatment, apoptosis was dramatically increased from 6.6 to 85.4 % in control vs. GPAT2 KO MDA-MB-231 cells (p<0.001) and this effect was also evident at 2 hours of treatment with an increase from 36.9 to 96.1 % (p<0.01). GPAT2 expression in breast carcinomas and cell lines shows an on-off pattern. This suggests an epigenetic regulation, which is characteristic of CT genes. To test this, we incubated MDA-MB-231, Hela, and MCF7 cells with 5?aza-deoxycitidine (DAC) and analyzed GPAT2 expression by qPCR.  We found that DAC did not affect GPAT2 expression in MDA-MB-231 cells but increased GPAT2 expression 7.5 and 25-fold in HeLa  and MCF7 cells, respectively. By bisulfite sequencing experiments we found different methylation status of the GPAT2 promoter in cells expressing or not the gene. Our results confirm that GPAT2 contributes to tumorigenicity of MDA-MB-231 cells and like other CT genes, its expression is epigenetically regulated