Avaliação do papel da metilação de DNA na repressão gênica, restrita à hipóxia, do supressor tumoral SAV1 e seu potencial antitumoral

Abstract

Tumor hypoxia has been associated with a worse prognosis, especially in breast cancer. It induces global genomic hypomethylation and hypermethylation of specific loci in DNA. DNA methylation is an epigenetic, context-dependent, reversible mechanism that occurs in CpGs islands located in the promoter and/or first gene exon, promoting gene silencing. However, little is known about which genes are regulated by methylation in hypoxia. Preliminarily, this work identified by DNA microarray, that the tumor suppressor gene SAV1, belonging to the Hippo signaling pathway, is a candidate for regulation by DNA methylation, in hypoxia. In addition, SAV1 has CpG Island in its promoter region and its gene expression is increased due to treatment with DNA demethylating agent. Activation of the Hippo pathway culminates with the inhibition of cell proliferation and the promotion of apoptosis, acting as a tumor suppressor. However, the importance of SAV1 in breast cancer, especially in hypoxia, is little known. Therefore, this work analyzed, in vitro and in silico, the expression and methylation profile of the SAV1 gene, in normoxia and hypoxia, and the prognostic potential of SAV1 for breast cancer, as well as changes in the transcriptome in response to the demethylating agent. (5-Aza-dC), in normoxia and hypoxia, by DNA microarray. The MDA-MB-231 and MCF-7 cell lines were used as an experimental model. The analysis of protein subcellular expression/location was performed by ICQ and gene expression by RT-qPCR. SAV1 promoter methylation was evaluated by bisulfite conversion and DNA sequencing. In silico analyzes were performed using various bioinformatics tools. Methylation analysis revealed a methylated cytosine, in hypoxia, a binding site for the transcription factors, THAP1 and NRF1, indicating that this methylation can regulate the SAV1 gene expression. The inverse relationship between SAV1 and CYR61/CTGF expressions, Hippo pathway targets, in the MDA-MB-231, suggests that this pathway may be more directed towards the pro-tumor face, in this cell line, than in the MCF-7 , which may be associated with its greater aggressiveness. SAV1 is less expressed in breast tumor tissues than in normal ones and methylation may contribute to the regulation of SAV1 gene expression, in the claudin-low and luminal A subtypes in normoxia. The lower SAV1 gene expression in samples from the hypoxia group suggests lesser tumor suppressive action in hypoxia than in normoxia, and may be associated with the worse prognosis related to hypoxia. The SAV1 levels expression decreased in a stage-dependent manner and had an impact on the survival curve. The data suggested SAV1 as a tumor suppressor, with a potential prognosis for breast cancer, in which low expression of SAV1 is associated with a worse prognosis. In addition, pro-inflammatory pathways were overexpressed after 5-Aza-dC. However, 5-Aza-dC in normoxia showed a greater amount of overexpressed pro-inflammatory pathways than in hypoxia and increased expression of NF-κB, suggesting that 5-Aza-dC induces a greater inflammatory change, at molecular levels, in normoxia than hypoxia