A expressão de SOX3 modula as vias de apoptose e transição epitélio-mesênquima em carcinoma ductal invasivo de mama humano

Abstract

Breast cancer is one of the diseases that kill most women worldwide. It is considered a complex disease with dynamic behavior, it is necessary to search for new tumor markers and therapeutic targets. Proteins that act as transcription factors have been studied in neoplasms due to their important role in the transcription of genes that encode oncogenic proteins or tumor suppressors. Among the transcription factors important in the cell fate, differentiation, and organ and tissues development, the SOX family proteins have been highlighted as a target in the study of cancer, among them SOX2 and SOX14, members of the same subgroup of the SOX3 protein, still little studied. To study the role of the SOX3 protein in the regulation of apoptosis in the epitheliummesenchymal transition (EMT) in mammary neoplasms, the cell line MDAMB213, from invasive ductal breast carcinoma, was selected as an experimental model because it does not express the protein SOX3, and show a more aggressive phenotype (Triplenegative). Therefore, to fulfill this objective, the region encoding the human SOX3 protein was subcloned into the pEF1/MycHis expression vector using a SOX3 sequence amplified from human genomic DNA. MDAMB231 cells were cultured and transfected with the pEF1/MycHis/ SOX3 vector, and untreated cells and cells transfected with empty pEF1/MycHis vector were used as controls. After 24 hours of transfection, cytotoxicity was evaluated by viability testMTT, the percentage of cells undergoing apoptosis was determined by flow cytometry using the Annexin VFITC/ PI protocol, and qPCR was performed to evaluate gene expression of apoptotic and epithelialmesenchymal pathways: SOX3, CASP3, CASP8, CASP9, BAX, BCL2, ECAD, NCAD, SNAIL, ZEB1 and ZEB2. Results indicated a reduction of 55% in cells transfected with pEF1/MycHis/ SOX3 viability, in relation to their controls, corroborating the results of the percentage of apoptosis by flow cytometry in cells transfected with pEF1/MycHis/ SOX3 (52%) when compared to the untransfected control (10%) and the empty vector control (13%). Realtime PCR results for apoptosisrelated genes demonstrated upregulation of mRNA expression of CASP3 (3.26fold), CASP8 (2.24fold), CASP9 (18.54fold) BAX (4.11fold), and downregulation of BCL2 mRNA expression (0.40fold) compared to the untransfected control. The evaluation of genes related to the epitheliummesenchymal transition showed downregulation of NCAD (0.06fold), SNAIL (0.18fold), ZEB1 (0.15fold), ZEB2 (0.11fold), mRNA expression, and upregulation of ECAD mRNA expression (27fold). Thus, the present study results support the hypothesis that SOX3 is a transcription factor involved in the regulation of proapoptotic genes and epithelial phenotype maintenance, with a possible tumor suppressor action. So, the loss of its expression indicates resistance to apoptosis and favoring EMT. Future functional studies and the investigation of the expression of this protein in breast tumor tissues, correlated with clinical characteristics and tumor prognosis, should be performed to better elucidate the role of this protein in breast cancer.