Análise da heterogeneidade celular estrutural e ultraestrutural, imunofenotipagem e via de sinalização EGRF em subpopulações de células-tronco de linhagens de câncer de mama triplo- negativo e suas implicações no mecanismo de resistência quimioterápica

Abstract

Triple negative breast cancer (TNBC) is characterized by high molecular and cellular heterogeneity, which influences the therapeutic response and it makes difficult the discovery of effective targets. This heterogeneity is attributed to the presence of breast cancer stem cells (BCSCs), which determines resistance to chemotherapy and subsequently disease recurrence and metastasis. In this context, this work aimed to evaluate the morphological and phenotypic cellular heterogeneity and the EGFR signaling pathway gene expression in cell subpopulations obtained from TNBC cell lines, as well as determined their implications in chemotherapy resistance. The BT-549 and Hs 578T TNBC cell lines and HMT-3522 S, a nonmalignant breast cell line, were cultured in monolayer and in BCSCs enrichment culture (tumorspheres-TS assay). Furthermore, the BT-549 and Hs578T were characterized by fluorescence and electron microscopy and flow cytometry. In addition, the cell lines cultured in two models were treated with paclitaxel (PTX) and doxorubicin (DOX). BT-549 TS was used to evaluate EGFR-associated gene expression before and after DOX treatment. The results showed that BT-549 TS after BCSCs enrichment, identified by the CD44+/CD24- and ALDH+ phenotype, showed subexpression a majority of the evaluated genes (64 genes) and only 3 genes were overexpressed. Among the overexpressed genes (MAPK3, PRKCZ and STAT3), STAT3 had the highest expression level. The BT-549 and Hs 578T analysis demonstrated large morphological and phenotypic heterogeneity between these cell lines, as well as between the cell subpopulations that compose them, as well as in PTX and DOX response. For BT-549, it is suggested that the plasticity of CD44+/CD24+/CD146+ hybrid tumor cells, characterized by a mesenchymal and epithelial phenotype, and CD44/CD24+/CD146+ epithelial tumor cells originated CD44+/CD24-/CD146- BCSCs EMT-like, through the phenotypic conversion induced by epithelial-mesenchymal transition (EMT). In addition, these cells could originate BCSCs identified ALDH+ epithelial-like. BCSCs can selfrenewal and differentiate into CD44-/CD24-/CD146- tumor cells. Same phenotypic plasticity was not observed for Hs 578T and HMT-3522 S1. Cytotoxicity data showed that BT-549 was more resistant to PTX, which appears to be associated with the CD24+ phenotype. In contrast, BT-549 TS was extremely resistant to DOX probably due to the enrichment of BCSCs (CD44+/CD24-/CD146- and ALDH+ cells). These results showed the high BT-549 EMTassociated heterogeneity and plasticity, being able to completely altered the phenotype of the cellular subpopulations under CSC enrichment culture. This finding appears to play an important role in TNBC resistance to DOX treatment. In addition, STAT3 was highlighted as DOX-resistance related TNBC molecular signature and as a possible therapeutic target.