Estudo comparativo do perfil proteômico de linhagens celulares em diferentes estágios do processo de tumorogênese do câncer de mama

Abstract

Breast cancer is the leading cause of deaths in women worldwide. Genetic and environmental conditions support the outbreak of carcinomas with varying degrees of aggressiveness and tumorogenesis, leading to the cell transformation to primary cancer cells. Such factors can also give rise to metastasis. Even though breast cancer is one of the most studied type of cancer, yet pharmacological and therapeutic treatments are not efficient to reduce death rate. The ways to study breast cancer are through fragments of solid tumors removed in surgical procedures or in vitro studies using cell lines, though both strategies have limitations. For example, fragment of solid tumor is composed by several cell populations, which leads to high heterogeneity. On the other hand, in vitro studies usually are based on metastatic tumor lines, reflecting only the end of the tumor development process. The main objective of this work was to evaluate the proteome profile of breast cancer cell lines at different tumorigenic stages to better understand the molecular mechanisms underlying the tumorigenic process. Through the comparative analysis of the proteomes of MCF-10A (non-tumor origin), MGSO-3 and MACL-1 (primary tumor origin), and MDA-231-MD (metastatic origin) cell lines, we evaluated the functional state of each cell profile. In total, we identified and quantified 3,008 proteins and 1,709 regulated proteins (P < 0.05 and fold-change = 1.3). The PCA analysis and heat map showed a correlation between tumorigenic profile and proteome profile. Besides, the Venn diagrams demonstrated that similar tumorigenic profiles also shared higher number of proteins. Through gene ontology analysis, several positive and negative regulated biological processes were found across the cell lines. Similar biological processes were grouped into large clusters. In this work, we focused our analyses in terms related to energy metabolism, alterations in the cell cycle, and alterations in the cytoskeleton. Concerning energy metabolism, the tumor cell lines showed downregulation of lipid biosynthesis and the metastatic cell (MDA-231-MD) was the only one showing upregulation of β-oxidation. On the other hand, proteins related with the carbohydrate metabolism were mainly upregulated in the primary cells (MGSO-3 and MACL-1) and downregulated in the metastatic cell, mainly the proteins associated with the aerobic phase. These results are in line with an aerobic metabolism for the primary tumor lines and anaerobic metabolism for the metastatic one. This metabolic dichotomy seems to reflect on mitochondrial activity, as the primary tumor cells had lower mitochondrial density compared with the normal and metastatic cell lines. Furthermore, regulation profile of proteins associated with the mitochondrial BAX-dependent apoptotic pathways suggest that the primary cells are more prone for apoptosis compared with metastatic and normal cells. Regarding the cell cycle, the tumor cell lines have the highest number of upregulated processes. Besides, primary tumor cells seem to have upregulated biological processes related to the 26s proteasome, which is responsible for controlling oxidative processes and signaling pathways, mainly on the MACL-1. Regarding the cytoskeleton, the MDA-231-MD was the only tumor cell line to show upregulated biological processes, mainly related to cell adhesion mechanisms. Moreover, some upregulated proteins associated with cytoskeleton were also related with the glycolytic pathway, demonstrating that the role of cytoskeleton proteins are not only structural and for defense, but also for the survival of the metastatic tumor cell. All the results presented here are important for the better understanding of the molecular grounds of breast cancer tumorigenesis.