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Author(s)

Daniele Albuquerque

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2014-06-24

Examining board members

Vitor Marcel Faça; Claudio Miguel da Costa Neto; Leo Kei Iwai

• Advisor: Vitor Marcel Faça

Abstract

Breast cancer is a major worldwide public health problem because it is the most common malignancy among women. Despite the scientific understanding of the disease and methods of diagnostic imaging advances one of the major challenges is still the resolution of its high clinical and molecular heterogeneity. Breast cancer is caused by various genetic and epigenetic alterations, such as mutations in BRCA-1 and BRCA-2 genes that are considered the most frequent. And mutations in TP53, PTEN, ATM, CDH1, CHEK2 and STK11 (LKB1) gene. ln addition to these mutations, the self-sufficiency of the neoplastic cells in growth factors allows the tumor to evolve into more advanced stages and develop the metastatic process. Growth factors such as TGFβ are known to induce a process called epithelial-mesenchymal transition (EMT) in which normal epithelial cells during embryogenesis or malignant cells during tumor progression and metastasis lose their intracellular contacts and acquire migratory character. TGFβ induced EMT is considered as one of the mechanisms of tumor progression in adenocarcinomas as well as the process of generating cancer stem cells. ln this study, we used the induction of EMT by TGFβ2 in normal breast epithelial cell line MCF-10A as a controlled model of metastasis in order to evaluate the molecular changes during the EMT process. The assessment of the induction was performed initially by RT-PCR, Western blotting, multiple reaction monitoring (MRM) and migration assay (Wound healing). Techniques for quantitative proteomics analysis based on metabolic labeling with isotopes of amino acids in cell culture (Stable lsotope Labeling by Amino Acids in Cell culture, SILAC) combined with liquid chromatography coupled to mass spectrometry (LC-MS/MS) were used to characterize morphological changes during EMT of MCF-10A cells. Cell extracts were subjected to subcellular fractionation by differential centrifugation and then protein were further fracttonated by SDS-PAGE to reduce the complexity of the samples prior LC-MS/MS analysis. Twenty-four fractions were generated corresponding to 2 main comparisons of molecular changes in MCF-10A cells by treatment with TGFβ2: alterations in cytoplasmic proteins and in nuclear proteins. A total of 2603 proteins were identified in the proteomic analysis and of those, 18% were detected only in cytoplasmic fractions and 24% only in nuclear, indication the importance of subcellular fractionation to increase the coverage of the proteomic analysis. Of this total of proteins identified, 169 were detected in differential abundance after EMT induction. The 80 proteins that have changed the most were selected for characterization in more details. The ontology analysis for the molecular function showed that the up-regulated proteins after the induction of EMT are related to binding to other biomolecules, whereas down-regulated proteins exert catalytic activity. The bioinformatic analysis of interaction networks showed that the differentially expressed proteins are related to the processes of splicing and ribosome biogenesis, regulation of protein synthesis, vesicular transport and cell division. ln conclusion, this study revealed proteins that might be important for the process of progression and metastasis in cancer and after extensive analysis and validation may potentially serve as targets for inhibition of metastasis or diagnosis of metastatic disease. (AU)

• FAPESP's process: 12/02518-4 - PROTEOMIC STUDY OF EPITHELIAL TO MESENCHYMAL TRANSITION IN BREAST CANCER
• Grantee: Daniele Albuquerque
• Support Opportunities: Scholarships in Brazil - Master