title: Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells.
abstract: Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.
authors.affiliation: From the Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Camila de Souza
authors.initials: Cde S
authors.lastname: Palma
authors.affiliation: From the Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Mariana Lopes
authors.initials: ML
authors.lastname: Grassi
authors.affiliation: From the Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Carolina Hassibe
authors.initials: CH
authors.lastname: Thomé
authors.affiliation: the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Germano Aguiar
authors.initials: GA
authors.lastname: Ferreira
authors.affiliation: From the Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil;
authors.forename: Daniele
authors.initials: D
authors.lastname: Albuquerque
authors.affiliation: the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Mariana Tomazini
authors.initials: MT
authors.lastname: Pinto
authors.affiliation: the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Fernanda Ursoli
authors.initials: FU
authors.lastname: Ferreira Melo
authors.affiliation: the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Simone
authors.initials: S
authors.lastname: Kashima
authors.affiliation: the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
authors.forename: Dimas Tadeu
authors.initials: DT
authors.lastname: Covas
authors.affiliation: the Department of Radiology, Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, Stanford, California 94305-5101.
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Pitteri
authors.affiliation: From the Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and vitor.faca@fmrp.usp.br.
authors.forename: Vitor M
authors.initials: VM
authors.lastname: Faça
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D044127:Epigenesis, Genetic; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D053263:Gene Regulatory Networks; D056284:Histone Deacetylase 1 / Q000378:metabolism*; D006801:Humans; D061986:MCF-7 Cells; D009361:Neoplasm Invasiveness; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D000071250:Snail Family Transcription Factors / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome; D000071250:Snail Family Transcription Factors; D051057:Proto-Oncogene Proteins c-akt; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1