title: Proteomic analysis of ovarian cancer cells during epithelial-mesenchymal transition (EMT) induced by epidermal growth factor (EGF) reveals mechanisms of cell cycle control.
abstract: Epithelial to mesenchymal transition (EMT) is a well-orchestrated process that culminates with loss of epithelial phenotype and gain of a mesenchymal and migratory phenotype. EMT enhances cancer cell invasiveness and drug resistance, favoring metastasis. Dysregulation of transcription factors, signaling pathways, miRNAs and growth factors including EGF, TGF-beta and HGF can trigger EMT. In ovarian cancer, overexpression of the EGFR family is associated with more aggressive clinical behavior. Here, the ovarian adenocarcinoma cell line Caov-3 was induced to EMT with EGF in order to identify specific mechanisms controlled by this process. Caov-3 cells induced to EMT were thoroughly validated and a combination of subcellular proteome enrichment, GEL-LC-MS/MS and SILAC strategy allowed consistent proteome identification and quantitation. Protein network analysis of differentially expressed proteins highlighted regulation of metabolism and cell cycle. Activation of relevant signaling pathways, such as PI3K/Akt/mTOR and Ras/Erk MAPK, in response to EGF-induced EMT was validated. Also, EMT did not affected the proliferation rate of Caov-3 cells, but led to cell cycle arrest in G1 phase regulated by increased levels of p21Waf1/Cip1, independently of p53. Furthermore, a decrease in G1 and G2 checkpoint proteins was observed, supporting the involvement of EGF-induced EMT in cell cycle control.
  
  
  
  Cancer is a complex multistep process characterized by accumulation of several hallmarks including epithelial to mesenchymal transition (EMT), which promotes cellular and microenvironmental changes resulting in invasion and migration to distant sites, favoring metastasis. EMT can be triggered by different extracellular stimuli, including growth factors such as EGF. In ovarian cancer, the most lethal gynecological cancer, overexpression of the EGFR family is associated with more aggressive clinical behavior, increasing mortality rate caused by metastasis. Our proteomic data, together with specific validation of specific cellular mechanisms demonstrated that EGF-induced EMT in Caov-3 cells leads to important alterations in metabolic process (protein synthesis) and cell cycle control, supporting the implication of EGF/EMT in cancer metastasis, cancer stem cell generation and, therefore, poor prognosis for the disease.
authors.affiliation: Dept. Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil; Cell-Based Therapy Center, Ribeirão Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Mariana Lopes
authors.initials: ML
authors.lastname: Grassi
authors.affiliation: Dept. Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil; Cell-Based Therapy Center, Ribeirão Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Camila de Souza
authors.initials: CS
authors.lastname: Palma
authors.affiliation: Dept. Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil; Cell-Based Therapy Center, Ribeirão Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Carolina Hassibe
authors.initials: CH
authors.lastname: Thomé
authors.affiliation: Dept. Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil; Cell-Based Therapy Center, Ribeirão Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Guilherme Pauperio
authors.initials: GP
authors.lastname: Lanfredi
authors.affiliation: Dept. Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Aline
authors.initials: A
authors.lastname: Poersch
authors.affiliation: Dept. Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil; Cell-Based Therapy Center, Ribeirão Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil. Electronic address: vitor.faca@fmrp.usp.br.
authors.forename: Vitor Marcel
authors.initials: VM
authors.lastname: Faça
mesh_terms: D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D002465:Cell Movement; D002853:Chromatography, Liquid; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005260:Female; D059585:G1 Phase Cell Cycle Checkpoints; D006801:Humans; D009361:Neoplasm Invasiveness; D010051:Ovarian Neoplasms / Q000473:pathology*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004815:Epidermal Growth Factor
keywords: Cell cycle; Epidermal growth factor; Epithelial to mesenchymal transition; Ovarian cancer