Proteomic analysis of ovarian cancer cells during epithelial-mesenchymal transition (EMT) induced by epidermal growth factor (EGF) reveals mechanisms of cell cycle control

Author(s)

Grassi, Mariana Lopes ; Palma, Camila de Souza ; Thome, Carolina Hassibe ; Lanfredi, Guilherme Pauperio ; Poersch, Aline ; Faca, Vitor Marcel

• Document type: Journal article
• Source: JOURNAL OF PROTEOMICS; v. 151, p. 10-pg., 2017-01-16.

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 p21Wafl/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. Biological significance: 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. (C) 2016 Elsevier B.V. All rights reserved. (AU)

• FAPESP's process: 13/07675-3 - Study of PI3K/AKT signaling pathways in acute myeloid leukemia using selective ion monitoring (SRM).
• Grantee: Carolina Hassibe Thomé
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 13/08135-2 - CTC - Center for Cell-Based Therapy
• Grantee: Dimas Tadeu Covas
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 12/09682-4 - Proteomic analysis of Epithelial to Mesenchymal Transition in Breast Cancer Promoted by Overexpresison of Transcription Factors.
• Grantee: Camila de Souza Palma
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 13/08755-0 - Targeted proteomic analysis of potential ovarian cancer biomarkers during tumoral progression.
• Grantee: Mariana Lopes Grassi
• Support Opportunities: Scholarships in Brazil - Doctorate