Proteomics analysis reveals the role of ubiquitin specific protease (USP47) in Epithelial to Mesenchymal Transition (EMT) induced by TGF beta 2 in breast cells

Author(s):Show less -

Silvestrini, Virginia Campos ^{[1, 2]} ; Thome, Carolina Hassibe ^{[1, 2]} ; Albuquerque, Daniele ^{[1, 2]} ; Palma, Camila de Souza ^{[1, 2]} ; Ferreira, Germano Aguiar ^{[1, 2]} ; Lanfredi, Guilherme Pauperio ^[2] ; Masson, Ana Paula ^[2] ; Alberici Delsin, Lara Elis ^{[1, 2]} ; Ferreira, Fernanda Ursoli ^[1] ; de Souza, Felipe Canto ^[1] ; Franco de Godoy, Lyris Martins ^[3] ; Aquino, Adriano ^[4] ; Carrilho, Emanuel ^[4] ; Panepucci, Rodrigo Alexandre ^[1] ; Covas, Dimas Tadeu ^[1] ; Faca, Vitor Marcel ^{[1, 2]}

Affiliation

^[1] Hemotherapy Ctr Ribeirao Preto, Ctr Cell Based Therapy, Rua Tenente Catao Roxo 2501, BR-14051140 Ribeirao Preto, SP - Brazil

^[2] FMRP Univ Sao Paulo, Dept Biochem & Immunol, Av Bandeirantes 3900, BR-14049900 Ribeirao Preto, SP - Brazil

^[3] Fiocruz Parana, Inst Carlos Chagas, BR-81350010 Curitiba, Parana - Brazil

^[4] Univ Sao Paulo, Inst Quim Sao Carlos, Av Trabalhador Sao Carlense 400, BR-13566590 Sao Carlos, SP - Brazil

• Document type: Journal article
• Source: JOURNAL OF PROTEOMICS; v. 219, MAY 15 2020.
• Web of Science Citations: 0

Abstract

Epithelial to Mesenchymal Transition (EMT) is a normal cellular process that is also triggered during cancer progression and metastasis. EMT induces cellular and microenviromental changes, resulting in loss of epithelial features and acquisition of mesenchymal phenotypes. The growth factor TGF beta and the transcription factor SNAIL1 (SNAIL) have been described as inducers of EMT. Here, we carried out an EMT model with non-tumorigenic cell line MCF-10A induced with the TGF beta 2 specific isoform of TGF protein family. The model was validated by molecular, morphological and functional experiments and showed correlation with the up-regulation of SNAIL. In order to identify additional regulators of EMT in this non-tumorigenic model, we explored quantitative proteomics, which revealed the Ubiquitin carboxyl-terminal hydrolase 47 (USP47) as one of the top up-regulated proteins. USP47 has a known role in cell growth and genome integrity, but not previously correlated to EMT. After validating USP47 alterations using MRM and antibody-based assays, we demonstrated that the chemical inhibition of USP47 with the inhibitor P5091 reduced expression of EMT markers and reverted morphological changes in MCF-10A cells undergoing EMT. These results support the involvement of USP47 in our EMT model as well as potential applications of deubiquitinases as therapeutic targets for cancer progression management. Biological significance: Metastasis is responsible for most cancer-associated mortality. Additionally, metastasis requires special attention, as the cellular transformations make treatment at this stage very difficult or occasionally impossible. Early steps in cancer metastasis involve the ability to detach from the solid tumor mass and invade the surrounding stromal tissues through cohesive migration, or a mesenchymal or amoeboid invasion. One of the first steps for metastatic cascade is denominated epithelial to mesenchymal transition (EMT), which can be triggered by different factors. Here, our efforts were directed to better understand this process and identify new pathways that contributes for acquisition of EMT, mainly focused on post translational modifications related to ubiquitin proteasome system. Our model of EMT induction by TGF beta 2 mimics early stage of metastatic cancer in epithelial breast cells and a proteomic study carried out for such model demonstrates that the deubiquitinase enzyme USP47 acts in SNAIL stabilization, one of the most important transcription factors for EMT phenotype acquisition and consequent metastasis. In addition, the inhibiton of USP47 with P5091, reverted the EMT phenotype. Together the knowledge of such processes of cancer progression and regulation can help in designing new strategies for combined therapies for control of cancer in early stages. (AU)

• FAPESP's process: 17/03960-6 - Characterization of enzymes involved in post-translational modifications that modulate protein stability and translocation during the Epithelial-Mesenchymal transition
• Grantee: Virgínia Campos Silvestrini
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 16/03809-3 - Targeted proteomics of subcellular translocation during epithelial to mesenchymal transition (EMT)
• Grantee: Vitor Marcel Faça
• Support Opportunities: Regular Research Grants
• 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