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Characterization of the involvement of WNK2 protein in autophagic and endocytic vesicle traffic in glioma

Grant number: 16/18907-0
Support type:Scholarships in Brazil - Master
Effective date (Start): November 01, 2017
Effective date (End): May 31, 2019
Field of knowledge:Biological Sciences - Genetics - Human and Medical Genetics
Principal researcher:Rui Manuel Vieira Reis
Grantee:Ana Laura Vieira Alves
Home Institution: Hospital do Câncer de Barretos. Fundação Pio XII (FP). Barretos , SP, Brazil

Abstract

The autophagic process is a recycling and degradation system, suggested as an important pathway of cell death in tumor cells. The autophagic pathway is connected with the endocytic pathways from the same final destination, lysosomal. Lysosomes in turn are crucial regulators of cell homeostasis, responsible for downregulate the signaling receptors and their turnover, contributing to tumor progression. WNK2 a member of the subfamily WNK (With No Lysine (K)) are serine / threonine kinases, it has been reported to be negatively regulated through hypermethylation of its promoter, and has been proposed to act as a suppressor gene in specific brain tumors. Studies in our group have shown that inhibition of WNK2 in gliomas associated with cell proliferation, migration, and death metalloproteinase-2 expression levels. Moreover, there is increasing evidence of additional functions WNK kinases in vesicular traffic. Preliminary results from our group suggest that the restoration of WNK2 protein in gliomas does not interfere in the process of cell death after treatment with temozolomide chemotherapy, but may be related in some way to vesicle traffic, autophagic and endocytic. Studies reported so far are contradictory while displaying WNK2 as a modulator of autophagy, and its role in tumor cell death largely unknown. In this context, given the importance of autophagic mechanisms, endocytic and lissomal in tumor progression and contradictory association with WNK2 protein, is fundamental importance a better characterization of the mechanisms that governing this protein in the cancer context, and particularly in gliomas, tumors high mortality rate and innate resistance to current therapeutic options. This project aims therefore to characterize the role of WNK2 protein in autophagic, endocytic and lysosomal vesicle traffic in glioma cell lines. For this we will: i) evaluate the expression of the gene encoding WNK2 in a panel of commercial cell lines and primary gliomas by Western blot using 3 commercial antibodies; ii) edit WNK2 gene into a commercial line using the CRISPR vector; iii) validating the overexpression and silencing of WNK2 by RT-PCR and Western blot in the edited and silenced cell lines; iv) identify the gene profile of autophagic, endocytic and lysosomal pathways associated with WNK2 by in silico analysis using the Oncomine database; v) evaluate the autophagy process and key proteins related to this route using the WNK2 edited cell lines and silenced; vi) evaluate the process of endocytosis baseline, and its inhibition in the transfected cell lines; vii) evaluate the presence of lysosomal markers on the edited cell lines. This work could improve a better understanding of the involvement of WNK2 protein in vesicular traffic and its relation to gliomagenesis. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DE MENEZES, WEDER PEREIRA; OLIVEIRA SILVA, VIVIANE ALINE; FARIA GOMES, IZABELA NATALIA; ROSA, MARCELA NUNES; CORCOLL SPINA, MARIA LUISA; CARLONI, ADRIANA CRUVINEL; VIEIRA ALVES, ANA LAURA; MELENDEZ, MATIAS; ALMEIDA, GISELE CARAVINA; DA SILVA, LUCIANE SUSSUCHI; CLARA, CARLOS; DA CUNHA, ISABELA WERNECK; MAROSO HAJJ, GLAUCIA NOELI; JONES, CHRIS; BIDINOTTO, LUCAS TADEU; REIS, RUI MANUEL. Loss of 5 `-Methylthioadenosine Phosphorylase (MTAP) is Frequent in High-Grade Gliomas; Nevertheless, it is Not Associated with Higher Tumor Aggressiveness. CELLS, v. 9, n. 2 FEB 2020. Web of Science Citations: 0.
VIEIRA ALVES, ANA LAURA; COSTA, ANGELA MARGARIDA; MARTINHO, OLGA; DA SILVA, VINICIUS DUVAL; JORDAN, PETER; OLIVEIRA SILVA, VIVIANE ALINE; REIS, RUI MANUEL. WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line. CELLS, v. 9, n. 2 FEB 2020. Web of Science Citations: 0.

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