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Proteomic Analysis of Extracellular Vesicles from Saliva and Plasma of Patients with Squamous Cells Carcinoma and the Study of Post-Translational Modifications and Identification of Disease Markers

Grant number: 18/11958-4
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): November 01, 2018
Effective date (End): October 31, 2021
Field of knowledge:Health Sciences - Medicine
Principal Investigator:Adriana Franco Paes Leme
Grantee:Leandro Xavier Neves
Home Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia, Inovações e Comunicações (Brasil). Campinas , SP, Brazil

Abstract

The squamous cell carcinoma (SCC) is a malignancy derived from stratified squamous epithelium and represents the most common type of oral cancer. In Brazil, 600,000 new cases of cancer are expected to occur in 2018, of which 14,700 would be affections of oral cavity. The prognosis is mostly based on the tumour, node and metastasis (TNM) staging system. However, the TNM classification assumes that tumours in the same stage and similar morphology are biologically alike although evidences reinforce that subtle molecular aspects may affect the disease progression. For example, extracellular vesicles (VEs) are strongly related with intercellular communication processes by carrying signalling molecules. By means of their secretions, cancer cells are able to reprogram both adjacent and distant cells creating a pro-oncogenic environment and preparing premetastatic niches. Unfortunately, the knowledge regarding the composition and role of VEs in tumour development is scarce. Data obtained by this research group support the findings published in the literature reporting that the proteome of VEs is complex and prone to post-translational modifications (PTMs), such as phosphorylation, ubiquitination and proteolysis. Therefore, it is of utmost urgency a deep proteomic characterisation of VEs in SCC patients to the better understanding of their physiological role. The present project propose the application of MS-based quantitative proteomics of proteins and endogenous peptides from VEs isolated from patients saliva divided in four groups: (a) No oral SCC, (b) premalignancy lesion, (c) SCC pre-surgery and (d) SCC post-surgery. The employment of enrichment techniques to phosphorylated and ubiquitinylated peptides will increase analytical sensitive towards these group of molecules allowing for the profiling of these PTMs in VEs from oral SCC patients. Peptide identification by sequence deduction independent of protein databases (de novo) will constitute a strategy to study alternative splicing, point mutation and polymorphisms. Moreover, bioinformatics analyses will predict regulated cellular processes, active proteases involved in endogenous peptide production, and other functional and structural features. At last, biomarkers of biomedical interest will be proposed and validated using targeted proteomics after development of method with adequate sensitivity, selectivity and throughput. We expect our data will (i) reveal the profile of proteins and endogenous peptides present in VEs isolated in saliva and plasma in different SCC stages, (ii) expand the understanding about the role of PTMs and alternative sequences in VEs isolated from patients with oral SCC, (iii) correlate these information to clinical-pathology aspects, (iv) provide a quantitative method for the analysis of SCC markers using targeted proteomics.