Role of low molecular weight protein tyrosine phosphatase in the biogenesis of extracellular vesicles secreted by colorectal cancer cells

Author(s)

Stefano Piatto Clerici

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-02-23

Examining board members

Carmen Veríssima Ferreira; Wagner José Fávaro; Claudio Saddy Rodrigues Coy

• Advisor: Carmen Veríssima Ferreira

Abstract

Cancer is a problem that affects world public health. According to global reports, in 2012 there were more than 14 million new cases of cancer with approximately 8.2 million deaths. According to Brazilian reports for the biennium of 2016-17, almost 600 000 new cases of this disease among which 34280 affected colon and rectum. This type of neoplasm originates in any area of the colon and rectum and in most cases is confined to the wall of these segments being curable if early detection through surgical resection with or without adjuvant chemotherapy. However, 15-20% of cases will develop metastases in other organs, reducing the efficiency of current therapeutic regimens and reducing patients' survival. Recently, research groups became interested in the study of extracellular vesicles as integral parts of tumor biology. Extracellular vesicles have become relevant as a structure that permits cell-cell communication and the transport of biomolecules, such as proteins, lipids, nucleic acids and oncogenic factors. Protein phosphatases, especially tyrosine phosphatases are presented as tumor suppressors, but low molecular weight protein tyrosine phosphatase (LMWPTP) has been identified as oncogenic and related to aggressiveness and tumor resistance to chemotherapeutics. The aim of this work was to investigate if the level of LMWPTP expression is related to the biogenesis of extracellular vesicles in human colorectal cell lines and to evaluate if the LMWPTP can be one of the components carried by the extracellular vesicles. We used as model two human colorectal cells line: HCT116 and HT29 under two distinct conditions: cultured for 48 hours in culture medium with fetal bovine serum and cells cultured for 48 hours in medium with fetal bovine serum and for 12 following hours in medium without fetal bovine serum, which is a necessary step for the isolation of the extracellular vesicles. Analysis of the protein expression showed that LMWPTP is overexpressed in HT29 compared to HCT116 as well as EVs biogenesis related proteins LAMP-1, LAMP-2, HRS, STAM-2, Rab5, Rab7, Rab11, Rab 25, Rab27A and Rab35, however, TSG101 and ALIX were lower expressed in HT29. In the lysate of cells submitted to fetal bovine serum for the isolations of extracellular vesicles, we observed that the expression of HRS, STAM-2, Rab5 and Rab 7was reduced in HT29 when compared to HCT116. Extracellular vesicles were isolated by two methods: ultracentrifugation and a polymer precipitation based kit. By both methods, the culture media from HT29 displayed higher concentration of extracellular vesicles than HCT116. Our results shown that or initial hypothesis is valid, LMWPTP might be associated to EVs biogenesis and/or trafficking mechanisms. These findings indicate an application potential of this enzyme in the EVs research (AU)

• FAPESP's process: 16/02770-6 - Role of the low molecular weight protein tyrosine phosphatase in the biogenesis of exosomes secreted by colon rectal cells
• Grantee: Stefano Piatto Clerici
• Support Opportunities: Scholarships in Brazil - Master