Finding binding parterns of Agrin fragments to study their role on the Oral Cancer progression

Abstract

Agrin is a high molecular weight heparan sulfate proteoglycan found with high protein expression in various types of cancer, such as squamous cell carcinoma (SCC), liver cancer, lung cancer, among others. We have recently shown that agrin knockdown (shAgrin) in oral SCC cell lines (SCC9 and HSC3) decreases about 50% the cell proliferation, migration and invasion, as well as decreases the formation of cell colonies and the tumour spheres. These processes are essential for the development, progression and tumor aggressiveness. These results were further corroborated by the in vivo orthotopic model, performed by the injection of control and shAgrin HSC3 cells on the side of the tongue of mice. The results showed that the decrease of agrin expression interferes negatively on angiogenesis and vascular invasion, cell morphology, cell growth pattern and keratinization, slowing down the tumor progression, decreasing its severity. The same study shows that some interaction partners of agrin, especially the C-terminal portion may be associated with the development of cancer. However, it has been observed that agrin undergoes a post-translational proteolytic processing generating 90 kDa (1103-1863 aa) and 22 kDa (1864-2067 aa) proteins (neoproteins) of the C-terminal region. These neoproteins were observed overexpressed in the secretome of the SCC9 and HSC3 cell lines, and they have been suggested to play an independent role on tumour development, although their role is unclear. In this way, this work seeks to continue the studies of our group (Kawakara et al., 2015 and Rivera et al, under evaluation), understanding the mechanisms of neoproteins signalling in the progression in oral cancer, as well as to investigate if the N-terminal region also plays a role in this process. For this, we will use strategies by structural proteomics to: i) identify interaction partners for the different constructions of agrin; ii) interfere with agrin direct binding partner through synthetic peptides that modulate agrin activity using protein-protein interaction experiments and functional experiments on cells. Thus, we expect that this project i) explain the mechanisms by which agrin in its different processed forms acts on tumour development and progression in oral cancer and ii) advance in the search for molecules that may interfere in the effect of agrin.