Study of the molecular mechanisms by protein SET with impact on tumorigenesis and progression of oral cancer

Abstract

This project aims to continue the study of the molecular and biochemical actions of SET protein in tumorigenesis and progression and spread of solid tumors using as model/experimental approach head and neck squamous cell carcinoma (HNSCC). The main front research is based on the impact of this protein in intracellular signaling pathways important for tumorigenesis. At the molecular level, the mechanisms of action of this protein in cancer are not yet completely understood, although several functions have already been described. Recently, our group showed strong accumulation of SET in HNSCC samples, its involvement in the Akt signaling pathway in response to oxidative stress and its potential role in response to DNA damage. Moreover, in the previous project, we obtained evidence that the knockdown for SET in HNSCC cell line changes the level of some miRNAs and RNAs of several transcription factors altered in cancer, a fact that suggests its action in controlling expression. We also obtained evidence of his involvement in cell invasion, epithelial-mesenchymal differentiation, with a strong immune response against tumor xenografts in nude mice from a lineage HNSCC with SET silenced. All these data point to new functions SET protein that may contribute to oral cancer. However, the molecular mechanisms related to the actions described above have not been determined because it depends on further studies. Therefore, in this project we propose to define some of the molecular mechanisms by which the protein SET contributes to tumorigenesis and progression of oral cancer. To answer this question will be used HNSCC cell lineages (HNSCC: HN13, HN12, HN6, CAL-27), NOK-SI (spontaneously immortalized keratinocyte) and lineage kidney HEK293 (all provided by Dr. J.Silvio Gutkind, NIH, USA) after knockdown or overexpression of the protein of interest. Strategies for cellular and molecular biology and biochemistry will be employed in the study of protein functional model using in vitro and in vivo. Western blotting and immunofluorescence methods are used to determine the status (level and phosphorylation) of proteins and distribution in the cell. Various methods and strategies will be used: RNA interference systems, real-time array PCR, real time PCR for miRNA, PCR screening system using a phosphorylated kinase Antibody Array, protein-protein interactions, ELISA, flow cytometry and proliferation cell invasion methylation analysis by PCR and real-time PCR, microscopy, formation of xenograft tumors in nude mice, among others. It is hoped this proposal to identify the molecular mechanisms associated with SET that favor the development and progression of oral cancer, with emphasis on the following specific actions: genomic instability, DNA methylation, miRNA expression, phosphorylation of kinases, epithelial-mesenchymal differentiation, response tumor immune. The approach is multidisciplinary and will expand our understanding on the functions of the SET in oral cancer. (AU)