Genome-wide search for regulome mutations in squamous cell carcinoma of the larynx

The project is carried out as part of the scientific consortium. The leader of the project is the Institute of Human Genetics of the Polish Academy of Sciences, while the partners are the M. Nencki Institute of Experimental Biology of the Polish Academy of Sciences and the Medical University of Warsaw, 1st Faculty of Medicine. In the proposed project, we assume that in tumors, apart from the already well-documented gene damage, there are also unknown mutations within the regulome (non-coding sequences responsible for the regulation of gene expression), which significantly contribute to the pathogenesis of these diseases. The aim of this project is to identify potential mutations within the regulome of laryngeal squamous cell carcinoma (LSCC). In this project, we plan targeted NGS sequencing of the regulome, which includes a defined region of 46 min base pairs, covering the proximal enhancers and promoters, identified experimentally with DNase l-seq and CAGE methods in the ENCODE and FANTOM projects; both constitutive (active in many cell types) and epithelial specific. Sequencing will be performed on the HiSeq 1500 genomic platform (Illumina) using the TruSeq SBS Kit (Illumina).All libraries will be sequenced in paired end mode (2 x 100 bp) with an average coverage of 90x (minimum 20x) for reference samples and 190x (minimum 40x) for cancer cell lines and tumors. The tested material, 94 samples in total, will consist of 7 pairs: LSCC cell line - belonging culture of non-neoplastic fibroblasts and 40 pairs: pharyngeal tumor of the larynx - peripheral blood (from the same patient). At the same time, genome-wide analysis of gene expression using microarrays will be carried out in a group of 7 tested cell lines (a total of three repetitions) and in a group of 15 non-cancer reference samples (epithelial cells). The obtained results will be subjected to bioinformatics analysis, the purpose of which will be to identify somatic changes by comparing the cell line - fibroblasts, cancer - blood, and then integrating the identified changes with expression data. Such an approach will allow the emergence of mutations in regulatory regions in the vicinity of genes with significantly altered expression. For this purpose, a constantly updated database of regulatory areas and motives (Nencki Genomics Database - NGD) will be used. Thus, the selection of the most important changes in regulatory sequences will be carried out on the basis of: (a) the impact of the changes found on the expression of neighboring genes (comparison of expression in groups of cell lines - control non-cancer tissues, microarray data), (b) frequency of changes in a given regulatory sequence ( in a group of 40 tumors). In the last stage of the work, the most important, identified changes will be validated by other techniques (pyrosequencing, Sanger sequencing, qRT-PCR) in the group of tested cell lines and primary tumors, as well as by Western blot at the protein level. In addition, targeted testing of the identified loci with the above techniques in an additional group of 50 primary laryngeal tumors will be performed to estimate the frequency of lesions. As of today, there are no reports in the literature on the role of regulome changes in the pathogenesis of LSCC. Hence, the planned research is the first research approach on a global scale that integrates data on the presence of regulatory sequence variants with global gene expression in these tumors. We believe that this research approach may contribute to the description of significant changes in the regulom, which will contribute to a better understanding of the genetic basis of LSCC pathogenesis.