Genomic and functional characterization of genes with expression restricted to melanoma (RMELs)

Abstract

Melanoma is the cancer that arises from melanocytes and constitutes a highly aggressive disease, which has elevated mortality rates. Its most common etiology, a constitutive activating mutation in codon 600 of BRAF protein, which encodes a serine -threonine kinase protein, responsible for the initiation of the cascade of MAPK , comprise the major therapeutic target in the treatment of this disease. The initial efficiency of mutated BRAF inhibitors leading to a considerable remission of tumor size is replaced, usually after 6 months of treatment, by rapid progression of the disease after the onset of acquired resistance to the therapy. Seen that, the identification of new genes involved in melanoma progression and disease recurrence is essential. Within this context, previous studies by our research group identified three new genes, named RMEL1, 2 and 3 as having restricted expression to melanocytes and/or melanoma and that are highly associated with BRAFV600E. Subsequent studies confirmed, by pharmacological inhibition of mutated BRAF using PLX4032 that blocks the MAPK pathway, leads to a drastic reduction in the RMELs expression levels (results also observed with inhibition of MEK). Seen that and because of the fact that RMELs possibly constitute Long Non-coding RNAs, it is believed that they are regulated by the MAPK pathway and could exert control over effectors controlled by this pathway, being object of interest its role in maintaining aggressive phenotype of melanoma . The silencing mediated by siRNAs suggests that RMEL1 inhibits the clonogenic capacity in vitro, whilst RMEL3 promotes this property and RMEL2 induces the anchorage independent growth. Preliminary results obtained by processing large-scale data show an inverse correlation of RMEL1 with pro-tumorigenic factors such as NUAK1 and NRP1, supporting the results obtained in the clonogenic assay in consequence of RMEL1 depletion. Furthermore, the observed correlation of RMEL1 with proteins involved in melanogenesis and melanocyte differentiation, such as MITF, MLANA and MREG, is justified by their restricted expression in melanocytes and melanoma. It was also shown a strong correlation with SOX10 that recently was involved in a mechanism of resistance, which was confirmed by preliminary data from microarray performed by our laboratory. This project aims to use the large-scale data as provided by the TCGA (The Cancer Genome Atlas) in conjunction with microarray data of cells silenced for RMELs, obtained through international collaborations, to correlate them with various genomic and clinical factors. Examples of such factors are: methylation, mutation, SNPs, exome, trascriptome, miRNA, and information such as metastasis presence, depth of tumor, mitotic index, disease stage and patient survival. These results will provide more insights into the involvement of RMELs in signaling pathways such as MAPK, PI3K and AKT, transcription factors and components of cell cycle control and apoptosis, essential in the maintenance of melanoma. The correlation with these proteins will be evaluated in the context of overexpression and silencing of RMELs, as well, evaluate their role in sensitive and resistant cell lines to PLX4032, as a way to identify new mechanisms of therapeutic resistance to the development of a possible application in treatment resensitization. The development of this work will provide important tools, through the analysis of large-scale data, for the other ongoing projects in the laboratory as well to contribute the understanding of tumor progression and therapeutic resistance against mutated BRAF in melanoma by genomic and functional characterization of RMELs. (AU)