Genomic and functional characterization of putative melanoma-restricted lncRNAs (RMELs)

Abstract

RMEL genes, first described by our group in 2010, are potential lncRNAs with expression restricted to melanoma. High expression of RMELs 2, 3 and 4 (HCCAT5) is associated with the presence of BRAFV600E, the major driver mutation of tumorigenesis in melanoma and other cancers. Ongoing research in our laboratory demonstrates that RMEL3 is a target of MAPK and PI3K pathways, and the transcript, conversely, appears to take part in a feedback positive looping of these pathways. Knockdown and overexpression approaches indicate that all four lncRNA, subject of this study, in different ways and intensity, act by promoting survival and proliferation of melanoma cells. Transcriptomics, proteomics and bioinformatics integrative analyses show that knockdown of RMELs in melanoma cells influences the expression of major pathways involved in proliferation/survival and therapeutic resistance to BRAF/MEK inhibitors or immunotherapy. RMEL2 (annotated as OVAAL) is overexpressed in a smaller number of melanoma cases and cell lines than the other three RMELs and, although silencing of RMEL2 does not affect proliferation under normal adherent conditions, lead to significant reduction of the anchorage-independent growth, and we present evidence of an association of RMEL2 with therapeutic resistance. RMEL1 shows good correlation with MITF and a program of melanocytic differentiation/survival. RMEL4 appears to correlate with a molecular signature of immunotherapy non-responsive melanoma patients. Encouraged by these results, we propose here to characterize these lncRNAs regarding their structures, isoforms, molecular interactions, subcellular localization, coding capacity, and bi-functionality. Interactions with microRNAs and proteins will be analyzed using Luciferase reporter assay and pulldown assays, respectively. Additionally, we will pursue functional studies using overexpression and knockdown (or CRISPr knockout) approaches for in vitro and in vivo analysis to explore the functions of these lncRNAs and applicability in different biological context and therapeutic settings in mouse models. (AU)