Maiorino, Teresa (2023) Functional characterization of 11p11.2 risk locus identifies HSD17B12 as neuroblastoma susceptibility gene involved in lipid metabolism. [Tesi di dottorato]

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Abstract

Genome-wide association studies (GWAS) have given an essential contribution to the study of neuroblastoma genetic basis by identifying common risk variants that activate cancer-related biological processes. The risk locus 11p11.2 detected in our previous GWAS on 2101 neuroblastoma cases and 4202 controls of European American origin, remains to date functionally unexplored. The present study aims to functionally characterize the 11p11.2 neuroblastoma predisposition locus by evaluating how its regulatory variant and target gene may affect neuroblastoma development. To detect functional variants, we annotated candidate SNPs inside the 11p11.2 locus using data regarding chromatin accessibility, epigenetic features and presence of transcription factor binding motifs from neuroblastoma cell lines, and identified rs2863002 as the functional variant of the locus 11p11.2. The enhancer activity of the regulatory variant was validated by in vitro luciferase assays, demonstrating that the C allele of rs2863002 acts as enhancer. Searching for the presence of transcription factors binding sites in the genetic region surrounding the candidate variant, we found that the rs2863002 C allele alters a GATA3 transcription factor binding motif and we assessed GATA3 differential allele binding through in vitro ChIP q-PCR experiments. Public Hi-C data from neuroblastoma cells and expression quantitative trait loci (eQTL) analysis were used to identify the most likely target gene of the locus activity. Then, to evaluate the target gene proto-oncogenic role in neuroblastoma, we correlated gene expression to clinical and prognostic parameters. The C allele of rs2863002 showed to be associated with increased HSD17B12 expression and risk of neuroblastoma development, and higher HSD17B12 expression correlated with poor prognostic features and poor survival in neuroblastoma tumors. In vitro proliferation and invasion cellular assays following HSD17B12 silencing were performed, demonstrating that HSD17B12 silencing significantly impairs proliferation and invasion capacities of neuroblastoma cells. Finally, the biological activity of HSD17B12 was investigated by targeted Lipidomic analysis and RNA-sequencing. Impairment of HSD17B12 expression proved to alter lipid metabolism in neuroblastoma cells, leading to a decrease in lipid species related to energy production and to changes in membranes composition and properties. This lipid metabolic stress seemed to activate in neuroblastoma cells silenced for HSD17B12 different response processes, such as Interferon-induced inflammation and Endoplasmic Reticulum stress response. In conclusion, the functional investigation of the 11p11.2 risk locus supports the role of HSD17B12 as a susceptibility gene in neuroblastoma. We dissected the molecular mechanism by which the candidate variant rs2863002 at 11p11.2 risk locus regulates HSD17B12 oncogenic expression. Moreover, our results indicated that HSD17B12 promotes neuroblastoma tumorigenesis by altering the availability of lipid energy sources and the properties of cellular membranes through its involvement in lipid metabolism. Altogether, the present study demonstrated that post-GWAS strategies are essential for the identification of causal functional variants at cancer-risk loci and for the detection of predisposition genes with key roles in tumor biology.

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