Endocrine Abstracts

Endocrine-metabolic alterations such as obesity and metabolic syndrome are closely linked to the development of chronic liver diseases, from metabolic-associated fatty liver (MAFLD) to non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). An emerging hallmark of endocrine-metabolic and hepatic diseases is the appearance or altered expression of pathological splicing variants (SVs). In fact, the components of the cellular machinery involved in the splicing process (spliceosome) represent pivotal players in the generation of SVs in different pathologies, suggesting their putative contribution to liver disease development/progression. Here, we aimed to explore the putative dysregulation and pathophysiological role of key spliceosome components (SCs) in liver disease. Specifically, expression (mRNA/protein) of key (n = 72) SCs and clinical implications were assessed in HCC patients with different aetiologies (alcohol consumption, viral infection, MAFLD/NASH) from two retrospective (n = 154 and n = 172) and six in silico [TCGA (n = 369), Wurmbach (n = 45), Roessler (n = 43), Roessler-2 (n = 445), Chen (n = 179) and Mas (n = 57)] cohorts. Functional and molecular consequences of siRNA-mediated EIF4A3-silencing were evaluated in liver-derived cell-lines (HepG2/Hep3B/SNU-387). Gene Set Enrichment Analysis (GSEA) and mutational landscape were analysed in TCGA patients with high EIF4A3 expression. Moreover, RNAseq from EIF4A3-silenced HepG2 cells were analysed. The results showed a profound dysregulation in the expression of 41, 42% SCs analyzed (at mRNA/protein levels) in HCC vs control-tissues in all cohorts studied. Among them, EIF4A3, SLM2, ESRP2, SRPK1 and HNRNPA2B1 were the SCs more profoundly dysregulated in tumor samples, being EIF4A3 the element most consistently altered in the different cohorts. Expression levels of these SCs were associated with clinical (survival, recurrence) and molecular (oncogenic SVs expression) parameters of aggressiveness. Particularly, EIF4A3 expression was higher in NASH-related HCC compared to other aetiologies, and high EIF4A3 expression was associated with alterations in key cancer-related pathways (Cell Cycle Mitotic, DNA-Repair, etc.) and with relevant mutations (CTNNB1, TP53). Furthermore, EIF4A3-silencing in vitro resulted in reduced proliferation, migration, tumorsphere-/colony-formation and higher sensitivity to standard clinical treatments in HCC (e.g. lenvatinib). Finally, RNAseq showed that EIF4A3-silencing altered the expression and splicing pattern of multiple genes involved in 3 functional clusters (RNA splicing, translational initiation and metabolic-processes), including key genes associated to chronic liver disease progression (e.g., FGFR4). Altogether, these results demonstrate that the splicing machinery is profoundly dysregulated in liver cancer, wherein it may represent a promising source of novel diagnostic, prognostic or therapeutic targets worth to be explored.

Fundings

ISCIII (ERDF/ESF, “Investing in your future”) (PI20/01301), JdA (BIO-0139) and CIBERobn.