Robert “Nate” Helsley | Gene Expression and Lipid Profiling of Metabolically-driven Human Hepatocellular Carcinoma

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the fastest-growing etiology of hepatocellular carcinoma (HCC). This work identifies novel gene and lipid associations in human MASLD-driven HCC that may be exploited for therapeutic benefit. Methods: Human HCC tumor (n=8) and adjacent non-tumor samples (n=8) were obtained from the Biospecimen Procurement and Translational Pathology Shared Resource Facility at the University of Kentucky Markey Cancer Center. All patients met cardiometabolic MASLD criteria and were negative for viral hepatitis. Hematoxylin and eosin (H&E) staining was used for pathological determination of tumor and adjacent nontumor tissue. Lipids were extracted using a methyl-tert-butyl ether extraction method and subjected to lipidomics by the West Coast Metabolomics Center. RNA was isolated and used for bulk sequencing. Data were analyzed using paired nonparametric analyses via a Wilcoxon or Mann-Whitney test, as appropriate. Results: Histological analysis by H&E showed significant lipid vacuole accumulation in HCC tumors relative to nontumor tissue. Lipidomic analysis revealed significant increases in long-chain nonesterified monounsaturated fatty acids (MUFAs; C16:1, C18:1, C20:1) and MUFA-enriched phospholipids (PC30:1, PC32:1, PE32:1, and PC36:1) in tumors relative to nontumor tissue. No significant differences were observed in nonesterified polyunsaturated fatty acids (PUFAs; C18:2, C20:4, and C22:6), PUFA-enriched phospholipids (C36:4, C38:4, C38:6, C40:6), or in fatty acid esters of hydroxy fatty acids (FAHFAs; C38:2, C38:4, C38:6). However, both MUFA- (C14:1, C18:1) and PUFA-enriched acylcarnitines (C18:2, C18:3) were collectively reduced in human tumors. Differential analysis of RNA sequencing revealed fatty acid oxidation genes (CPT1A, CPT2, ACADL, ACADM, ACADS, HADHA) were significantly reduced in tumor versus nontumor tissue. Further, genes involved in de novo lipogenesis were largely dysregulated (e.g. no differences in SREBF1 or FASN; increases in ACLY, ACACA, and SCD1; decreases in ACSL1) in tumor versus nontumor tissue. Conclusions: These results suggest human HCC tumors exhibit a reduced capacity to undergo mitochondrial β-oxidation resulting in accumulation of free and esterified MUFAs with concomitant reductions in MUFA-carnitines. Current studies are underway to determine the mechanisms by which impairment of hepatic MUFA catabolism via FAO promotes the development of HCC in mice.