A multi-center study on the role of ACSL4 in invasion and metastasis of hepatocellular carcinoma.

Abstract

e16213 Background: Hepatocellular carcinoma (HCC) has an easy recurrence and poor prognosis. Tumor invasive front (TIF) is the source of tumor recurrence and micro-metastasis. Therefore, mining of key biomarkers of TIF, promotes the development of new therapeutic strategies for HCC. This study was to explore the key biomarkers of TIF, which are closely associated with invasion and metastasis of HCC and its clinical value. Methods: Firstly, we based on the spatial transcriptomic sequencing data of HCC clinical specimens, uses a set of innovative gene screening algorithms to identify long-chain acyl-CoA synthetase 4 (ACSL4) as a molecular marker closely related to the invasion and metastasis of HCC. Since then, the histopathological characteristics of ACSL4 in whole processes of HCC invasion and metastasis was confirmed, including the primary lesions, circulating tumor cells (CTCs) microvascular invasion (MVI), intrahepatic metastatic lesions and extrahepatic distant metastases. Next, the clinical value of biomarker demonstrated through multi-center cohort that recruited 243 HCC patients from 3 clinical centers in China. Finally, the functional mechanism was revealed through in vivo, in vitro and multi-omics joint analysis. Results: ACSL4 has significant tissue specificity and spatial specificity in HCC, and was strongly positive in the whole process of HCC invasion and metastasis, such as CTCs, MVI, intrahepatic satellite nodules and distant metastases lesions. Multicenter cohort study shows that the overall survival rate (p < 0.05) and recurrence-free survival rate (p < 0.05) of the high ACSL4 expression patients were lower than the low ACSL4 expression patients. Correspondingly, the MVI occurrence rate of patients with high ACSL4 expression was higher than that of patients with low ACSL4 expression in tumor tissues (p < 0.05), and the expression level of ACSL4 in serum can effectively predict the occurrence of MVI in HCC patients (AUC = 0.832, p < 0.01). The results of in vivo and in vitro experiments indicate that the ACSL4 is closely related to the invasive and metastatic abilities of HCC cells. Transcriptomics and metabolomics joint analysis observed that after knocking down ACSL4, key enzymes (CPT1A, CPT2, ACOX1) and metabolic products (arachidonic acid, Acetyl-CoA) involved in fatty acid beta-oxidation process underwent significant changes (p < 0.01). Conclusions: ACSL4 promotes the invasion and metastasis of HCC cells, and can used as a potential prediction biomarker for invasion and metastasis of HCC. Critical follow-up data of two cohorts. Multi-center cohort TCGA cohort High ACSL4 (n=121) Low ACSL4 (n=121) P value High ACSL4 (n=137) Low ACSL4 (n=137) P value Microvascular invasion 0.039 0.033  Yes 38 24 50 35  None 83 97 87 102 Satellite nodule 0.032 -  Yes 17 8 - -  None 104 113 - - Recurrence 0.026 0.035  Yes 45 31 79 64  None 76 90 58 73 Survival status 0.021 0.009  Alive 48 30 93 108  Dead 73 91 44 29