The Expression And Molecular Mechanism Of ACSL4 In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is a highly heterogeneous,multigene-driven malignant tumor,which still lacks effective therapeutic targets.As an important serum biomarker of HCC,the high expression of AFP is closely related to the unique biological characteristics of HCC.Revealing the molecular characteristics of patients with high AFP subtypes of HCC is helpful for personalized precision therapy.Long chain acyl Co A synthetase 4(ACSL4)is a member of acyl-Co A synthetases(ACS)family and is a key enzyme involved in arachidonic acid(AA)metabolism,whose expression dysregulation is closely related to malignant tumors.However,its function and the underlying molecular mechanisms in HCC are still not fully elucidated.Methods:Transcriptomic microarray assay was performed to identify novel markers for AFP highly expressed HCC.Quantitative reverse transcription polymerase chain reaction(q RT-PCR),western blotting and immunohistochemistry(IHC)were used to detect ACSL4 expression in HCC and adjacent tumor tissues.Functional analysis,CHX and MG132 intervention experiments,ubiquitination experiments,rescue experiments and nude mouse xenograft models were used to determine the potential mechanism by which ACSL4 promotes tumorigenesis in vitro and in vivo.Correlation of ACSL4 and c-Myc in HCC were proved by IHC staining.Further,RNA-seq analysis was carried out to characterize the global changes in ACSL4-dependent transcriptome.Gas chromatography–mass spectrometry(GC-MS)was performed and intracellular lipid accumulation in HCC cells was tested to verify the pivotal role of ACSL4 in fatty acid metabolism regulation.The key lipogenic enzymes and the master transcription factors involved in de novo FA synthesis were detected by q RT-PCR and western blotting.Chromatin immunoprecipitation(Ch IP)-q PCR and luciferase reporter assays were used to validate that c-Myc can transcriptionally regulate the expression of SREBP1.Functional rescue experiments in vitro,nude mouse xenograft models and tail vein metastatic assay in vivo were used to demonstrate that SREBP1-related lipid metabolism was essential for ACSL4-mediated tumor progression.Moreover,the relevance and clinical significance of ACSL4 and SREBP1 in HCC were also proved using the method of IHC.Results:Through transcriptome profiling,ACSL4 exhibited a high expression level in HCC tissues with high serum AFP concentration,and was identified as a novel marker for AFP high subtype HCC.ACSL4 was frequently upregulated in HCC samples and associated with poor prognosis.Functionally,ACSL4 knockdown resulted in decreased cell growth,whereas ectopic ACSL4 expression facilitated tumor formation in vitro and in vivo.Mechanistically,ACSL4 stabilized the oncoprotein c-Myc through ubiquitin-proteasome system in an ERK/FBW7-dependent manner,thereby mediating the phenotype that promotes cell proliferation.c-Myc si RNA or its inhibitor 10058-F4 can rescue the promoting cell proliferation effect mediated by ACSL4 elevation.In contrast,the inhibitory effect of ACSL4 silencing on cell growth were partially reversed by c-Myc overexpression via FBW7 knockdown.Clinically,ACSL4 expression was positively correlated with c-Myc expression in HCC.In addition,a new function of ACSL4 in the reprogramming of fatty acid metabolism in HCC was identified.ACSL4 can modulate de novo lipogenesis by accumulating intracellular triglycerides,cholesterols,and lipid droplets in HCC.Mechanistically,ACSL4 upregulates the master lipogenesis regulator sterol regulatory element binding protein 1(SREBP1)and its downstream lipogenic enzymes in HCC cells via c-Myc,thus promoting de novo lipogenesis.Moreover,SREBP1 is crucial for ACSL4-mediated lipid metabolism reprogramming as well as HCC cell proliferation and metastasis,as SREBP1 overexpression rescues lipogenic deficiency and decreased oncogenic capabilities associated with ACSL4 suppression in vitro and in vivo.Clinically,our data showed that the expression of ACSL4 was positively correlated with that of SREBP1 in patients with HCC,and the combinational biomarkers showed strong predictive value for HCC.Conclusion:ACSL4 is a novel marker for AFP high subtype HCC.Its expression is significantly up-regulated in hepatocellular carcinoma,and the high expression of ACSL4 is related to the malignant phenotype and the poor survival prognosis of HCC.Our data uncovered novel mechanisms of ACSL4 promoting HCC tumor progression:stabilizing the expression of proto-oncoprotein c-Myc at a post-transcriptional level in an ERK/FBW7-dependent manner,and modulating aberrant lipid metabolism via c-Myc/SREBP1 signaling,thus mediating metabolic reprogramming and malignant phenotype of HCC cells.Our findings suggest that ACSL4 plays a key role in the development of hepatocellular carcinoma and could be a valuable prognostic biomarker and potential therapeutic target in HCC.