Deletion Of Acyl-CoA Thioesterase 12 Promotes YAP/TAZ-Mediated Hepatocarcinogenesis By Enhancing Glycerolipid Production

Background&Aim:Cancer cells use acetate to support the higher demand for energy and lipid biosynthesis during uncontrolled cell proliferation,as well as for acetylation of regulatory proteins.In the liver,acyl-Co A thioesterase 12（Acot12）is the primary cytosolic enzyme that hydrolyzes acetyl-Co A to acetate and Co ASH.Acot12 is downregulated in human HCC,with lower expression levels predictive of poor prognosis.The study was designed to access the function of Acot12 on liver tumorigenesis and explore underlying the molecular mechanism.Method:Acot12^-/-and wild-type control male mice were treated with DEN at 2 w,followed by biweekly CCl₄administration for 11 w.Acot12^-/-and wild-type control female mice were treated with DEN at 2 w,followed by biweekly CCl₄administration for 16 w.HCC was also induced using the NASH diet:6 w old Acot12^-/-and control male mice were fed this diet for 18 mo.Hepatic lysophosphatidic acid（LPA）molecular species and concentrations were measured by liquid chromatography-mass spectrometry.Lipids were extracted from liver tissue and measured by kits;RT-PCR was used to detect the m RNA levels of lipid biosynthesis pathways genes;the protein expression levels of cancer-related pathways were detected by western blot.Primary hepatocytes from wild-type mice were treated with purified synthetic LPA and vehicle,as well as conditioned media prepared from cultured Acot12^-/-and wild-type primary hepatocytes.Hippo pathway-related proteins were assessed by immunoblot analyses.LPA signaling was assessed by loss of function studies using si RNA-mediated knockdown of the G-protein subunits Gna12 and Gna13 associated with LPA receptors in wild-type hepatocytes.Results:1.Relative to wild-type mice,DEN/CCl₄-treated Acot12^-/-male mice exhibited accelerated liver tumor formation that was characterized by increased tumor number and tumor volumes.The non-tumor tissue from Acot12^-/-male mice exhibited decreased hepatic acetate levels,while increased hepatic TG levels.We also found increased TG and phosphlipids levels in tumor tissues from Acot12^-/-male mice compared to wild-type mice,as well as increased plasma concentrations of cholesterol and phospholipids.2.Relative to wild-type mice,DEN-CCl₄treated Acot12^-/-male mice exhibited increased expression of glycerolipids biosynthesis pathway-related genes（such as Gpat3,Gpat4 and Dgat1）.Consistent with this,LPA,a direct product of GPAT,was increased in non-tumor tissue from Acot12^-/-male mice.We also found increased expression of LPAR3 in tumor tissue from Acot12^-/-male mice.3.Relative to wild-type mice,DEN/CCl₄-treated Acot12^-/-female mice exhibited accelerated liver tumor formation.We found increased hepatic phospholipids levels in non-tumor tissue from Acot12^-/-female mice and increased plasma phospholipids levels from Acot12^-/-female mice compared to wild-type mice.Acot12^-/-female mice exhibited increased expression of glycerolipids biosynthesis pathway-related genes（such as Gpat1,Gpat3 and Gpat4）.The m RNA level of Lpar3 was increased in tumor tissue from Acot12^-/-female mice.4.Relative to wild-type mice,NASH-diet fed Acot12^-/-male mice exhibited accelerated liver tumor formation.We found a trend to increase in hepatic TG levels and increased phospholipids from NASH-diet fed Acot12^-/-male mice.NASH-diet fed Acot12^-/-mice showed increased expression of LPAR3 in tumor tissue,compared to wild-type mice.5.DEN/CCl₄-treated or NASH-diet fed Acot12^-/-mice showed reduced activity of Hippo signaling and increased YAP/TAZ nuclear localization,along with increased hepatic m RNA expression of Ctgf、Birc5 or Ccnd1.6.Knockdown of YAP or TAZ inhibited hepatocarcinogenesis and YAP activation in DEN/CCl₄treated Acot12^-/-male mice.Restoration of Acot12 expression by AAV9-Acot12 inhibited hepatocarcinogenesis and YAP activation in Acot12^-/-male mice.7.P-Yap/Yap ratios in cultured wild-type hepatocytes were decreased by treatment with 16:0-LPA in a dose-dependent manner,as well as by conditioned media from Acot12^-/-hepatocytes.Indicative of signaling through an LPA receptor,the knockdown of Gna12 and 13 in hepatocytes abolished the capacity of LPA or Acot12^-/-conditioned medium to suppress Yap phosphorylation.Conclusion:These mechanistic studies reveal that up-regulated LPA levels in the livers of Acot12^-/-mice act through LPA receptor coupled to Gα12/13 subunits to promote YAP/TAZ activation,promoting its nuclear localization and transcriptional activity.Our findings identify a crucial role for Acot12 in maintaining lipid homeostasis by limiting glycerolipid biosynthesis,which in turn preserves Hippo signaling and forestalls hepatocarcinogenesis.We speculate that Acot12 plays a crucial role in suppressing hepatocarcinogenesis and provide a potent target for the treatment of HCC.