| Background and Aims:Primary liver cancer consists predominantly of hepatocellular carcinoma(HCC)originating in liver cells,intrahepatic cholangiocarcinoma originating in intrahepatic bile duct cells and combined hepatocellular cholangiocarcinoma.HCC is the major histological type of primary liver cancer.HCC mainly occurs in established chronic liver diseases due to infections of hepatitis B(HBV)and/or hepatitis C virus(HCV),heavy alcohol drinking,and metabolic dysfunction-associated fatty liver disease(MAFLD).Most HCCs in China were due to chronic HBV infection,that is tested seropositive for hepatitis B surface antigen(HBsAg).With implementation of hepatitis B vaccination and antiviral therapy to the patients with viral hepatitis,the major risk factors of HCC are in changing.Currently,MAFLD caused by obesity,diabetes or high-fat diets has become an important HCC risk factor in China as observed in many Western countries.With the development of HBV nucleic acid detection technology,HBsAg seronegative occult HBV infection(OBI)was frequently reported in HCC patients,and even in some asymptotic blood donors,some individuals who receive hepatitis B vaccination.OBI could spread HBV through blood transfusion,organ transplantation and mother to child transmission,which is the potential source of HBV infection.The relationship between OBI and HCC need to be paid more attention.A meta-analysis indicated that OBI was significantly associated with an increased risk of HCC(adjusted RR=2.86,95%CI=1.59-4.13),indicating that OBI could result in HCC in some situations.However,the factors and underlying mechanisms in HCC development after OBI infection are not well known.Clinical studies documented that metabolic factors synergistically promote HCC development among HBsAg-positive patients.It is not clear whether and how MAFLD promotes HCC development after OBI.Abnormal accumulation of ceramides is a hallmark in the manifestation of metabolic-related disorders.Ceramides are central molecules of sphingolipid metabolism,with an amide-linked fatty acid chain varying in the length range of C14-C26.The de novo pathway commences with the condensation of serine and palmitoyl coenzyme A(catalysed by serine palmitoyltransferase,the rate-limiting enzyme of de novo synthesis pathway)to generate 3-keto-dihydrosphinganine.3-keto-dihydrosphinganine is reduced to form dihydrosphinganine.Six ceramide synthases(CERSs)generate dihydroceramides of specific fatty acyl chain lengths.Dihydroceramides are then desaturated by dihydroceramide desaturase(DES1,encoded by DEGS1)to form ceramides with different acyl chain lengths.Hepatocellular endoplasmic reticulum(ER)is the main organelle of ceramide generation.Abnormal generated ceramides are important endogenous "danger signals" to stimulate the innate immune system,and can aggravate the progression of metabolic-related disorders,such as diabetes mellitus,by activating NOD-like receptor protein 3(NLRP3)inflammasomes.HBV virions are released/secreted from the hepatocellular ER,which is also an organelle of ceramide generation.Many OBIs are associated with infections with HBV genetic variants that have PreS mutations,which may lead to ER dysfunction.Therefore,our study aimed to analyze the impacts of HBV PreS-mutant infection on ceramides generation by hepatocytes,and whether and how the increased ceramides accelerate HCC development after OBI.Approach and Results:1.Tumor-adjacent hepatocytes of HBsAg-seronegative HCCs retained HBsAg and replication-component HBV.Based on our previous study,we reviewed 1823 cases of histology-confirmed HCCs who were diagnosed in National Cancer Center of China.Of the HBV-related HCCs,236 were HBsAg-negative but positive for anti-HBc and HB V-DNA in their sera.We sampled 35 of the 236 HBsAg-seronegative HCCs,immunohistochemistry was used to analyze the expression of HBsAg in HCC and HCC-adjacent tissues.Meanwhile,the co-localization of HBsAg and hepatocellular ER was analyzed.HBsAg was detected in all HCC-adjacent,occasionally in HCC tissues.HBsAg was colocalized with hepatocellular ER.Replication-competent HBV-DNA was detected in all HCC-adjacent and only in 6/35(17%)paired tumorous tissues.HBV rcDNA,the genome of HBV infectious virion in blood,was detected in all case sera.Hepatic steatosis with inflammation in the HCC-adjacent liver tissues was also observed in most cases.Sequencing analysis of the PreS-S regions showed that all isolates from HBsAg-seronegative HCCs were HBV genotype C(GenBank accession#MW422170:MW422204).The isolates from blood and from liver tissue of each case were identical in PreS-S region,but different from case to case.2.HBsAg-seronegative HCCs harbored extensive mutations in PreS-S regions,changed the conformation of HBV envelop protein.Across the PreS1,PreS2 and S regions,the isolates from 28 of above 35 HCCs were recognized different from the reference GU434374,which was isolated from a chronic hepatitis B patient.In total,180 mutated animo acids(AAs)were identified.Most mutations displayed alterations in AA properties,including polarity and charge modifications.RaptorX structure prediction showed that the HBV envelope protein exhibited different 3D structures from GU434374 when mutations occurred in the PreS1 or PreS2.For further study the effects of these mutations on hepatocytes,two isolates harbored E54K,V90A and G102R mutations in PreS1,one isolate harbored deletion of 16-22(RVRGLYF)with V32L and I42T mutations in PreS2 were screened out.3.Hepatocytes transfected with PreS mutants retained envelope proteins within the ER,which increased the expression of CERS and DEGS1,and promoted ceramide generation.Human hepatocytes were transfected with the two representative sequences of PreSl mutants or PreS2 mutants.We observed HBsAg retained in hepatocellular ER and induced ER stress.HBsAg intracellular retention enhanced the expression of CERS2,CERS6 and DEGS1 after being transfected with HBV,particularly with PreS mutants.Treatment of palmitic acid on these cells,the transcription of CERS2,CERS6 and DEGS1 was further augmented significantly,that depended on the treated dose and time.High Performance Liquid Chromatography-Tandem Mass Spectrometry(HPLC-MS/MS)analysis showed that the cells generated significantly more ceramides,particularly CI 6 ceramide increased 2 folds after transfection of HBV plasmids,and 5 folds after PreS mutants transfection.Treatment with palmitic acid,more amounts of ceramides were detected.Inclusion of myriocin to inhibit the de novo pathway of ceramides synthesis reduced the elevation.4.Increased ceramides from HBV-transfected hepatocytes activated macrophage NLRP3 inflammasome.Inflammatory macrophages were stimulated with C6-ceramide and/or HBV proteins(HBsAg and HBcAg)or cultured with the conditioned medium collected from PreS mutants transfected-HepG2 cells with palmitic acid treatment.Increased ceramides and HBV proteins synergistically activated NLRP3 inflammasome with the increased NLRP3 production,caspase-1 p20 generation and the increased IL-1 βand I L-18 secretion.5.High-fat diets accelerated HCC in a murine autochthonous liver cancer model transfected with PreS mutants by enhancing ceramide generation.To induce murine autochthonous HCC,male C57BL/6J mice at 2 weeks old were intraperitoneally injected with carcinogen DEN and received HBV plasmids via tail vein hydrodynamic injection at 6 weeks old.High-fat diets were administered at their 8 weeks old.Examined at their 13 weeks old,all the mice received HBV plasmids displayed hepatic steatosis with mild infiltration of inflammatory cells,particularly those with PreS mutants.High-fat diets accelerated murine autochthonous HCC development only when hepatocytes were transfected with HBV,especially with PreS mutants.Compared with the mice without HBV,C16 ceramide increased 10 folds and 15 folds in the mice livers with HBV and PreS mutants respectively after high-fat diet feeding.6.Inhibiting ceramide de novo synthesis or neutralizing IL-1β activities reduced autochthonous HCC development.Alb-1 HBV mice that have reduced HBsAg secretion with HBsAg retention within hepatocytes were also injected with carcinogen DEN at 2 weeks old and fed high-fat diets for 13 weeks.Some high-fat diets feeding mice received myriocin or IL-1β neutralizing antibodies.High-fat diets boosted HCC development when liver harbored premalignant/malignant cells.Liver inflammatory macrophages,in response to HBV proteins and increased ceramides,secreted large amounts of IL-1β and IL-18 with NLRP3 inflammasome activation.Inhibiting ceramide de novo synthesis or blocking IL-1β activities repressed autochthonous HCC progression.Summary:HBV PreS mutant-transfected hepatocytes retained HBsAg within the ER from secretion to generate more ceramides,particularly C16 ceramide.HCC development was accelerated due to ceramide overproduction,which most likely activated the liver inflammatory macrophage NLRP3 inflammasome in response to HBV antigens.With antiviral therapy to suppress HBV,the strategies to reduce the generation of ceramides or to block the effect of ceramides may be considered to control HCC development among HBV infected individuals. |
