| Background and Aims:Primary liver cancer(PLC)is one of the most common malignant tumors globally.In males,PLC ranks the second leading cause of cancer-related deaths and in females ranks the sixth.The most common histological type of PLC is hepatocellular carcinoma(HCC)which accounts for about 85%of PLC.Hepatitis B virus(HBV)and/or hepatitis C virus(HCV)are the most important infectious factors for promoting HCC development.Male sex increases HCC risk is an important epidemiological feature of HCC and the sex differences of HCC have been extensively investigated for decades.Recently,a multiple center study that analyzed a total of 13003 HCC cases in East China,South China,North China,and West China reported that 84.4%of HCC cases were associated with HBV infection.HCC caused by HBV infection occurs 12 years earlier than that caused by HCV.The ratio of male to female HBV-related HCC cases is 6.6:1,higher than the ratio of male to female HCV-induced HCC cases,which is 4.0:1.In the 1980s,a cohort study of chronic hepatitis B patients without antiviral treatment in Taiwan reported that the sex difference was also reflected in the disease progression of HBV infected individuals,the ratio of male to female cases in asymptomatic carrier stage was 1.2:1,in chronic hepatitis B stage was 6.3:1,and in liver cancer stage was 9.8:1.Regarding on the mechanism of male sex in causing HBV-induced HCC.evidences from previous studies have pointed out that androgen receptor(AR)enhances HBV transcription and replication,promoting HCC.While estrogen receptor α(ERα)represses HBV transcription,reducing HCC risk.The seropositivity of hepatitis B e antigen(HBeAg)can reflect HBV transcription and replication.The cohort study of chronic hepatitis B without antiviral therapy in Taiwan reveled that the proportion of seroconversion from HBeAg-positive to HBeAg-negative in male individuals at over 30 years was more significantly than that in female individuals.Therefore,in addition to the sex hormones in promoting and suppressing HBV replication and transcription,some other undefined factors might contribute to promoting HCC development in male sex.The progression from chronic HBV infection into HCC is affected by variated factors.In addition to sex,HCC risk is synergistically enhanced by exposure to chemical carcinogens,including aflatoxin B1(AFB1).Most HCC cases in China are related to HBV infection.The data from population-based tumor registration reported that the male to female ratio of liver cancer incidence was 2.80:1 nationwide,but the ratio was 3.14:1 in Qidong from 1972 to 2011.Qidong is a classical region of high AFB1 exposure,where the liver cancer incidence and the male to female ratio are higher compared with that in other regions of China.These evidences suggest sex and sex hormones also play some roles in AFB1-related HCC and are not fully understood.Previous studies have shown that AFB1 is transported into cells through the aryl hydrocarbon receptor(AHR),and is activated by the cytochrome P450(CYP450)oxidase family members.Some AFB1 was metabolized to form the hydroxylated metabolites Aflatoxin M1(AFM1),which is discharged from urine,and some form aflatoxin-8,9-epoxide(AFBO),which becomes genotoxic by the formation of AFBi-N7-guanine(AFB1-DNA)adduct through binding to genomic DNA.It is clear that sex and sex hormones play important roles in the control of cancer-initiating cell populations,metabolism,formation of the tumor microenvironment,and immune response.Therefore,between males and females,the HCC development and the formed HCC mass in responses to different therapies might be differ,including the genotoxin-related chemotherapy and immune therapy.However,anti-androgen therapy or anti-estrogen therapy in clinical trials for the treatment of advanced HCC conducted a decade ago failed to achieve the expected effect.Hence,it is necessary to recognize the sex differences in genomic features and elucidate the effects and potential mechanisms of sex and sex hormones on HBV-related HCC development and progression.The aims of this study for my thesis include:(1)To find out the sex differences in genomic landscape of HBV-related HCC;(2)To find out the sex differences in transcriptional dysfunction,particular in those HCC related to aflatoxin exposure subsequent to HBV infection.I would like pay more attention on the sex differences in antitumor immunity against the HBV-related HCC that is imposed by androgen signaling.(3)To explore the strategies against HCC for improving anti-PD-1 treatment effect based the sex differences.From Qidong Liver Cancer Institute/Qidong People’s Hospital in Jiangsu Province,we obtained tumorous tissues and matched nonneoplastic liver tissues of 126 HCC patients(72 males and 54 females)who were confirmed with pathological diagnosis.All the patients were local residents and the samples were snaped frozen and stored at-80℃ after hepatic section.All the patients had serological markers of HBV infection and the AFM1 was detected in their urine 5-17 years before HCC diagnosis.Therefore,these HCC were considered as HCC caused by aflatoxin exposure subsequent to HBV infection,and named as QD-HCC.Whole genome sequencing(WGS)or whole exosome sequencing(WES)was performed on the samples from 101 cases.RNA sequencing was performed on the samples of 87 cases.Of them 62 cases(18 males and 44 females)were sequenced for both genome and transcriptome.To understand the effect of genotoxicity agent,such as AFB1,on HCC,we obtained the 119 cases of HBV-related HCC from TCGA database,that was termed as TCGA-HCC in the current study.We recruited 113 cases(73 males and 40 females)by excluding 6 cases that harbored mutational signatures of AFB1 exposure.Previous studies have shown that sex differences reflected the sum of distinct sex hormone-related transcriptional regulation plus tissue cell-specific regulation.We chose HBV-positive HCC cell lines HepG2.2.15 and PLC/PRF/5 to analyze the effect of the combination effect of AFB1 and androgen on the expression of genes related to sex differences observed in HCC samples.We further used two syngeneic graft mouse models to explore the role and possible mechanism of enhanced androgen signaling in anti-tumor immunity against HCC.Methods and Results:1.Sex differences in mutations profilesWhole-genome or whole-exon sequencing was performed on 101 QD-HCC cases(47 males and 54 females),and the GATK Best Practices Pipeline was used to identify somatic variants.Results from the analysis showed no difference in the numbers of single base substitutions(SBSs)at the genomic or exome scale,tumor mutational burden(TMB),and mutation patterns between male and female QD-HCC samples.The genome sequencing data of TCGA-HCC was analyzed,the results also showed that no sex differences were observed in the numbers of SBSs,TMB,and mutation patterns between male and female TCGA-HCC samples.We compared the mutation frequencies of all protein-coding genes between male and female QD-HCC samples by Chi-square test or Fisher’s exact test.The mutation frequencies of 75 previously identified HCC driver genes were similar in both sexes.However,we identified 71 genes with significantly different mutation frequencies between the two sexes,some of which have been previously found as driver genes in other cancers such as lung cancer and melanoma.Among these 71 genes.62 genes were more frequently associated with males.Mutations in PAK7,ANP32E,CEP250,and ATXN2 detected in male QD-HCC samples were significantly associated with poor survival of male patients but not female patients based on the 5-year follow-up results.9 genes were more frequently associated with females.Among these genes,the GTF21 mutation was mainly occurred in the position chr.7:74159247 with G>A transversion.Mutation frequencies of genes in males also differed from in females with cirrhosis,without cirrhosis,or with serum AFP ≤20 ng/mL.The genome sequencing data of TCGA-HCC was analyzed.7 genes with significantly different mutation frequencies between two sexes.Of them,4 genes mutated more frequently in males,including the previous identified HCC driver gene TP53.The TP53 mutation frequency in male TCGA-HCC samples was 30%in males but only 10%in female TCGA-HCC samples.These results.indicated that there were sex differences in mutation profiles.2.Sex differences in HBV integrationWGS-analyzed 88 QD-HCC samples,including 36 males and 52 females,were examined HBV integration in the human genome.A total of 110 HBV integration breakpoints from 37 QD-HCC samples were detected in the tumorous tissues.No difference in HBV integration frequencies or in HBV breakpoint numbers was detected between males and females.However,only male QD-HCC samples harbored HBV breakpoints that were reported to bind AR and HNF4a.Across the HBV genome,the percentage of S-gene breakpoints were more in male QD-HCC samples,while the percentage of X-gene breakpoints were more in female QD-HCC samples.Across the human genome,HBV breakpoints in QD-HCC samples were mostly detected on chromosome 5 and no sex difference was observed.But the samples from males harbored more breakpoints on chromosomes 19 than those from females;the samples from females harbored more breakpoints on chromosome 3 than males.HBV-integration targeted genes(ITGs)and their functions in male QD-HCC samples also differed from those in female QD-HCC samples.Combined with the analysis of somatic variants,these results further indicated that there were sex differences in mutation profiles.3.Sex differences in multiple biological capabilities of HCCWe performed RNA sequencing(RNA-seq)on tumorous and matched nonneoplastic liver tissues from 87 QD-HCC samples(43 males and 44 females).Transcription levels of the genes were analyzed with Hisat,StringTie,and Ballgown,and analysis of differentially expressed genes(DEGs)was performed using the limma R package.3070 significantly DEGs were identified between male and female QD-HCC samples.In the samples from males,2352 DEGs were upregulated.Compared with female QD-HCC samples,male QDHCC samples expressed higher levels of AR and the gene sets related to estrogen response early.We then conducted gene set enrichment analysis(GSEA)and gene set variation analysis(GSVA)of all protein-coding genes.Results showed that male QD-HCC samples demonstrated different biological capabilities from those of females.Compared with female QD-HCC samples,several pathways related to Wnt/β-catenin signaling and Notch signaling were upregulated,the inflammation-related gene expression was augmented,DNA double-strand break(DSB)repair and specific antitumor immunity were repressed in male QD-HCC samples.The transcriptome sequencing data of TCGA-HCC was analyzed.Results showed that compared with female TCGA-HCC samples,male TCGA-HCC samples expressed higher levels of AR and ESR1.GSVA results showed the differences of gene signatures between males and females,most of which were similar with the results of QD-HCC samples.These results suggest that transcriptional regulatory mechanisms might be different between two sexes,which may affect the occurrence and biological capabilities of HCC.4.Sex differences in gene expression related to aflatoxin metabolismQD-HCC samples were from the patients with aflatoxin exposure subsequent to HBV infection.We,therefore,analyzed the genes associated with aflatoxin metabolism between two sexes.Compared with female QD-HCC samples,male QD-HCC samples expressed greater levels of AHR and CYP1A1.The enhanced protein expression of AHR and CYP1A1 in QD-HCC samples from males was confirmed by immunohistochemistry staining.Sexbased differences of these genes were also observed in the TCGA-HCC samples.Compared with female TCGA-HCC samples,AHR and CYP450 family genes related to AFB1 metabolism,including CYP1A1,CYP1A2,and CYP3A4,were significantly upregulated in male TCGA-HCC samples.We then used HBV-positive HepG2.2.15 cells and PLC/PRF/5 cells to test sex hormones in the regulation of AHR and CYP1A1,impacting AFB1 cytotoxicity.Following AFB1 treatment,the addition of testosterone to the cultures significantly enhanced the transcription levels of AHR and CYP1A1.The dose of AFB1 causing cell death of HepG2.2.15 cells and PLC/PRF/5 cells was reduced by 50%in the presence of testosterone.In addition,more AFB1-DNA adducts were detected in these cells treated with AFB1 plus testosterone than the cells treated with the same concentration of AFB1 alone.5.Sex differences in DNA damage responsesThe generation of AFB1-DNA adducts results in DNA damage.GSVA results showed that compared with female QD-HCC samples,male QD-HCC samples exhibited downregulation of DSB repair pathway.We further analyzed different pathways in response to DNA damage between male and female QD-HCC samples.The most downregulated pathway in male QD-HCC samples was nonhomologous end joining(NHEJ).Male QD-HCC samples expressed significantly lower levels of NHEJ factors than in female QD-HCC samples,including ATM,XRCC5,NBN,MRE11,and XRCC4.In the samples of TCGA-HCC,males also expressed significantly lower levels of ATM,MRE11,LIG4 than females.To test the effects of androgen on the regulation of NHEJ factors,we also used HBVpositive HepG2.2.15 cells and PLC/PRF/5 cells.The transcriptional levels of XRCC4,LIG4 and MRE11 were significantly reduced in these cells that were treated with AFB1 plus testosterone compared with those treated with the same concentration of AFB1 alone.Notably,the addition of 17β-estradiol estrogen partially reversed the reduction of XRCC4 and MRE11 expression.We used web-based MEME Suite to predict whether AHR,CYP1A1,XRCC4,LIG4,and MRE11 are the AR target genes.Results indicated that androgen responsive elements(AREs)were presented in the promoter areas of these specified genes.Combined with the analysis of aflatoxin metabolism,these results suggested that the gender differences in biological capabilities of HCC might be influenced by sex hormones.6.Sex differences in cGAS-STING pathway of tumorous tissuesIt has been demonstrated that chronic genotoxic stress induces nuclear DNA leakage into the cytosol,where double-strand DNA(dsDNA)is intrinsically detected by cGAS(cyclic GMP-AMP synthase)to activate stimulator of interferon gene(STING)-dependent cytokine production.We found that the genes related to IFN-I signaling and response were significantly upregulated in males but not in females.Notably,significantly higher levels of STING and IRF3 were detected in male QD-HCC samples.To validate this observation,HepG2.2.15 cells and PLC/PRF/5 cells were used for analysis.Compared with the cells that were treated with the same concentration of AFB1 alone,HBV-positive cells that were treated with AFB1 plus testosterone expressed more y-H2AX.Analysis of immunofluorescent staining showed that more dsDNA was detected in the cytoplasm,and analysis of immunoblot showed that significantly higher levels of cGAS,phosphorylated STING,and phosphorylated IRF3 were detected in the cells treated with AFB1 plus testosterone than in the cells treated with AFB1 alone.A greater transcription levels of IFNB1 were also detected in HepG2.2.15 cells treated with AFB1 plus testosterone as compared to those treated with AFB1 alone,but not in PLC/PRF/5 cells.These results indicated that androgen androgen signaling in HCC could repress the DSB repair,thereby promote the activation of the cGAS-STING pathway in HCC cells by increased nuclear DNA leakage into the cytosol.7.Sex differences in antitumor immunity against HCCBased on the above results,we analyzed the antitumor immunity of HCC between two sexes.Significantly increased CD8+ T-cell infiltration was detected in tumorous tissues of QD-HCC samples from the males but not from the females.Compared with adjacent nonneoplastic tissues,tumor tissues from male QD-HCC samples but not from female samples displayed significantly higher transcriptional levels of PD-1,CTLA-4,B7-H2,B7H3,and lower transcriptional levels of CD80(B7.1),CD86(B7.2).The enhanced expression of PD-1 and B7-H2 in the tumor tissues of male QD-HCC samples was confirmed by immunohistochemistry staining.To validate this observation,HBV-positive HepG2.2.15 cells and PLC/PRF/5 cells were used for analysis.The expression levels of B7-H2 and B7-H3 in the two HCC cell lines were significantly elevated after AFB1 plus testosterone(AFB1/Tes)treatment compared with the same concentration of AFB1 alone(AFB1/Veh)treatment.We collected HCC cell medium(CM)from the different treatment and conducted a chemotaxis assay to examine the effect of soluble factors on immune cells,mimicking the infiltration of immune cells into tumor tissues.In response to the CM from either AFB1/Tes-treated HCC cells,more CD8+PD-1+T cells migrated into the lower chambers than the cells in response to the CM from AFB1/Veh-treated or only testosteronetreated HCC cells.These results indicated that the expression levels of immune checkpoints were significantly elevated in HCC cells after AFB1/Tes treatment.Meanwhile,the soluble factors derived from these HCC cells could recruit more CD8+T cells into tumorous tissues.Therefore,although more CD8+ T-cell infiltration was detected in tumorous tissues,male QD-HCC samples existed in an immunosuppressive microenvironment,due to the high expression levels of immune checkpoints.8.Favorable AR signaling increased CD8+T-cell infiltration in the tumor mass and improved the anti-PD-1 effectWe then used two murine HCC cells,Hepal-6 and H22,and blocked ER signaling via tamoxifen administration to favor the AR signaling in the syngeneic tumor-bearing mouse models in both male and female mice,in order to examined the impact of favorable androgen pathway on anti-PD-1 treatment effects.Without tamoxifen administration and injection of anti-PD-1 antibody(control),male mice displayed greater tumor CD8+T-cell infiltration than females.Compared with mice treated with one dose of anti-PD-1 injection only,tamoxifen administration synergistically enhanced the anti-PD-1 effects to eradicate the established tumor quickly regardless of the mouse strain and sex.However,in male mice,tumors grew faster in the flutamide-treated mice than in the vehicle-treated mice.We further processed the mouse tumors and analyzed.With one dose of anti-PD-1 injection,tamoxifen administration significantly increased tumor CD8+T-cell infiltration.The expression levels of NHEJ-related genes in tumors were significantly reduced,while genes related to cGAS-STING pathway were augmented.However,the tumors of flutamidetreated male mice demonstrated enhanced expression of NHEJ-related genes,decreased expression of genes related to cGAS-STING pathway and reduced CD8+T-cell infiltration compared with those of the control male mice.These results indicated that favorable AR signaling inhibited the expression levels of NHEJ-related genes in HCC tissues,therefore activating the cGAS-STING pathway in tumors,promoting tumor CD8+T-cell infiltration,and thereby improved the anti-PD-1 effect.Conclusions:In HBV-related HCC,mutation profiles are different between males and females.Males also demonstrated different biological capabilities from those of females.Subsequent to exposed to genotoxicity agent,such as AFB1,androgen stimulation elevated aflatoxin metabolism-related genes and immune checkpoints,reduced NHEJ-related genes for DSB repair,potentially enhancing aflatoxin-related hepatocarcinogenesis in HBV chronically infected males.On the other hand,androgen signaling in hepatoma increased the CD8+T-cell infiltration,probably by repressing DNA repair that causes nuclear dsDNA leakage into the cytosol to activate cGAS-STING in tumors,and thus might improve the anti-PD-1 effect of HCC.Androgen may play different roles in the progression from chronic HBV infection into HCC. |
PDF Full Text Request