In findings that point to a potential treatment strategy, researchers in China have discovered how two risk factors – male hormones and aflatoxin – may drive hepatocellular carcinoma (HCC). The liver cancer genetics and biology differ between men and women and help explain why aflatoxin exposure increases the risk of HCC in hepatitis B virus (HBV)–infected patients, particularly in men.
The researchers found evidence that androgen signaling increased aflatoxin metabolism and genotoxicity, reduced DNA repair capabilities, and quelled antitumor immunity, Chungui Xu, PhD, with the State Key Lab of Molecular Oncology at the National Cancer Center at Peking Union Medical College in Beijing, and colleagues wrote. The study was published in Cellular and Molecular Gastroenterology and Hepatology.
“Androgen signaling in the context of genotoxic stress repressed DNA damage repair,” the authors wrote. “The alteration caused more nuclear DNA leakage into cytosol to activate the cGAS-STING pathway, which increased T-cell infiltration into tumor mass and improved anti–programmed cell death protein 1 [PD-1] immunotherapy in HCCs.”
In the study, the researchers conducted genomic analyses of HCC tumor samples from people with HBV who were exposed to aflatoxin in Qidong, China, an area that until recently had some of the highest liver cancer rates in the world. In subsequent experiments in cell lines and mice, the team investigated how the genetic alterations and transcription dysfunctions reflected the combined carcinogenic effects of aflatoxin and HPV.
Dr. Xu and colleagues performed whole-genome, whole-exome, and RNA sequencing on tumor and matched nonneoplastic liver tissues from 101 HBV-related HCC patients (47 men and 54 women). The patients had received primary hepatectomy without systemic treatment or radiation therapy and were followed for 5 years. Aflatoxin exposure was confirmed by recording aflatoxin M1 in their urine 3-18 years before HCC diagnosis. For comparison, the research team analyzed 113 HBV-related HCC samples without aflatoxin exposure from the Cancer Genome Atlas database. They also looked at 181 Chinese HCC samples from the International Cancer Genome Consortium that had no record of aflatoxin exposure. They found no sex differences in mutation patterns for previously identified HCC driver genes, but the tumor mutation burden was higher in the Qidong set.
In the Qidong samples, the research team identified 71 genes with significantly different mutation frequencies by sex. Among those, 62 genes were associated more frequently with men, and 9 genes were associated with women. None of the genes have been reported previously as HCC drivers, although some have been found previously in other cancers, such as melanoma, lung cancer, and thyroid adenocarcinoma.
From whole-genome sequencing of 88 samples, the research team detected HBV integration in 37 samples and identified 110 breakpoints. No difference in HBV breakpoint numbers was detected between the sexes, though there were differences in somatic mutation profiles and in HBV integration, and only men had HBV breakpoints binding to androgen receptors.
From RNA sequencing of 87 samples, the research team identified 3,070 significantly differentially expressed genes between men and women. The transcription levels of estrogen receptor 1 and 2 were similar between the sexes, but men expressed higher androgen receptor levels.
The researchers then analyzed the variation in gene expression between the male and female gene sets to understand HCC transcriptional dysfunction. The samples from men showed different biological capabilities, with several signaling pathways related to HCC development and progression that were up-regulated. The male samples also showed repression of specific antitumor immunity.
Men’s HCC tumor samples expressed higher levels of aflatoxin metabolism-related genes, such as AHR and CYP1A1, but lower levels of GSTM1 genes.
Turning to cell lines, the researchers used HBV-positive HepG2.2.15 cells and PLC/PRF/5 cells to test sex hormones in the regulation of AHR and CYP1A1 and how their interactions affected aflatoxin B1 cytotoxicity. After aflatoxin treatment, the addition of testosterone to the cultures significantly enhanced the transcription levels of AHR and CYP1A1. The aflatoxin dose needed to cause cell death was reduced by half in the presence of testosterone.
DNA damage from aflatoxin activates DNA repair mechanisms, so the research team analyzed different repair pathways. In the male tumor samples, the most down-regulated pathway was NHEJ. The male samples expressed significantly lower levels of NHEJ factors than did the female samples, including XRCC4, MRE11, ATM, HRCC5, and NBN.
In cell lines, the researchers tested the effects of androgen alone and with aflatoxin on the regulation of NHEJ factors. The transcriptional levels of XRCC4, LIG4, and MRE11 were reduced significantly in cells treated with both aflatoxin and testosterone, compared with those treated with aflatoxin alone. Notably, the addition of 17beta-estradiol estrogen partially reversed the reduction of XRCC4 and MRE11 expression.
The tumor samples from men also showed different gene signatures of immune responses and inflammation from the samples from women. The genes related to interferon I signaling and response were up-regulated significantly in male samples but not in female samples. In addition, the samples from men showed repression of antigen-specific antitumor immunity. The research team detected significantly increased CD8+T-cell infiltration in tumor tissues of men but not women, as well as higher transcriptional levels of PD-1 and CTLA-4, which are two immune checkpoint proteins on T cells that keep them from attacking the tumor. The data indicate that androgen signaling in established HBV-related HCCs contribute to the development of an immunosuppressive microenvironment, the authors wrote, which could render the tumor sensitive to anti–PD-1 immunotherapy.
In mice, the researchers examined the impact of a favorable androgen pathway on anti–PD-1 treatment effects against hepatoma. They administered tamoxifen to block ER signaling in syngeneic tumor-bearing mice. In both male and female mice, tamoxifen enhanced the anti–PD-1 effects to eradicate the tumor quickly. They also administered flutamide to tumor-bearing mice to block the androgen pathway and found no significant difference in tumor growth in female mice, but in male mice, tumors grew faster in the flutamide-treated mice.
“Therapeutics that favor androgen signaling and/or blocking estrogen signaling may provide a new strategy to improve the efficacy of immune checkpoint inhibitors against HCC in combination with radiotherapy or chemotherapy that induced DNA damage,” the authors wrote. “The adjuvant effects of tamoxifen for favorable androgen signaling to boost the anti–PD-1 effect in HCC patients needs future study in a prospective HCC cohort.”
The study was supported by the National Natural Science Foundation Fund of China, Innovation Fund for Medical Sciences of Chinese Academy of Medical Sciences, State Key Project for Infectious Diseases, and Peking Union Medical College. The authors disclosed no conflicts.
To read an editorial that accompanied this study in Cellular and Molecular Gastroenterology and Hepatology, go to https://www.cmghjournal.org/article/S2352-345X(22)00234-X/fulltext.
