Do hepatitis B virus surface antigens have any role in viral carcinogenesis? (2018)

Potential conflict of interest: Dr. Block advises for and owns stock in Contravir. He owns stock in and received grants from Arbutus. He advises for Glycotest. Dr. Guo owns stock in, received grants from Arbutus. SEE ARTICLE ON PAGE 815 Do the hepatitis B virus (HBV) envelope proteins have any role in viral pathogenesis? This is a question that has dogged the hepatitis B research community since their discovery. HBV expresses three structurally related envelope proteins, designated as large (LHBs), middle (MHBs), and small (SHBs) surface proteins, from a single open reading frame (ORF; Fig. 1A). The SHBs protein is 226 amino acids (aa) in length, the MHBs has a N‐terminal extension of 55 aa of the pre‐S2 domain, and LHBs has the additional N‐terminal extension of 108 aa of the pre‐S1 domain. LHBs mediates binding of the virus to its receptor on hepatocytes. SHBs is the major component of viral envelope and subviral particles. Although a role for SHBs in induction of immune tolerance and carcinogenic activity for LHBs has been the subject of speculation for decades, the report from Yen et al., in this issue of Hepatology, provides circumstantial evidence for a role of LHBs and SHBs in hepatocarcinogenesis in humans.1Figure 1: Gene structure and expression strategy of HBV envelope proteins and possible consequences of their aberrant expression in hepatocytes. (A) The region of the HBV genome specifying the envelope proteins is shown, as if integrated (//) into host chromosomes. The major (most abundant) envelope protein, SHBs, is expressed under the control of the pre‐S2/S promoter, which directs a 2.1‐kb mRNA transcription. LHBs is translated from a 2.4‐kb mRNA, transcribed under the control of the pre‐S1 promoter. (B) Continued expression of LHBs and loss of SHBs expression results in the retention of LHBs in the ER and activates ER stress. The unresolved ER stress drives a liver pathological process characterized by hepatocyte death and proliferation under the inflammatory liver condition, which fosters the accumulation of transforming mutations in hepatocytes and eventually development of HCC. The red color indicates liver inflammation. Please see text for more detailed interpretation.Specifically, analyses of LHBs and SHBs expression in tumor and adjacent liver tissues from 34 hepatocellular carcinoma (HCC) patients with chronic HBV infection by immunohistochemical staining (12 cases) and western blotting assays (22 cases) revealed that, compared to nontumorous tissue from the same patients, SHBs expression was greatly diminished in the tumors examined, whereas LHBs expression was not significantly decreased in the tumors of 95% patients. These data imply that while LHBs expression is maintained during HCC development, SHBs expression is lost or down‐regulated. This is consistent with the previous findings that expression of either wild‐type or pre‐S2 mutant LHBs, in the absence of other viral proteins, is hepatocarcinogenic in mice.2 Taken together, the results support a hypothesis that, in the absence of SHBs expression, LHBs production may be cytopathic and pro‐oncogenic, as illustrated in Fig. 1B. There is a growing literature regarding mutant HBV envelope proteins and their role in pathogenesis. Briefly, HBV variants with deletions in pre‐S1 and/or pre‐S2 regions have been reported to correlate with progression of liver disease in chronic HBV.4 In‐frame deletions of the C‐terminal pre‐S1 region and the N‐terminal pre‐S2 region are the most common mutations found in HBV‐infected individuals with advanced liver diseases and HCC.5 These regions contain multiple T‐ and B‐cell epitopes, and their deletion might help the viruses to escape host antiviral immune responses. In a cohort of 175 HBV‐related HCC patients, Yen et al. found that the increased peripheral blood levels of HBV mutants, with in‐frame deletions in the pre‐S2 region, are associated with elevated risk of HCC recurrence after curative hepatic resection.1 Expression of pre‐S2 mutant LHBs was previously reported to be associated with type II ground glass hepatocytes, which are characterized by clustered nodule formations and suggested as preneoplastic lesions.6 Moreover, previous studies demonstrated that the misfolded pre‐S2 mutant LHBs triggers endoplasmic reticulum (ER) stress‐dependent DNA damage, disruption of cell‐cycle progress, induction of genomic instability, and angiogenesis. This may promote HCC development.6 However, although the possibility that pre‐S2 mutant LHBs is a “driver” of oncogenic transformation is attractive, and the hypothesis favored by Yen et al., their study did not definitively show that it is the preS2 mutant LHBs protein, and not the wild‐type LHBs, that is expressed in the tumors. Indeed, deep DNA sequence analyses indicated that pre‐S2 mutant HBV DNA was usually present only as a minor fraction of the total detected HBV DNA and coexisted with wild‐type HBV DNA in the blood, liver tissues, and even tumors. Therefore, it is possible that it is the wild‐type LHBs that is expressed in the majority of tumors. Direct sequence analysis of HBV DNA integrated into the chromosomes of the tumor cells would settle this question. The major question is then: How can expression of LHBs, either wild type or pre‐S2 mutant, in the absence of, or reduced amounts of, SHBs be oncogenic? The LHBs protein is normally secreted from the infected hepatocytes, complexed with MHBs and SHBs, as envelope components of virions. However, as illustrated in Fig. 1B, in the absence of SHBs, LHBs proteins accumulate within the ER. The investigators reasoned that this was also the case, perhaps even in an exaggerated way, for the pre‐S2 mutant LHBs, attributed to its misfolding. Retention of LHBs proteins (including pre‐S2 mutant LHBs) in the ER activates ER stress pathways that predispose the cell to induction of oxidative stress, DNA damage/genomic instability, and, ultimately, cell death.6 The LHBs‐driven hepatocyte death, liver inflammation, and regeneration would place large numbers of hepatocytes at risk for acquisition of transforming mutations. It can be imagined that the cells that acquire mutations permitting escape of LHBs‐induced cell death would gain a growth advantage, and would be selected for. The multiple rounds of ensuing clonal expansion could lead to malignancy.7 Moreover, the continued expression of LHBs in tumor cells suggests that this viral protein may not only initiate the hepatocarcinogenic process, but may also play roles at multiple, postinitiation, stages of HCC development. It would then be important to know whether the tumor cells have actually become “addicted” to the viral protein, and need it for continued growth. This hypothesis assumes that a significant reduction or loss of SHBs expression is required for LHBs to drive HCC development. How can SHBs expression be selectively down‐regulated or abolished in an infected hepatocyte, while LHBs expression is maintained? As depicted in Fig. 1A, although the three HBV envelope proteins are encoded in a single ORF, they are translated from two different mRNAs transcribed under the control of pre‐S1 and pre‐S2/S promoters, respectively. While LHBs is translated from 2.4‐kilobase (kb) mRNA, MHBs and SHBs are translated from the 2.1‐kb mRNA. Therefore, selective down‐regulation of SHBs expression can be achieved genetically through mutations in the pre‐S2/S promoter region that down‐regulate or abolish transcription or translation from the 2.1‐kb mRNA. Of course, in order to maintain LHBs expression, mutations in the pre‐S1 and pre‐S2 regions must not disrupt the envelope protein ORF. It is thus possible that, for at least some of the prevalent pre‐S1 and pre‐S2 in‐frame deletion mutations, their underlining pathogenic mechanism may be the down‐regulation of SHBs expression, but not production of mutant LHBs proteins. In addition, selective down‐regulation of SHBs could also occur as a consequence of unknown hepatocyte physiology changes and epigenetic silence of pre‐S2/S promoter.8 The work by Yen et al. should stimulate further investigation into possible causal relationships between pre‐S1/S2 mutation and HCC. In fact, others have reported that SHBs and pre‐S2 mutants are not detected in clonally expanded liver nodules.9 This could reflect differing techniques applied in the studies or multiple pathways of viral carcinogenesis. Getting these questions resolved is important for reasons of both basic and clinical science. This is because if wild‐type or mutant LHBs expression does drive—and perhaps even maintain—HCC, antivirals that even repress viral covalently closed circular DNA may be insufficient to completely reduce the risk of HCC. Therapies that target the LHBs gene and its transcript may be needed by those with chronic HBV long after viral replication has been stopped with other antivirals.