Hepatitis B virus (HBV) infection remains the most important risk factor for hepatocellular carcinoma (HCC) worldwide and nonalcoholic fatty liver disease (NAFLD) has developed as major etiology of chronic liver diseases, cirrhosis and eventually HCC in the last decades. Although nucleos(t)ide analogs are recommended as the first-line drug for patients with chronic hepatitis B, incomplete eradication of HBV serves as an obstacle for effective cure of chronic hepatitis B and even HCC. NAFLD refers to a spectrum of hepatic metabolic disorders, compromised with multi-system diseases. Considering the specificity of hepatocytes and enrichment of immune cells in liver, this review aims to summarize the mechanisms of direct pro-tumorigenesis to hepatocytes induced by HBV infection and abnormal lipid metabolism, and indirect oncogenic processes mediated by immune cells. We also discuss similarities and differences of immune cells between HBV- and NAFLD-HCC and finally focus on the novel immunotherapies concerning preclinical and clinical studies for liver cancer.
Liver cancer is one of the most common causes of cancer-related deaths worldwide, with an estimated of 1.1 million new cases to be diagnosed in 10 years (GLOBOCAN, 2018). Hepatocellular carcinoma (HCC) accounts for nearly 80% of primary liver cancer and the occurrence is globally gender and geography biased. The estimated age-standardized incidence rates in men are mostly 3 times than that of women (13.9/1,000,000
HCC is prominently induced by chronic hepatitis B virus (HBV)/hepatitis C virus (HCV) infection, alcoholic or nonalcoholic fatty liver disease, aflatoxin intake and parasitic infection, the incidence of which are related with geographic variance. About 80% of HCC arises from persistent HBV infection in Asia-Pacific and Africa countries, while non-alcoholic fatty liver disease (NAFLD) is the major contributor in North America, followed by HCV infection. The direct-acting antiviral therapy with high rates of safety and sustained virologic response (> 95%), is considered as the contributing factor for eliminating HCV[
Although patients diagnosed with early stage of HCC undergo surgical resection or radiofrequency ablation for curative intent, up to 70% of them are susceptible to recurrence within 5 years[
Genomic features are significantly different in the group of HBV-HCC compared with non-infected patients. These results suggest that HBV-related HCC use alternative mechanisms for tumorigenesis to some extent. A high frequency of p53 inactivation and stem cell genes overexpression provide a potential pathogenic link between impaired cell reprogramming and HBV infection. Notably, these observations have clinical implication since TP53 mutations were associated with poor prognosis only in HBV-related tumors.
HBV genome contains four overlapping open reading frames, including preS1/preS2/S, preCore/Core, X and Pol, encoding viral proteins. Among them, hepatitis B X protein (HBx) is the most important oncogenic protein. Mutated or deleted HBx is frequently detected in HCC and plays critical role in liver carcinogenesis[
Direct oncogenic mechanisms of HBV- and NAFLD- HCC. HBV-encoded proteins lead to cellular heterogeneity via multiple manners. HBx stabilizes oncoproteins by suppressing ubiquitination or regulates transcription of host genes through transactivation. Cytoplasmic accumulation of HBs induces ER stress and thereby initiates cellular hippo pathways. Besides ER response, NF-κB, Src/PI3K/Akt and TRAIL/Fas pathways are involved in HBc-induced abnormal proliferation and metabolism of hepatocytes. Furthermore, HBV DNA integration and viral protein result in DNA damage and genomic instability, which drive cellular heterogeneity. Excessive lipid accumulation stimulates ER stress and dysregulates apoptosis and fibrogenesis via STAT3 and hippo pathways. The STAT5 and Akt pathways are involved in upregulation of the lipogenetic genes and downstream targets. Moreover, gene polymorphism is significantly related with NAFLD progression. HBV: hepatitis B virus; NAFLD: nonalcoholic fatty liver disease; HCC: hepatocellular carcinoma; ER: endoplasmic reticulum
As the most abundant proteins, HBV surface (HBs) proteins are composed of three proteins, the large (L), middle (M), and small (S) HBs, encoded by the preS1/preS2/S gene respectively[
As the major capsid protein and important viral replication mediator, HB core-related antigen (HBcrAg) is reported to be an independent risk factor for HCC development[
HBV DNA integration occurs early once HBV entering into hepatocytes and remains stable throughout tumor progression, which explains the existence of monoclonal or polyclonal origins of HCC from the view of virus infection[
In infected hepatocytes, both HBV DNA integration and viral proteins lead to genomic instability. Sequencing analysis of 373 liver cancer samples demonstrated ultra-high structural instability and preserved un-methylation in HBV integrated regions[
NAFLD and its complication nonalcoholic steatohepatitis (NASH) are becoming the leading cause of HCC. Multiple pathways, such as abnormal metabolism, dysbiosis of gut microbiota and dysregulated immune responses, are involved in NAFLD initiated hepatocarcinogenesis and have been well summarized recently[
Other than metabolic disorder, gene polymorphism is significantly responsible for NAFLD. Recent genome-wide association studies (GWASs) in European ancestry suggest a robust relationship of
Multiple resident or migratory immune cells, including innate and adaptive immune cells, are responsible for maintaining immune homeostasis in the liver. In addition, the dysregulation of hepatic immune cells play critical roles in liver cancer pathogenesis. Liver is rich of innate immune cells including myeloid cells and innate immune cells. The myeloid cells contain macrophages, dendritic cells and myeloid-derived suppressor cells (MDSCs). Liver innate lymphocytes are represented by natural killer (NK) cells, NKT cells, mucosal associated invariant T cells and γδT cells. CD4+ T, CD8+ T and B cells comprise major adaptive cells in liver[
Alteration of immune cells in HBV- and NAFLD- HCC and corresponding immunotherapies. In HBV-infected liver microenvironment, MDSCs are recruited and macrophages are educated to polarization of pro-tumor phenotype, which constitute immunosuppressive microenvironment. However, excessive lipid accumulation and lipotoxic hepatocytes induce macrophages to pro-inflammatory phenotype which promotes liver damage. In both etiologies- associated HCC, cytotoxic T cells and NK cells inhibit progression of tumor formation through cytokines secretion or phenotype switch. B cells control progression of HBV infection by antibodies production and cytokines secretion, whereas promote NAFLD development towards fibrosis via inducing CD4+ T cells. Overall, activation and function of effector immune cells are suppressed by MDSCs and macrophages through interaction of checkpoint molecules, such as PD-1, PD-L1, CTLA-4, TIM-3 and LAG-3. Furthermore, several drugs blocking inhibitory checkpoint receptors have been developed and approved for treatment of cancer diseases. Collaboratively, CAR-T and CAR-NK cell therapy are investigated to be promising in treatment of liver cancer. HBV: hepatitis B virus; NAFLD: nonalcoholic fatty liver disease; HCC: hepatocellular carcinoma; MDSCs: myeloid-derived suppressor cells; CAR: chimeric antigen receptor; NK: natural killer; Mφ: macrophage; Arg1: arginase 1; MMP9: matrix metallopeptidase 9; IL: interleukin; IFN: interferon; TNF-α: tumor necrosis factor-alpha; TCR: T-cell receptor; BCR: B-cell receptor
Macrophages compromise almost 20% of overall immune cells in liver, containing residential Kupffer cells (KCs) and migratory monocyte-derived macrophages. Hepatic macrophages are extremely plastic and accommodative to signals from the microenvironment. Both inflammatory stimuli and viral proteins reprogram macrophages towards M2-like tumor macrophages, which in turn promote HCC progression[
Contrarily, pro-inflammatory macrophages are essential for progression of NASH-associated HCC. In a high-fat diet (HFD) zebrafish model, hepatic macrophage morphology is changed and number of TNF-α positive positive macrophages is increased, and liver size is subsequently reduced in macrophage-ablation NAFLD/NASH associated HCC larvae but not in HCC or NAFLD alone[
MDSCs, generated as immature myeloid cells from bone marrow, are the major immunosuppressive cells which inhibit T cells proliferation and function and enhance induction of Treg cells and tumor-associated macrophages in chronic inflammation and cancers. Multiple studies have reported that MDSCs are closely related with HBV persistence and HCC. In patients with chronic HBV infection (CHB), HBeAg induces expansion of monocytic MDSCs (mMDSCs) via indoleamine-2,3-dioxygenase (IDO), to suppress autologous T cell proliferation and IFN-γ production, favoring the establishment of tolerant immune environment[
Lymphocytes represent another crucial population in intrahepatic innate immune cells, including NKT cells, ILCs (NK cells and other ILC subpopulations) and γδT cells. NKT and NK cells, which mainly contain conventional circulating NK and liver-resident NK cells, comprise about 50% of lymphocytes in liver. Generally, hepatic NK cells recognize and restrain virus-infected cells via direct cytotoxicity or secreting immunoregulatory cytokines, such as IFN-γ and TNF-α, to activate T cells[
IL-15 is crucial for homeostasis of NK cells. Deficiency of IL-15 or IL-15Rα inhibits high fat diet-induced accumulation of lipids and inflammation in liver[
T cells represent the major adaptive lymphocytes in transformation from inflammation to cancer. CD8+ T cells frequently show restricted proliferation and exhausted function with high expression of inhibitory checkpoint molecules such as CTLA-4, PD-1 and TIM-3 in CHB and HBV-HCC. High expression of PD-1 on HBV-specific T cells or B cells induces exhaustion of T cells or reduced antibodies production, which is partially reversed by PD-1 blockade[
Abundant exhausted CD8+ T cells and Tregs exist and potentially clonally expand in cancerous tissues from HCC patients[
Compared to CD8+ T cells, HBV-specific CD4+ cytotoxic cells in PBMCs from HBV-associated HCC present at the similar level, but display weaker cytolysis capability and suppress cytotoxicity of CD8+ T cells in the absence of Tregs[
HBV viral load as well as HBsAg and HBeAg levels differed with progression of distinct phases of chronic HBV infection, accompanied by alteration of immune cells. Immunoglobulin-encoding genes and B cell function-related genes are more enriched in immune active patients than those in immune tolerant and inactive carrier patients[
Intrahepatic B cells are highly associated with NAFLD. CD20+ B cells are increased in liver tissues from NAFLD patients with activity score higher than 5, compared with those of 0 or 1~4[
As discussed above, a long duration is taken for both HBV- and NAFLD-related HCC, therefore, treatments aiming at eliminating HBV infection or blocking NASH progression and fibrogenesis can prevent the development of tumors. Abundant alterations of hepatic immune cells during liver carcinogenesis provide wide-range targets. Several immunotherapies have been approved for clinical application, nonetheless, the induced resistance and adverse events remain to be further investigated. The following sections will discuss immunotherapies in HCC, such as immune checkpoint inhibitors, virus or tumor vaccines, and adoptive cells transfer.
Inhibitory checkpoint receptors (ICRs) including CTLA-4, PD-1 and PD-L1, are recognized as master regulators for anti-tumor immunity. Immune checkpoint inhibition (ICI) therapy using antibody against PD-1, PD-L1 and CTLA-4 have been since been approved by the Food and Drug Administration[
CTLA-4 is another inhibitory molecule that attenuates activation and function of T cells. Upregulation of CTLA-4 promote the apoptosis, secretion of anti-inflammatory cytokines and exhaustion of CD4+ Th and CD8+ T cells in liver tissues from CHB and HCC patients[
Chimeric antigen receptor (CAR) therapy is a promising strategy for cancer immunotherapy. Accumulating evidence has demonstrated CAR-T cell therapy as effective strategy against lymphoid leukemia and multiple myeloma[
However, side effects to CAR-T therapy contain high manufacturing and fatal toxicities, such as cytokine release syndrome[
Chronic HBV infection-induced progression of HCC mainly depends on direct intrusion of viral components in hepatocytes, which disturbs its normal cellular metabolism and function. This subsequently stimulates immune imbalance in liver. Frequently accompanied by systemic metabolic syndrome, advancement of NALFD- associated HCC encompasses excessive lipid accumulation in hepatocytes and immune cells, insulin resistance and inflammation induced malignant transformation. The hepatic complexity of immune cells mediates tumorigenesis in situation of anergy of cytotoxic cells and enhancement of immunosuppressive cells. Cytotoxic T cells are the major factor which eradicates HBV-infected cells or malignant cells. The exhaustion mediated by MDSCs or Tregs leads to persistence of HBV and evasion of cancer cells. Flexibility of macrophages facilitates themselves to polarize according to different signals from microenvironment. Macrophages mostly function as suppressive cells to inhibit proliferation and activation of CTLs in HBV-related HCC, while promotes liver damage and fibrosis via releasing pro-inflammatory cytokines.
Since there still remains lack of effective drug specifically targeting eradication of HBV or controlling development of NAFLD, illustrating molecular mechanisms of immune cells and establishing novel immunotherapies will provide promising options for treatment of HCC. Unbridling cytotoxic cells (ICI) and stimulating the cells specifically engineered with tumor-associated antigens (CAR-T and CAR-NK) mutually contribute to effectively restrain progression of HBV- and NAFLD-induced HCC. Taken together, the drug resistance and adverse effect induced by immunotherapies should be further recorded and investigated in future studies.
Conceived of the presented idea: Ma CH, Song XJ
Performed the basic writing: Song XJ
Developed the further revision: Ma CH
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This work was supported by grants from the National Science Foundation of China (Key project 81830017 and 81902443), Taishan Scholarship (No.tspd20181201), National Key Research and Development Program (2018YFE0126500), Shandong Provincial Key Innovation project (No.2018FYJH0503), Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong.
All authors declared that there are no conflicts of interest.
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© The Author(s) 2020.