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Obesity is a major risk factor for cancers including hepatocellular carcinoma (HCC) that develops from a background of non-alcoholic fatty liver disease (NAFLD). Hypercholesterolemia is a common comorbidity of obesity. Although cholesterol biosynthesis mainly occurs in the liver, its role in HCC development of obese people remains obscure. Using high-fat high-carbohydrate diet-associated orthotopic and spontaneous NAFLD-HCC mouse models, we found that hepatic cholesterol accumulation in obesity selectively suppressed natural killer T (NKT) cell-mediated antitumor immunosurveillance. Transcriptome analysis of human liver revealed aberrant cholesterol metabolism and NKT cell dysfunction in NAFLD patients. Notably, cholesterol-lowering rosuvastatin restored NKT expansion and cytotoxicity to prevent obesogenic diet-promoted HCC development. Moreover, suppression of hepatic cholesterol biosynthesis by a mammalian target of rapamycin (mTOR) inhibitor vistusertib preceded tumor regression, which was abolished by NKT inactivation but not CD8(+) T cell depletion. Mechanistically, sterol regulatory element-binding protein 2 (SREBP2)-driven excessive cholesterol production from hepatocytes induced lipid peroxide accumulation and deficient cytotoxicity in NKT cells, which were supported by findings in people with obesity, NAFLD and NAFLD-HCC. This study highlights mTORC1/SREBP2/cholesterol-mediated NKT dysfunction in the tumor-promoting NAFLD liver microenvironment, providing intervention strategies that invigorating NKT cells to control HCC in the obesity epidemic.

Legend for Supplemental Figures S1-S8.

HDAC8 expression positively correlates with its signaling components in primary human NAFLD-associated HCCs.

Description of additional methods and procedures used in the study.

Clinicopathological information of the NAFLD-associated HCC patients.

The growing epidemic of obesity, which causes nonalcoholic fatty liver disease (NAFLD) and the more severe phenotype nonalcoholic steatohepatitis (NASH), has paralleled the increasing incidence of hepatocellular carcinoma (HCC). Accumulating evidence demonstrates that overnutrition and metabolic pathways can trigger modifications of DNA and histones via deregulation of chromatin modifiers, resulting in aberrant transcriptional activity. However, the epigenetic regulation of HCC development in NAFLD remains obscure. Here, we uncover key epigenetic regulators using both dietary and genetic obesity-promoted HCC models through quantitative expression profiling and characterize the oncogenic activities of histone deacetylase HDAC8 in NAFLD-associated hepatocarcinogenesis. HDAC8 is directly upregulated by the lipogenic transcription factor SREBP-1 where they are coexpressed in dietary obesity models of NASH and HCC. Lentiviral-mediated HDAC8 attenuation in vivo reversed insulin resistance and reduced NAFLD-associated tumorigenicity. HDAC8 modulation by genetic and pharmacologic approaches inhibited p53/p21-mediated apoptosis and G2–M phase cell-cycle arrest and stimulated β-catenin–dependent cell proliferation. Mechanistically, HDAC8 physically interacted with the chromatin modifier EZH2 to concordantly repress Wnt antagonists via histone H4 deacetylation and H3 lysine 27 trimethylation. In human NAFLD-associated HCC, levels of SREBP-1, HDAC8, EZH2, H4 deacetylation, H3K27me3, and active β-catenin were all correlated positively in tumors compared with nontumor tissues. Overall, our findings show how HDAC8 drives NAFLD-associated hepatocarcinogenesis, offering a novel epigenetic target to prevent or treat HCC in obese patients. Cancer Res; 75(22); 4803–16. ©2015 AACR.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive hepatic lipid accumulation, which can progress to nonalcoholic steatohepatitis (NASH). Histone deacetylase Sirtuin 6 (SIRT6) regulates NAFLD by regulating metabolism-related gene expression, but an extrachromosomal role for SIRT6 in NAFLD development remains elusive. We investigated whether SIRT6 functions on NAFLD in the cytoplasm. We found that SIRT6 binds saturated fatty acids, especially palmitic acid. This binding leads to its nuclear export, where it deacetylates long-chain acyl-CoA synthase 5 (ACSL5), thereby facilitating fatty acid oxidation. High-fat diet-induced NAFLD is suppressed by ACSL5 hepatic overexpression but is exacerbated by its depletion. As confirmation, overexpression of a deacetylated ACSL5 mimic attenuated NAFLD in Sirt6 liver-specific knockout mice. Moreover, NASH-hepatic tissues from both patients and diet-fed mice exhibited significantly reduced cytoplasmic SIRT6 levels and increased ACSL5 acetylation. The SIRT6/ACSL5 signaling pathway has a critical role in NAFLD progression and might constitute an avenue for therapeutic intervention.

ObjectiveAltered metabolites are important for the tumourigenicity of hepatocellular carcinoma (HCC). We performed integrative metabolomics analysis of the metabolites changes in portal venous blood and in comparison with the metabolites changes in liver tissues and stool samples of HCC patients and healthy liver donors.DesignSerum (portal and central vein), liver tissue (HCC tumour and adjacent non-tumour, normal liver) and stool samples were collected from 102 subjects (52 HCC patients and 50 healthy controls) in the discovery cohort; and 100 subjects (50 HCC patients and 50 healthy controls) in an independent validation cohort. Untargeted metabolomic profiling was performed using high-performance liquid chromatography-mass spectrometry. The function of candidate metabolites was validated in hepatocyte cell lines.ResultsDetailed metabolomic evaluation showed distinct clusters of metabolites in serum, liver tissue and stool samples from patients with HCC and control individuals (pConclusionsThe integrative metabolome analysis of serum, tissue and stool metabolites revealed unreported metabolic alterations in HCC patients. In portal vein, we identified elevated and depleted metabolites signifying that they might play a role in HCC development.

ObjectiveHepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy.DesignFunctional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined.ResultsAccumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model.ConclusionOur results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy.

Background Tumor-associated lymphangiogenesis is considered significant in number of solid malignancies. However, its impact on prognosis of intrahepatic cholangiocarcinoma (ICC) after resection remains further confirmation. Herein, we conducted this study to evaluate prognostic impact of tumor-associated lymphangiogenesis in patients with ICC. Methods Extent of tumor-associated lymphangiogenesis of ICC was evaluated by quantifying microlymphatic vessel density (MLVD) from immunohistochemical staining of a lymphatic endothelial-specific antibody (podoplanin). Clinicopathological characteristics were comprehensively analyzed to identify MLVD-associated factors. The patients were stratified into high and low MLVD groups according to the distinctive correlation between the MLVD and overall survival using the Spearman’s correlation test. Kaplan-Meier estimation was performed to confirm prognostic impact of MLVD in patients with ICC. Univariate and multivariate analyses were performed using the Cox proportional hazard model. Results The MLVD between 4 to 12 counts showed inverse proportion to the overall survival (Spearman’s r = − 0.66; 95% confidence interval [CI], − 0.82 to − 0.39; p

Obesity increases the risk of hepatocellular carcinoma (HCC) especially in men, but the molecular mechanism remains obscure. Here, we show that an androgen receptor (AR)-driven oncogene, cell cycle-related kinase (CCRK), collaborates with obesity-induced pro-inflammatory signaling to promote non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Lentivirus-mediated Ccrk ablation in liver of male mice fed with high-fat high-carbohydrate diet abrogates not only obesity-associated lipid accumulation, glucose intolerance and insulin resistance, but also HCC development. Mechanistically, CCRK fuels a feedforward loop by inducing STAT3-AR promoter co-occupancy and transcriptional up-regulation, which in turn activates mTORC1/4E-BP1/S6K/SREBP1 cascades via GSK3β phosphorylation. Moreover, hepatic CCRK induction in transgenic mice stimulates mTORC1-dependent G−csf expression to enhance polymorphonuclear myeloid-derived suppressor cell recruitment and tumorigenicity. Finally, the STAT3-AR-CCRK-mTORC1 pathway components are concordantly over-expressed in human NASH-associated HCCs. These findings unveil the dual roles of an inflammatory-CCRK circuitry in driving metabolic and immunosuppressive reprogramming through mTORC1 activation, thereby establishing a pro-tumorigenic microenvironment for HCC development., Obesity increases the risk of hepatocellular carcinoma (HCC) especially in men. Here the authors find a potential mechanistic explanation by showing that, in mice, obesity-induced STAT3 cooperates with the androgen receptor to activate the mTORC pathway through up regulation of CCRK, resulting in hepatic steatosis worsening and HCC development via metabolic and immune reprogramming.

Background The insufficient T cell infiltration into non-inflamed tumors such as hepatocellular carcinoma (HCC) restricts the effectiveness of immune-checkpoint blockade (ICB) to a minority of patients. Epigenetic therapy provides new opportunities to rewire cancer transcriptional programs, but whether and how selective epigenetic inhibition counteracts the immune-excluded phenotype to sensitize ICB therapy remain incompletely defined. Here, we aimed to investigate the therapeutic efficacy and mechanistic basis of histone deacetylase 8 (HDAC8), a histone H3 lysine 27 (H3K27)-specific isoform, in HCC development and ICB responsiveness. Methods The immune-modulatory and anti-tumor effects of HDAC8 inhibition via a HDAC8-selective inhibitor, PCI34051, were determined in orthotopic HCC mouse models. Molecular mechanisms and functional significance of HDAC8 inhibition were conducted by genome-wide H3K27ac ChIP-seq and RNAseq in HCC patient specimens, cancer cell lines, NOD-SCID and humanized mouse models. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and PCI34051 was determined in orthotopic and spontaneous HCC mouse models. Results Pharmacological inhibition of HDAC8 thwarted HCC tumorigenicity in immunocompetent but not immunodeficient mice. The tumor-suppressive effect of PCI34051 was abrogated by CD8+T cell depletion or regulatory T cell adoptive transfer. Chromatin profiling of human HDAC8-expressing HCCs revealed genome-wide H3K27 deacetylation in 1,251 silenced enhancer-target gene pairs that were enriched in metabolic and immune regulators. Mechanistically, down-regulation of HDAC8 increased global and enhancer levels of H3K27 acetylation to reactivate T cell-trafficking chemokine production from HCC cells, thus relieving T cell exclusion in both NOD-SCID and humanized mouse models. In the HCC preclinical model, selective HDAC8 inhibition significantly increased tumor-infiltrating CD8+T cells and potentiated eradication of established hepatoma by anti-PD-L1 therapy without a sign of toxicity. Importantly, mice treated with HDAC8/PD-L1 co-blockade were protected against subsequent tumor re-challenge with the induction of memory T cells and remained tumor-free for ≥15 months. Conclusions Our study demonstrates that selective HDAC8 inhibition elicits effective and durable ICB responses by co-opting adaptive immunity via enhancer reprogramming, thereby providing a new strategy for effective combined epigenetic immunotherapy.

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

Background & aims: VEGFR-3 has been shown of great significance in lymph node metastasis and some malignancies, however, its expression in tumors and impact on outcome of intrahepatic cholangiocarcinoma (iCCA) remains unknown. The aim of this study was to assess the role of VEGFR-3 positive tumors for prognosis of iCCA and tumor-associated lymphangiogenesis. Methods: Clinicopathological features, prognostic factors and survival rate were analyzed to evaluate the influence of VEGFR-3 positive expression on prognosis of iCCA. In addition, tumor-associated lymphangiogenesis quantified as micro-lymphatic vessel density (MLVD) was assessed to explore the correlation between VEGFR-3 expression and lymph node metastasis for iCCA. Results: Patients with VEGFR-3 positive tumors had increased lymph node metastasis (p=0.025) and were more likely to suffer from tumor recurrence compared with VEGFR-3 negative tumors (p

ObjectiveMyeloid-derived suppressor cells (MDSCs) contribute to tumour immunosuppressive microenvironment and immune-checkpoint blockade resistance. Emerging evidence highlights the pivotal functions of cyclin-dependent kinases (CDKs) in tumour immunity. Here we elucidated the role of tumour-intrinsic CDK20, or cell cycle-related kinase (CCRK) on immunosuppression in hepatocellular carcinoma (HCC).DesignImmunosuppression of MDSCs derived from patients with HCC and relationship with CCRK were determined by flow cytometry, expression analyses and co-culture systems. Mechanistic studies were also conducted in liver-specific CCRK-inducible transgenic (TG) mice and Hepa1–6 orthotopic HCC models using CRISPR/Cas9-mediated Ccrk depletion and liver-targeted nanoparticles for interleukin (IL) 6 trapping. Tumorigenicity and immunophenotype were assessed on single or combined antiprogrammed death-1-ligand 1 (PD-L1) therapy.ResultsTumour-infiltrating CD11b+CD33+HLA-DR− MDSCs from patients with HCC potently inhibited autologous CD8+T cell proliferation. Concordant overexpression of CCRK and MDSC markers (CD11b/CD33) positively correlated with poorer survival rates. Hepatocellular CCRK stimulated immunosuppressive CD11b+CD33+HLA-DR− MDSC expansion from human peripheral blood mononuclear cells through upregulating IL-6. Mechanistically, CCRK activated nuclear factor-κB (NF-κB) via enhancer of zeste homolog 2 (EZH2) and facilitated NF-κB-EZH2 co-binding to IL-6 promoter. Hepatic CCRK induction in TG mice activated the EZH2/NF-κB/IL-6 cascade, leading to accumulation of polymorphonuclear (PMN) MDSCs with potent T cell suppressive activity. In contrast, inhibiting tumorous Ccrk or hepatic IL-6 increased interferon γ+tumour necrosis factor-α+CD8+ T cell infiltration and impaired tumorigenicity, which was rescued by restoring PMN-MDSCs. Notably, tumorous Ccrk depletion upregulated PD-L1 expression and increased intratumorous CD8+ T cells, thus enhancing PD-L1 blockade efficacy to eradicate HCC.ConclusionOur results delineate an immunosuppressive mechanism of the hepatoma-intrinsic CCRK signalling and highlight an overexpressed kinase target whose inhibition might empower HCC immunotherapy.

Epidemiological studies have demonstrated a close association of type 2 diabetes and hepatocellular carcinoma (HCC). Exenatide (Ex-4), a potent diabetes drug targeting glucagon-like peptide-1 receptor (GLP-1R), is protective against non-alcoholic fatty liver disease (NAFLD). However, the Ex-4 function and GLP-1R status have yet been explored in HCC. Herein we investigated the effect of Ex-4 in diethylnitrosamine (DEN)-treated mice consuming control or high-fat high-carbohydrate diet. Administration of Ex-4 significantly improved obesity-induced hyperglycemia and hyperlipidemia and reduced HCC multiplicity in obese DEN-treated mice, in which suppressed proliferation and induced apoptosis were confined to tumor cells. The tumor suppression effects of Ex-4 were associated with high expression of GLP-1R and activation of cyclic AMP (cAMP) and protein kinase A (PKA). Importantly, Ex-4 also downregulated epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription 3 (STAT3), which lie downstream of cAMP-PKA signaling, resulting in suppression of multiple STAT3-targeted genes including c-Myc, cyclin D1, survivin, Bcl-2 and Bcl-xl. The growth inhibitory effects of Ex-4 were consistent in GLP-1R-abundant hepatoma cell lines and xenograft mouse model, wherein both PKA and EGFR had obligatory roles in mediating Ex-4 functions. In addition, Ex-4 also effectively suppressed inflammatory and fibrotic phenotypes in mice fed with methionine-choline-deficient (MCD) diet and choline-deficient ethionine-supplemented (CDE) diet, respectively. In summary, Ex-4 elicits protective functions against NAFLD and obesity-associated HCC through cAMP-PKA-EGFR-STAT3 signaling, suggesting its administration as a novel approach to reduce HCC risk in diabetic patients.

Insufficient T cell infiltration into noninflamed tumors, such as hepatocellular carcinoma (HCC), restricts the effectiveness of immune-checkpoint blockade (ICB) for a subset of patients. Epigenetic therapy provides further opportunities to rewire cancer-associated transcriptional programs, but whether and how selective epigenetic inhibition counteracts the immune-excluded phenotype remain incompletely defined. Here, we showed that pharmacological inhibition of histone deacetylase 8 (HDAC8), a histone H3 lysine 27 (H3K27)-specific isozyme overexpressed in a variety of human cancers, thwarts HCC tumorigenicity in a T cell-dependent manner. The tumor-suppressive effect of selective HDAC8 inhibition was abrogated by CD8+ T cell depletion or regulatory T cell adoptive transfer. Chromatin profiling of human HDAC8-expressing HCCs revealed genome-wide H3K27 deacetylation in 1251 silenced enhancer-target gene pairs that are enriched in metabolic and immune regulators. Mechanistically, down-regulation of HDAC8 increased global and enhancer acetylation of H3K27 to reactivate production of T cell-trafficking chemokines by HCC cells, thus relieving T cell exclusion in both immunodeficient and humanized mouse models. In an HCC preclinical model, selective HDAC8 inhibition increased tumor-infiltrating CD8+ T cells and potentiated eradication of established hepatomas by anti-PD-L1 therapy without evidence of toxicity. Mice treated with HDAC8 and PD-L1 coblockade were protected against subsequent tumor rechallenge as a result of the induction of memory T cells and remained tumor-free for greater than 15 months. Collectively, our study demonstrates that selective HDAC8 inhibition elicits effective and durable responses to ICB by co-opting adaptive immunity through enhancer reprogramming.

Background Metastasis is a prominent cause of cancer-related death governed by both cancer cell-intrinsic mechanisms and extrinsic microenvironment. Clinical observations demonstrated liver as a common metastatic site for various cancers, which may be due to its immune tolerant environment. Myeloid-derived suppressor cell (MDSC) is a heterogeneous cell population of immature myeloid cells that contribute to the formation of a favorable metastatic environment partially via suppression of immune effector cells. However, the underlying mechanisms in liver tropism of tumor metastasis remain poorly understood. We have previously discovered that cell cycle-related kinase (CCRK) can promote primary hepatocellular carcinoma (HCC) development via MDSCs. Here we hypothesize that the accumulation of hepatic MDSCs induced by CCRK may contribute to the formation of a favorable metastatic liver microenvironment. Methods We constructed a liver-specific CCRK inducible transgenic (TG) mouse model by a Cre/loxP system. Orthotopic and metastatic mouse models were used to investigate the role of CCRK in promoting tumor growth and metastasis. PMN-MDSC was depleted by anti-Ly6G antibody. Cytokine, chemokine, and immune cells profiling were performed after sacrifice. Results Induction of CCRK expression by tamoxifen injection could increase CD11b+Gr-1+Ly6G+Ly6Clow polymorphonuclear (PMN)-MDSCs liver accumulation specifically in male mice with upregulated Cxcl1 and Gcsf expression. Intrahepatic injection of a mouse hepatoma cell line Hep1–6 in male TG mice developed larger tumors compared to control and positively associated with increased PMN-MDSCs levels in liver. Moreover, the tumorigenicity was abolished by PMN-MDSC depletion. Notably, intrasplenic injection of a mouse melanoma cell line B16F10 exhibited an increased level of liver-infiltrating PMN-MDSCs and enhanced liver metastasis in male TG mice compared to control mice. Moreover, depletion of PMN-MDSCs suppressed metastasis in liver. Mechanistically, anti-tumor NKT cells, rather than NK cells and CD8+T cells, were negatively correlated with tumor weight and MDSC proportion, indicating involvement of cross-talk between MDSC and NKT in liver metastasis. Conclusions Our findings suggest that hepatic CCRK expression create a tumor growth- and metastasis-supportive liver microenvironment via enhancing immunosuppression.

Background Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune-checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy. Methods Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet, and verified by clinical specimens. Mechanistic studies were conducted in the human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically-trialed BET bromodomain inhibitor i-BET762 was determined. Results Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumor-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumor-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumor phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signaling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumor eradication and prolonged survival in the fibrotic-HCC mouse model. Conclusions Our results signify how non-tumor intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy (figure 1). Acknowledgement This project is supported by the University Grants Committee through the Collaborative Research Fund (C4045–18W) and the Theme-based Research Scheme (T11–706/18-N), the Health and Medical Research Fund (16170451), the Terry Fox Foundation, the Focused Innovations Scheme-Scheme B (1907309) from the Chinese University of Hong Kong and the Li Ka Shing Foundation (Canada). Man Liu is supported by China Postdoctoral Science Foundation.

Background The heterogeneous responses to immune-checkpoint blockade (ICB) therapy e.g. anti-programmed death-ligand 1 (PD-L1) antibody are attributable to the complex interplay between a range of cancer-cell-autonomous cues and immunosuppressive tumor microenvironment. Two recent phase I/II trials of PD-1 checkpoint inhibitors in patients with advanced hepatocellular carcinoma (HCC) have produced promising results, yet the objective response rates were relatively low ( Methods The effect of PCI-34051 on tumorigenicity was investigated in orthotopic HCC mouse models. Immune profiling in tumor microenvironment was determined by multi-color flow cytometry. The underlying mechanism was investigated by integrative epigenomics analysis. The anti-tumor efficacy of combined therapy with PCI-34051 and anti-PD-L1 antibody was further determined. Results PCI-34051 significantly suppressed tumorigenicity in immunocompetent but not immunodeficient mice, which was accompanied by increased effector T cell tumor infiltration. Mechanistically, tumor-intrinsic HDAC8 epigenetically silenced chemokine CCL4 expression via H3K27 deacetylation at the enhancer to inhibit T cell infiltration to tumor. Furthermore, PCI-34051 dramatically improved the therapeutic efficacy of anti-PD-L1 checkpoint blockade, leading to eradication of large tumor. More importantly, HDAC8 and PD-L1 co-blockade resulted in long-term survival (more than 1 year) with the induction of T cell memory responses. Conclusions Our finding delineates that selective chromatin modifications by HDAC8 can augment the therapeutic efficacy of PD-L1 blockade therapy to fully unleash T cell responses, leading to long-term remission of HCC. This study highlights a new epigenetic target for immune potentiation in HCC, providing a rational combinatorial epigenetic immunotherapy.

Increasing studies have demonstrated that neuroendocrine system is involved in the development and progression of cholangiocarcinoma. The neuroendocrine hormones, neurotransmitters and neuropeptides regulate cholangiocarcinoma via affecting pathophysiology of tumor cells. The developing interaction and interplay between neuroendocrine-associated factors and tumor cells provide novel insights into neural control of tumorigenesis and reveal potential therapeutic effect on patients with cholangiocarcinoma. Herein we reviewed the latest findings and achievements which demonstrate the close interactions between neuroendocrine regulation and progression of cholangiocarcinoma. Also, future therapeutic approaches targeting neuroendocrine-associated factors are discussed which may help improve management and treatment of cholangiocarcinoma.

Background Recent clinical trials of inhibition of immune-checkpoints, programmed death-ligand 1 (PD-L1)/PD-1 axis, which elicits anti-tumour T-cell responses in various cancers, have also produced durable efficacy in a fraction of patients with advanced hepatocellular carcinoma (HCC). The heterogeneous responses to immune-checkpoint blockade therapy result from the complicated interplay between cancer-cell-autonomous cues and the immunosuppressive tumour microenvironment. Our previous epigenomics and functional analysis have elucidated an oncogenic role of histone deacetylase 8 (HDAC8) in hepatic carcinogenesis (Cancer Research 2015;75:4803–16). In this study, we aimed to investigate the therapeutic potential of an HDAC8-specific inhibitor PCI-34051 in preclinical HCC model. Methods To investigate the immune-modulatory and anti-tumour effects of PCI-34051, we established an orthotopic HCC mouse model via intrahepatic implantation of syngeneic Hepa1–6 hepatoma cells in immunocompetent C57BL/6 and immunodeficient nude mice. The mice have been treated with vehicle, PCI-34051, anti-PD-L1 antibody, or combined therapy for 2 weeks. The tumorigenicity was assessed by in vivo imaging and correlated with immune profiling. Results We demonstrated that PCI-34051 significantly reduced HCC tumorigenicity in C57BL/6 (p 300 days), which was associated with elevated CD8+T memory cells. Conclusions Our data suggest that selective chromatin modifications by HDAC8 alter the tumour immune surveillance program and demonstrate the potential of rational combinatorial epigenetic immunotherapy to fully unleash T-cell responses, leading to long-term remission of HCC.

Pioglitazone (PGZ), a synthetic PPARγ ligand, is known to have anti-tumor activity. However, it is unclear how it acts against hepatocellular carcinoma (HCC). We hypothesized that the pathological receptor for advanced glycation end products (RAGE) is involved in the PGZ anti-tumor process. To test this notion, human primary HCC tissues and corresponding adjacent non-cancerous tissues (ANCT) from 75 consecutive cases were analyzed. The expression and clinical significance of RAGE was assessed by immunohistochemical assay through tissue microarray. After HCC cells were pretreated with different concentrations of PGZ, cell proliferation, apoptosis, cell invasion, and cell cycle distribution were evaluated by multiple assays. The results showed that, the positive expression of RAGE was significantly higher in HCC tissues than in ANCT (66.7% vs. 36.0%, P < 0.001), and was closely associated with pathological staging (P = 0.014) and lymph-vascular space invasion (P = 0.003). Moreover, PGZ inhibited proliferative activity and invasive potential, and induced apoptosis and cell cycle arrest in HCC cells resulting in increased expression of PPARγ and decreased expression of RAGE, NF-κB, HMGB1, p38MAPK, Ki-67, MMP-2, and CyclinD1. Furthermore, knockdown of RAGE or NF-κB by siRNA effectively suppressed cell proliferation and invasion, and mediated the inhibitory effects of PGZ in HCC cells. Taken together, our findings suggest that, RAGE is overexpressed in human HCC tissues, and is closely associated with the pathological staging and tumor invasion of HCC. In addition, PGZ as a PPARγ agonist may inhibit growth and invasion of HCC cells via blockade of the RAGE signaling.

Metastasis is a prominent cause of cancer-related death governed by both cancer cell-intrinsic mechanisms and extrinsic microenvironment. Clinical observations demonstrated liver as a common metastatic site for various cancers, which may be due to its immune tolerant environment. Myeloid-derived suppressor cell (MDSC) is a heterogeneous cell population of immature myeloid cells that contribute to the formation of a favorable metastatic environment partially via suppression of immune effector cells. However, the underlying mechanisms in liver tropism of tumor metastasis remain poorly understood. We have previously discovered that cell cycle-related kinase (CCRK) can promote primary hepatocellular carcinoma (HCC) development via MDSCs (Gut 2018; Nat Commun accepted). Here we hypothesize that the accumulation of hepatic MDSCs induced by CCRK may contribute to the formation of a favorable metastatic liver microenvironment. By the construction of a liver-specific CCRK inducible transgenic (TG) mouse model by a Cre/loxP system, induction of CCRK expression by tamoxifen injection could increase CD11b+Gr-1+Ly6G+Ly6Clow polymorphonuclear (PMN)-MDSCs liver accumulation specifically in male mice with upregulated Cxcl1 and Gcsf expression. Intrahepatic injection of a mouse hepatoma cell line Hep1-6 in male TG mice developed larger tumors compared to control and positively associated with increased PMN-MDSCs levels in liver. Moreover, the tumorigenicity was abolished by PMN-MDSC depletion. Notably, intrasplenic injection of a mouse melanoma cell line B16F10 exhibited increased level of liver-infiltrating PMN-MDSCs and enhanced liver metastasis in male TG mice compared to control mice. Moreover, depletion of PMN-MDSCs suppressed metastasis in liver. Mechanistically, anti-tumor NKT cells, rather than NK cells and CD8+T cells, were negatively correlated with tumor weight and MDSC proportion, indicating the potential crosstalk between MDSC and NKT in liver metastasis. Our findings suggest that hepatic CCRK expression create a tumor growth- and metastasis-supportive liver microenvironment via enhancing immunosuppression. Additional colorectal cancer metastasis models will be evaluated in our established TG mice. Moreover, the roles and underlying mechanisms of CCRK-PMN-MDSC interactions in the establishment of liver metastasis await further investigation. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309. Citation Format: Xuezhen ZENG, Jingying Zhou, Zhewen Xiong, Hanyong Sun, Weiqin Yang, Wenshu Tang, Yu Feng, Alfred Sze-Lok Cheng. Hepatic cell cycle-related kinase shapes a metastatic-prone liver microenvironment via crosstalk between myeloid-derived suppressor cell and natural killer T cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1485.

Gankyrin is a critical oncoprotein overexpressed in human hepatocellular carcinoma (HCC). However, the mechanism underlying gankyrin-mediated hepatocarcinogenesis remains elusive. Herein, we provide evidence that gankyrin expression was progressively elevated in liver fibrosis, cirrhosis, and HCC. Levels of gankyrin expression were closely associated with the dedifferentiation status of hepatoma in patients. Decrease of hepatocyte characteristic markers and increase of cholangiocyte-specific markers were observed in rat primary hepatocytes with enforced gankyrin expression and diethylnitrosamine (DEN)-triggered rat hepatocarcinogenesis. Overexpression of gankyrin also attenuated the hepatic function of primary hepatocytes, which further suggests that gankyrin promotes the dedifferentiation of hepatocytes. Moreover, elevated expression of gankyrin closely correlated with the expression of HCC stem/progenitor cell markers in DEN-triggered hepatocarcinogenesis and human HCCs. Hepatoma cells derived from suspension-cultured spheroids exhibited a higher gankyrin level, and enforced gankyrin expression in hepatoma cells remarkably enhanced cluster of differentiation (CD)133, CD90, and epithelial cellular adhesion molecule expression, indicating a role of gankyrin in hepatoma cell dedifferentiation and the generation of hepatoma stem/progenitor cells. In contrast, down-regulation of gankyrin in hepatoma cells by lentivirus-mediated microRNA delivery significantly improved their differentiation status and attenuated malignancy. Interference of gankyrin expression in hepatoma cells also diminished the proportion of cancer stem/progenitor cells and their self-renewal capacity. Furthermore, gankyrin was found to bind hepatocyte nuclear factor 4α (HNF4α), which determines hepatocyte differentiation status and enhances proteasome-dependent HNF4α degradation in hepatoma cells. The inverse correlation of gankyrin and HNF4α was further confirmed in primary hepatocytes, DEN-induced hepatocarcinogenesis, and human HCCs. Conclusion: Gankyrin-mediated dedifferentiation of hepatocytes and hepatoma cells via, at least partially, down-regulation of HNF4α facilitates HCC development, and interference of gankyrin expression could be a novel strategy for HCC prevention and differentiation therapy. (HEPATOLOGY 2011;54:1259–1272)

One of the challenges surrounding nonalcoholic fatty liver disease (NAFLD) is to discover the mechanisms that underlie the initiation of it. The aim of the present study was to elucidate the effects of Toll-like receptor 4 (TLR4) signaling in liver parenchymal cells during the early stage of NAFLD. Male TLR4-wildtype, TLR4-knockout, TLR2-knockout, MyD88-knockout, and TRIF-knockout mice were fed a normal diet or high-fat diet (HFD). Liver steatosis, alanine aminotransferase levels, nuclear translocation of nuclear factor kappa B (NF-κB) (p65), macrophage accumulation, and neutrophil infiltration were assessed. Using Kupffer cell depletion or bone marrow transplantation, we examined the potential role of Kupffer cells and myeloid infiltrating cells during the initiation of NAFLD. Immunohistochemistry and western blotting were implemented to determine the release of high-mobility group box1 (HMGB1). The neutral-antibody against HMGB1 was used to block the activity of free HMGB1. Here we report that the activation of TLR4 signaling in hepatocytes, accompanied with the relocation of P65 in nucleus, was proven to play an important role during the initiation of NAFLD. Importantly, HMGB1 releasing from hepatocytes in response to free fatty acid (FFA) infusion was first reported as the key molecule for the TLR4/MyD88 activation and cytokines expression in vitro and in vivo. Treatment with neutralizing antibody to HMGB1 protects against FFA-induced tumor necrosis factor alpha and interleukin-6 production. Conclusion: Our study supports the notion that TLR4/MyD88 signaling in liver parenchymal cells plays a pivotal role during the early progression of HFD-induced NAFLD, in which free HMGB1 served as a positive component mediating TLR4 activation. (HEPATOLOGY 2011;)

Background & Aims Reactive oxygen species (ROS) are considered to play a prominent causative role in the development of various hepatic disorders. Antioxidants have been effectively demonstrated to protect against hepatic damage. Hydrogen (H 2 ), a new antioxidant, was reported to selectively reduce the strongest oxidants, such as hydroxyl radicals ( OH) and peroxynitrite (ONOO − ), without disturbing metabolic oxidation–reduction reactions or disrupting ROS involved in cell signaling. In place of H 2 gas, hydrogen-rich saline (HS) may be more suitable for clinical application. We herein aim to verify its protective effects in experimental models of liver injury. Methods H 2 concentration in vivo was detected by hydrogen microelectrode for the first time. Liver damage, ROS accumulation, cytokine levels, and apoptotic protein expression were, respectively, evaluated after GalN/LPS, CCl 4 , and DEN challenge. Simultaneously, CCl 4 -induced hepatic cirrhosis and DEN-induced hepatocyte proliferation were measured. Results HS significantly increased hydrogen concentration in liver and kidney tissues. As a result, acute liver injury, hepatic cirrhosis, and hepatocyte proliferation were reduced through the quenching of detrimental ROS. Activity of pro-apoptotic players, such as JNK and caspase-3, were also inhibited. Conclusions HS could protect against liver injury and also inhibit the processes leading to liver cirrhosis and hepatocyte compensatory proliferation.

The growing epidemic of obesity, which causes nonalcoholic fatty liver disease (NAFLD) and the more severe phenotype nonalcoholic steatohepatitis (NASH), has paralleled the increasing incidence of hepatocellular carcinoma (HCC). Accumulating evidence demonstrates that overnutrition and metabolic pathways can trigger modifications of DNA and histones via deregulation of chromatin modifiers, resulting in aberrant transcriptional activity. However, the epigenetic regulation of HCC development in NAFLD remains obscure. Here, we uncover key epigenetic regulators using both dietary and genetic obesity-promoted HCC models through quantitative expression profiling and characterize the oncogenic activities of histone deacetylase HDAC8 in NAFLD-associated hepatocarcinogenesis. HDAC8 is directly upregulated by the lipogenic transcription factor SREBP-1 where they are coexpressed in dietary obesity models of NASH and HCC. Lentiviral-mediated HDAC8 attenuation in vivo reversed insulin resistance and reduced NAFLD-associated tumorigenicity. HDAC8 modulation by genetic and pharmacologic approaches inhibited p53/p21-mediated apoptosis and G2–M phase cell-cycle arrest and stimulated β-catenin–dependent cell proliferation. Mechanistically, HDAC8 physically interacted with the chromatin modifier EZH2 to concordantly repress Wnt antagonists via histone H4 deacetylation and H3 lysine 27 trimethylation. In human NAFLD-associated HCC, levels of SREBP-1, HDAC8, EZH2, H4 deacetylation, H3K27me3, and active β-catenin were all correlated positively in tumors compared with nontumor tissues. Overall, our findings show how HDAC8 drives NAFLD-associated hepatocarcinogenesis, offering a novel epigenetic target to prevent or treat HCC in obese patients. Cancer Res; 75(22); 4803–16. ©2015 AACR.

Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of immature myeloid cells that suppress anti-tumor immune responses. The accumulation of CD33+HLA-DR- MDSCs correlates with tumor stage and metastatic burden in various cancers including hepatocellular carcinoma (HCC) (1). Cancer cells can secrete a variety of cytokines and chemokines to facilitate the peripheral expansion and tumor infiltration of MDSCs. However, the cancer cell-specific signaling cascades that promote MDSC expansion remain poorly understood. We previously demonstrated that cell cycle-related kinase (CCRK) acts as a new oncogenic signaling hub in hepatocellular proliferation and transformation (2-4). To investigate whether CCRK regulates tumor microenvironment in HCC, we determined the role of CCRK signaling in the crosstalk between cancer cells and MDSCs. Our results showed that treatment of human peripheral blood mononuclear cells (PBMCs) with conditional medium of CCRK-over-expressing immortalized hepatocytes and HCC cells resulted in expansion of CD33+ HLA-DR-MDSCs with increased inhibitory effects on T cell responses. In contrast, knockdown of CCRK in hepatic cells downregulated MDSCs. Cytokine profiling analysis revealed that CCRK significantly induced hepatocellular interferon-6 (IL-6) expression and release, which mediated MDSC expansion as shown by IL-6 interfering experiments. Mechanistic studies demonstrated that CCRK triggered nuclear factor-kappa B (NF-κB) signaling in an enhancer of zeste homolog 2 (EZH2)-dependent manner. Simultaneously, the phosphorylation of p65 by CCRK facilitated the co-occupancy of IL-6 promoter by NF-κB-EZH2 complex for transcriptional activation. Using a Hepa1-6 orthotopic HCC model in immune-competent C57BL/6J mice, we showed that knockdown of Ccrk significantly decreased hepatic tumorigenicity and the levels of circulating and tumor-infiltrating MDSCs as well as related T cell responses. Notably, adoptive transfer of MDSCs rescued the effects of Ccrk knockdown. In a complementary experiment, we found that MDSC depletion by anti-Gr-1 antibody significantly reduced CCRK-induced tumorigenicity. Furthermore, transgenic over-expression of CCRK in mouse liver led to activation of EZH2/NF-κB signaling and elevated circulating levels of MDSC. As we also showed concordant over-expression of CCRK, IL-6 and MDSC markers (CD33, Arg-I) in human HCCs, our results uncover CCRK to be a critical immune regulator to promote MDSC, thereby providing a new mechanistic link between aberrant oncogenic signaling and tumor evasion for therapeutic exploitation. Acknowledgement: This project was supported by the University Grants Committee through the Collaborative Research Fund C4017-14G. References 1) Marigo I., et al., 2010. Immunity. 2) Feng H., et al., 2011. J Clin Invest. 3) Yu Z., et al., 2014. Gut. 4) Feng H., et al., 2015. J Hepatol. Citation Format: Jingying Zhou, Man Liu, Hanyong Sun, Zhiwei Chen, Alfred Sze Lok Cheng. A novel kinase connection between oncogenic signaling and myeloid-derived suppressor cell functions in tumor evasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 529.

Background Oxidative stress has been implicated in the pathogenesis of a wide spectrum of human diseases, including Hepatitis B virus (HBV)-related liver disease. Hepatitis B virus X protein (HBx) is a key regulator of HBV that exerts pleiotropic activity on cellular functions. Recent studies showed that HBx alters mitochondrial membrane potential, thereby sensitizing cells to pro-apoptotic signals. However, it remains largely unknown whether susceptibility of hepatocytes could be disturbed by HBx under oxidative stress conditions. The purpose of this study is to determine the apoptotic susceptibility of HBx-expressing hepatocytes upon exposure to pro-oxidant stimuli in vitro and in vivo and explore its underlying mechanism. Results Although expression of HBx itself did not activate apoptotic signaling, it significantly enhanced oxidative stress-induced cell death both in vitro and in vivo. Interestingly, this phenomenon was associated with a pronounced reduction of protein levels of Mcl-1, but not other anti-apoptotic Bcl-2 members. Importantly, enforced expression of Mcl-1 prevented HBx-triggered cell apoptosis; conversely, specific knockdown of Mcl-1 exacerbated HBx-induced apoptosis upon exposure to oxidative stress. Furthermore, inhibition of caspase-3 not only abrogated HBx-triggered apoptotic killing but also blocked HBx-induced Mcl-1 loss. Additionally, expression of HBx and Mcl-1 was found to be inversely correlated in HBV-related hepatocellular carcinogenesis (HCC) tissues. Conclusions Our findings indicate that HBx exerts pro-apoptotic effect upon exposure to oxidative stress probably through accelerating the loss of Mcl-1 protein via caspase-3 cascade, which may shed a new light on the molecular mechanism of HBV-related hepatocarcinogenesis.

Hepatitis B virus X protein (HBx) is implicated in the pathogenesis of hepatitis B virus (HBV)-associated liver diseases. However, whether HBx has the ability to disturb the susceptibility of hepatocytes to common chemotherapeutic agents remains incompletely understood. Here we demonstrate that HBx enhances cisplatin-induced hepatotoxicity by a mechanism involving degradation of Mcl-1, an antiapoptotic member of the Bcl-2 family. Ectopic expression of HBx sensitized hepatocytes to cisplatin-induced apoptosis, which was accompanied by a marked downregulation of Mcl-1 but not of Bcl-2 or Bcl-xL. Overexpression of Mcl-1 prevented HBx-induced proapoptotic and proinflammatory effects during cisplatin treatment both in vitro and in vivo . HBx-induced dysregulation of Mcl-1 resulted mainly from posttranslational degradation rather than transcription repression. Moreover, a caspase-3 inhibitor effectively abrogated HBx-enhanced Mcl-1 degradation and cell death. Importantly, antioxidants blocked activation of caspase-3 and acceleration of Mcl-1 loss, as well as cell death, in HBx-expressing hepatocytes upon cisplatin exposure in vitro and in vivo . Collectively, these data implicate oxidative stress-dependent caspase-3-mediated degradation of Mcl-1 as a mechanism contributing to HBx-mediated sensitization of cisplatin-induced hepatotoxicity. A combination of cisplatin and antioxidants might provide more advantage than cisplatin alone in the treatment of cancer patients with chronic HBV infection.