VSTM2A reverses immunosuppression in colorectal cancer by antagonizing the PD-L1/PD-1 interaction.

Immunoglobulin (Ig) VSTM2A (V-set and transmembrane domain containing 2A) is a top-ranked secretory protein frequently silenced during colorectal carcinogenesis; however, its role in immune modulation remains largely unknown. Bioinformatic and immunohistochemistry analysis of human colorectal specimens and Vstm2a+/- knockout mice indicated that VSTM2A positively correlated with CD8a and immune infiltration in both physiological and pathological conditions. We then utilized liquid chromatography-mass spectrometry to pinpoint programmed death ligand 1 (PD-L1) as a membrane receptor of VSTM2A. A series of in vitro biochemistry assays further revealed the binding pattern and kinetics between VSTM2A and PD-L1 proteins through their IgV domains at a dissociation constant of 0.7-2.5 nM. Recombinant VSTM2A protein inhibited the PD-1/PD-L1 interaction and induced NFAT response element (RE) luciferase activity dose dependently. Furthermore, interleukin (IL)-2 production from DO11.10 T cells upon co-culture with mouse non-T splenocytes was upregulated in the presence of VSTM2A conditioned medium. Finally, tumor killing assay and ex vivo data from human peripheral blood mononuclear cells and autologous dendritic cell-T cell co-culture demonstrated that VSTM2A significantly enhanced immune activation via the release of granzyme B and interferon (IFN)-γ cytokines. In conclusion, our study demonstrates the tumor-extrinsic role of VSTM2A in sterically blocking the PD-L1/PD-1 interaction at a picomole to nanomole affinity, which leads to the enhanced anti-tumor effect of cytotoxic T cells.

TP53 R249S mutation in hepatic organoids captures the predisposing cancer risk.

BACKGROUND AND AIMS: Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor-suppressive function from TP53 deficiency and gain-of-function activities from mutant p53. APPROACH AND RESULTS: Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor-like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP-seq analysis of HCC cell lines underscored gain-of-function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome-edited liver organoids, quantitative polymerase chain reaction corroborated ChIP-seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. CONCLUSIONS: We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

TUBB4B is a novel therapeutic target in non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

Non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) is an emerging malignancy due to the rising prevalence of NAFLD. However, no drug is available to target NAFLD-HCC. In this study, we aim to unravel novel therapeutic targets of NAFLD-HCC utilizing a high-throughput CRISPR/Cas9 screening strategy. We utilized the Epi-drug CRISPR/Cas9 library consisting of single-guide RNAs (sgRNAs) targeting over 1,000 genes representing the FDA-approved drug targets and epigenetic regulators to perform loss-of-function screening in two NAFLD-HCC cell lines (HKCI2 and HKCI10). CRISPR/Cas9 library screening unraveled TUBB4B as an essential gene for NAFLD-HCC cell growth. TUBB4B was overexpressed in NAFLD-HCC tumors compared with adjacent normal tissues (N = 17) and was associated with poor survival (p < 0.01). RNA-sequencing and functional assays revealed that TUBB4B knockout in NAFLD-HCC promoted cell apoptosis, cell cycle arrest, and cellular senescence, leading to suppressed NAFLD-HCC growth in vitro and in vivo. We identified that TUBB4B inhibitor mebendazole (MBZ), an FDA-approved drug, inhibited NAFLD-HCC growth by inducing apoptosis and cellular senescence. Since protein expression of pro-survival Bcl-xL was induced in TUBB4B knockout NAFLD-HCC cells, we examined combination of TUBB4B inhibition with navitoclax, a Bcl-xL inhibitor that selectively targets senescent cells. Consistent with our hypothesis, either TUBB4B knockout or MBZ synergized with navitoclax to inhibit NAFLD-HCC cell growth via the induction of intrinsic and extrinsic apoptosis pathways. In summary, TUBB4B is a novel therapeutic target in NAFLD-HCC. Inhibition of TUBB4B with MBZ in combination with navitoclax synergistically inhibited NAFLD-HCC cell growth, representing a promising strategy for the treatment of NAFLD-HCC. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Bifidobacterium pseudolongum-generated acetate suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

BACKGROUND & AIMS: Recent studies have highlighted the role of the gut microbiota and their metabolites in non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). We aimed to identify specific beneficial bacterial species that could be used prophylactically to prevent NAFLD-HCC. METHODS: The role of Bifidobacterium pseudolongum was assessed in two mouse models of NAFLD-HCC: diethylnitrosamine + a high-fat/high-cholesterol diet or + a choline-deficient/high-fat diet. Germ-free mice were used for the metabolic study of B. pseudolongum. Stool, portal vein and liver tissues were collected from mice for non-targeted and targeted metabolomic profiles. Two human NAFLD-HCC cell lines (HKCI2 and HKCI10) were co-cultured with B. pseudolongum-conditioned media (B.p CM) or candidate metabolites. RESULTS: B. pseudolongum was the top depleted bacterium in mice with NAFLD-HCC. Oral gavage of B. pseudolongum significantly suppressed NAFLD-HCC formation in two mouse models (p < 0.01). Incubation of NAFLD-HCC cells with B.p CM significantly suppressed cell proliferation, inhibited the G1/S phase transition and induced apoptosis. Acetate was identified as the critical metabolite generated from B. pseudolongum in B.p CM, an observation that was confirmed in germ-free mice. Acetate inhibited cell proliferation and induced cell apoptosis in NAFLD-HCC cell lines and suppressed NAFLD-HCC tumor formation in vivo. B. pseudolongum restored heathy gut microbiome composition and improved gut barrier function. Mechanistically, B. pseudolongum-generated acetate reached the liver via the portal vein and bound to GPR43 (G coupled-protein receptor 43) on hepatocytes. GPR43 activation suppressed the IL-6/JAK1/STAT3 signaling pathway, thereby preventing NAFLD-HCC progression. CONCLUSIONS: B. pseudolongum protected against NAFLD-HCC by secreting the anti-tumor metabolite acetate, which reached the liver via the portal vein. B. pseudolongum holds potential as a probiotic for the prevention of NAFLD-HCC. IMPACT AND IMPLICATIONS: Non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) is an increasing healthcare burden worldwide. There is an urgent need to develop effective agents to prevent NAFLD-HCC progression. Herein, we show that the probiotic Bifidobacterium pseudolongum significantly suppressed NAFLD-HCC progression by secreting acetate, which bound to hepatic GPR43 (G coupled-protein receptor 43) via the gut-liver axis and suppressed the oncogenic IL-6/JAK1/STAT3 signaling pathway. Bifidobacterium pseudolongum holds potential as a novel probiotic for NAFLD-HCC prevention.

Unique molecular characteristics of NAFLD-associated liver cancer accentuate ß-catenin/TNFRSF19-mediated immune evasion.

BACKGROUND & AIMS: The hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), can lead to the development of hepatocellular carcinoma (HCC). Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations. METHODS: Herein, tumor-normal pairs from 100 patients diagnosed with NAFLD-HCC were subject to next-generation sequencing. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of the underlying CTNNB1 S45P driver mutation. A syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mutand TNFRSF19 in reshaping the tumor microenvironment. RESULTS: Mutational processes operative in the livers of patients with NAFLD inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. Chromatin immunoprecipitation-sequencing integrated with transcriptome and immune profiling revealed a unique transcriptional axis, wherein CTNNB1mut leads to an upregulation of TNFRSF19 which subsequently represses senescence-associated secretory phenotype-like cytokines (including IL6 and CXCL8). This phenomenon could be reverted by the Wnt-modulator ICG001. CONCLUSIONS: The unique mutational processes in the livers of patients with NAFLD and NAFLD-HCC allude to a "field effect" involving a gain-of-function role of CTNNB1 mutations in immune exclusion. LAY SUMMARY: The increasing prevalence of metabolic syndrome in adult populations means that NAFLD is poised to be the major cause of liver cancer in the 21st century. We showed a strong "field effect" in the livers of patients with NAFLD, wherein activated ß-catenin was involved in reshaping the tumor-immune microenvironment.

Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

BACKGROUNDS & AIMS: Squalene epoxidase (SQLE) is the rate-limiting enzyme for cholesterol biosynthesis. We elucidated the functional significance, molecular mechanisms, and clinical impact of SQLE in nonalcoholic steatohepatitis (NASH). METHODS: We performed studies with hepatocyte-specific Sqle overexpression transgenic (Sqle tg) mice and mice given high-fat high-cholesterol (HFHC) or methionine- and choline-deficient (MCD) diet to induce NASH. SQLE downstream target carbonic anhydrase III (CA3) was identified using co-immunoprecipitation and Western Blot. Some mice were given SQLE inhibitor (terbinafine) and CA3 inhibitor (acetazolamide) to study the therapeutic effects in NASH. Human samples (N = 217) including 65 steatoses, 80 NASH, and 72 healthy controls were analyzed for SQLE levels in liver tissue and in serum. RESULTS: SQLE is highly up-regulated in human NASH and mouse models of NASH. Sqle tg mice triggered spontaneous insulin resistance, hepatic steatosis, liver injury, and accelerated HFHC or MCD diet-induced NASH development. Mechanistically, SQLE tg mice caused hepatic cholesterol accumulation, thereby triggering proinflammatory nuclear factor-κB signaling and steatohepatitis. SQLE directly bound to CA3, which induced sterol regulatory element-binding protein 1C activation, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase1 expression and de novo hepatic lipogenesis. Combined targeting SQLE (terbinafine) and CA3 (acetazolamide) synergistically ameliorated NASH in mice with superior efficacy to either drug alone. Serum SQLE with CA3 could distinguish patients with NASH from steatosis and healthy controls (area under the receiver operating characteristic curve, 0.815; 95% confidence interval, 0.758-0.871). CONCLUSIONS: SQLE drives the initiation and progression of NASH through inducing cholesterol biosynthesis, and SQLE/CA3 axis-mediated lipogenesis. Combined targeting of SQLE and CA3 confers therapeutic benefit in NASH. Serum SQLE and CA3 are novel biomarkers for the noninvasive diagnosis of patients with NASH.

A virome-wide clonal integration analysis platform for discovering cancer viral etiology.

Oncoviral infection is responsible for 12%-15% of cancer in humans. Convergent evidence from epidemiology, pathology, and oncology suggests that new viral etiologies for cancers remain to be discovered. Oncoviral profiles can be obtained from cancer genome sequencing data; however, widespread viral sequence contamination and noncausal viruses complicate the process of identifying genuine oncoviruses. Here, we propose a novel strategy to address these challenges by performing virome-wide screening of early-stage clonal viral integrations. To implement this strategy, we developed VIcaller, a novel platform for identifying viral integrations that are derived from any characterized viruses and shared by a large proportion of tumor cells using whole-genome sequencing (WGS) data. The sensitivity and precision were confirmed with simulated and benchmark cancer data sets. By applying this platform to cancer WGS data sets with proven or speculated viral etiology, we newly identified or confirmed clonal integrations of hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), and BK Virus (BKV), suggesting the involvement of these viruses in early stages of tumorigenesis in affected tumors, such as HBV in TERT and KMT2B (also known as MLL4) gene loci in liver cancer, HPV and BKV in bladder cancer, and EBV in non-Hodgkin's lymphoma. We also showed the capacity of VIcaller to identify integrations from some uncharacterized viruses. This is the first study to systematically investigate the strategy and method of virome-wide screening of clonal integrations to identify oncoviruses. Searching clonal viral integrations with our platform has the capacity to identify virus-caused cancers and discover cancer viral etiologies.

Personalized treatment for hepatocellular carcinoma: Current status and future perspectives.

Systemic treatment for hepatocellular carcinoma (HCC) has been advancing rapidly over the last decade. More novel agents, including both targeted agents and immune checkpoint inhibitors, are available for physicians to use sequentially or concurrently for patients with advanced HCC. Despite more options, only a proportion of patients benefit from each regimen. Therefore, clinicians are facing challenges on how to choose the right regimen for the right patient with HCC, which raises the importance of personalized treatment approach. To advance personalized treatment for HCC, one approach relies on the acquisition of biomarker data from clinical trials to evaluate clinical parameters or genotypes in association with outcomes of selected drugs. This approach has led to finding of high baseline alpha-fetoprotein levels in association with benefits of ramucirumab. Cumulative findings from multiple clinical trials and translational studies also suggest that selected etiology and/or genotype of HCC could predict resistance to immune checkpoint inhibitors. The second approach is to decipher the tumor heterogeneity of HCC with an aim to identify clinically relevant patterns to guide clinical decisions. Tumor heterogeneity could exist within a single tumor (intra-tumoral heterogeneity), among different tumors in the same patient (inter-tumoral heterogeneity) or between primary and recurrent tumors (temporal tumor heterogeneity). The analyses of tumor heterogeneity have also been powered by coverage of tumor immune environment and incorporation of circulating tumor nucleic acid technology. Emerging publications have been reported above tumor heterogeneity exist in HCC, which is potentially clinically impactful.

Manipulation of the Nanoscale Presentation of Integrin Ligand Produces Cancer Cells with Enhanced Stemness and Robust Tumorigenicity.

Developing strategies for efficient expansion of cancer stem-like cells (CSCs) in vitro will help investigate the mechanism underlying tumorigenesis and cancer recurrence. Herein, we report a dynamic culture substrate tethered with integrin ligand-bearing magnetic nanoparticles via a flexible polymeric linker to enable magnetic manipulation of the nanoscale ligand tether mobility. The cancer cells cultured on the substrate with high ligand tether mobility develop into large semispherical colonies with CSCs features, which can be abrogated by magnetically restricting the ligand tether mobility. Mechanistically, the substrate with high ligand tether mobility suppresses integrin-mediated mechanotransduction and histone-related methylation, thereby enhancing cancer cell stemness. The culture-derived high-stemness cells can generate tumors both locally and at the distant lung and uterus much more efficiently than the low-stemness cells. We believe that this magnetic nanoplatform provides a promising strategy for investigating the dynamic interaction between CSCs and the microenvironment and establishing a cost-effective tumor spheroid model.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas.

BACKGROUND & AIMS: Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. METHODS: Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. RESULTS: We determined the monoclonal origins of individual tumors using a single-sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phyloepigenetic analyses showed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis showed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features showed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin of recurrent HCCs allowed construction of a temporal map of genetic alterations that were associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that are associated with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution showed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the cancer genome atlas data set. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. CONCLUSIONS: We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that are associated with tumor progression and recurrence. We identified chromatin regulators that are up-regulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas.

BACKGROUND & AIMS: Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. METHODS: Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. RESULTS: We determined the monoclonal origins of individual tumors using a single sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phylo-epigenetic analyses revealed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis revealed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features revealed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin if recurrent HCCs allowed construction of a temporal map of genetic alterations that associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that associate with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution revealed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the Cancer Genome Atlas dataset. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. CONCLUSIONS: We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that associate with tumor progression and recurrence. We identified chromatin regulators that are upregulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Long-Read RNA Sequencing Identifies Alternative Splice Variants in Hepatocellular Carcinoma and Tumor-Specific Isoforms.

Alternative splicing (AS) allows generation of cell type-specific mRNA transcripts and contributes to hallmarks of cancer. Genome-wide analysis for AS in human hepatocellular carcinoma (HCC), however, is limited. We sought to obtain a comprehensive AS landscape in HCC and define tumor-associated variants. Single-molecule real-time long-read RNA sequencing was performed on patient-derived HCC cells, and presence of splice junctions was defined by SpliceMap-LSC-IDP algorithm. We obtained an all-inclusive map of annotated AS variants and further discovered 362 alternative spliced variants that are not previously reported in any database (neither RefSeq nor GENCODE). They were mostly derived from intron retention and early termination codon with an in-frame open reading frame in 81.5%. We corroborated many of these predicted unannotated and annotated variants to be tumor specific in an independent cohort of primary HCC tumors and matching nontumoral liver. Using the combined Sanger sequencing and TaqMan junction assays, unique and common expressions of spliced variants including enzyme regulators (ARHGEF2, SERPINH1), chromatin modifiers (DEK, CDK9, RBBP7), RNA-binding proteins (SRSF3, RBM27, MATR3, YBX1), and receptors (ADRM1, CD44v8-10, vitamin D receptor, ROR1) were determined in HCC tumors. We further focused functional investigations on ARHGEF2 variants (v1 and v3) that arise from the common amplified site chr.1q22 of HCC. Their biological significance underscores two major cancer hallmarks, namely cancer stemness and epithelial-to-mesenchymal transition-mediated cell invasion and migration, although v3 is consistently more potent than v1. Conclusion: Alternative isoforms and tumor-specific isoforms that arise from aberrant splicing are common during the liver tumorigenesis. Our results highlight insights gained from the analysis of AS in HCC.

Genomic landscape of lymphoepithelioma-like hepatocellular carcinoma.

Lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is a distinct variant of HCC that is characterized by dense tumor-infiltrating lymphocytes (TILs). Patients with LEL-HCC also show better clinical outcomes compared to conventional HCC (c-HCC), which is commonly presented with low TIL. Emerging evidence has begun to highlight tumor-intrinsic genetic abnormalities in the tumor-host immune interfaces. However, genome-wide characterization of LEL-HCC remains largely unexplored. Here, we defined the genomic landscape of 12 LEL-HCC using whole-exome sequencing, and further underpinned those genetic alterations related to an immune active microenvironment by comparing findings to 15 c-HCC that were sequenced in parallel. Overall, the mutational load between LEL-HCC and c-HCC was similar. Interestingly, SNV incidences of specific genes (CTNNB1, AXIN1, NOTCH1, and NOTCH2) were significantly higher in c-HCC than LEL-HCC, suggesting a plausible link between activated Wnt/ß-catenin and Notch signaling pathways and immune avoidance. Marked focal amplification of chromosome 11q13.3 was prevalent in LEL-HCC. Using The Cancer Genome Atlas dataset, we further established oncogenes expressed from chromosome 11q13.3 (CCND1, FGF19, and FGF4) to be strongly associated with the immune checkpoint signature (CD274, PDCD1, BTLA, CTLA4, HAVCR2, IDO1, and LAG3). Our results have illustrated for the first time the somatic landscape of LEL-HCC, and highlighted molecular alterations that could be exploited in combinatory therapy with checkpoint inhibitors in targeting HCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A comparability study of immunohistochemical assays for PD-L1 expression in hepatocellular carcinoma.

Programmed death ligand 1 (PD-L1) protein expression by immunohistochemistry is a promising biomarker for PD-1/PD-L1 blockade in hepatocellular carcinoma. There are a number of commercially available PD-L1 assays. Our study aimed to compare the analytical performance of different PD-L1 assays and evaluate the reliability of pathologists in PD-L1 scoring. Consecutive sections from tumor samples from 55 patients with surgically resected primary hepatocellular carcinoma were stained with four standardized PD-L1 assays (22C3, 28-8, SP142, and SP263). We also correlated the PD-L1 protein level by immunohistochemistry with the mRNA level of those genes associated with tumor immune microenvironment by the NanoString platform. Five pathologists independently assessed PD-L1 expression on tumor cells [tumor proportion score] together with tumor-infiltrating immune cells (combined positive score). The 22C3, 28-8, and SP263 assays had comparable sensitivity in detecting PD-L1 expression, whereas the SP142 assay was the least sensitive assay. The inter-assay agreement measured by intraclass correlation coefficients for the tumor proportion score and combined positive score were 0.646 and 0.780, respectively. The inter-rater agreement was good to excellent (the overall intraclass correlation coefficient for the tumor proportion score and combined positive score was 0.946 and 0.809, respectively). Pathologists were less reliable in scoring combined positive score than tumor proportion score, particularly when using the SP142 assay. Up to 18% of samples were misclassified by individual pathologists in comparison to the consensus score at the cutoff of combined positive score ≥ 1. The combined positive score by the 22C3 assay demonstrated the strongest correlation with immune-related gene mRNA signatures, closely followed by combined positive scores by the 28-8 and SP263 assays. In conclusion, the 22C3, 28-8, and SP263 assays are highly concordant in PD-L1 scoring and suggest the interchangeability of these three assays. Further improvement of the accuracy in assessing PD-L1 expression at a low cutoff is still necessary.

O-GlcNAc transferase promotes fatty liver-associated liver cancer through inducing palmitic acid and activating endoplasmic reticulum stress.

BACKGROUND & AIMS: O-GlcNAc transferase (OGT) is a unique glycosyltransferase involved in metabolic reprogramming. We investigated the functional role of OGT in non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). METHODS: The biological function of OGT in NAFLD-HCC was determined by gain- or loss- of OGT functional assays in vitro and in nude mice. OGT target factors and pathways were identified by liquid chromatography-tandem mass spectrometry (LC-MS), promoter luciferase assay, DNA binding activity assay and Western blot. RESULTS: OGT was upregulated in 12 out of 18 (66.7%) NAFLD-HCC tumor tissues by transcriptome sequencing, which was confirmed in additional NAFLD-HCC tumor tissues and cell lines. Biofunctional investigation demonstrated that OGT significantly increased cell growth (p<0.001), clonogenicity (p<0.01), migration and invasion (p<0.05) ability in vitro, and promoted xenograft tumor growth as well as lung metastasis in nude mice. The oncogenic effect of OGT was investigated, we found that OGT significantly induced palmitic acid production identified by LC-MS, which enhanced the protein expression of endoplasmic reticulum (ER) stress masters of glucose-regulated protein 78 and inositol-requiring enzyme 1α. Consequently, OGT significantly activated JNK/c-jun/AP-1 cascade by increasing protein expression of p-JNK, p-c-Jun and activation of AP-1; and induced NF-κB pathway through enhancing the protein levels of p-IKKα/ p-IKKß, p-p65, p-p50 and the NF-κB DNA binding activity. Notably, OGT inhibition by its antagonist (ST045849) suppressed cell proliferation in vitro (p<0.001) and in xenograft mice models (p<0.05). CONCLUSIONS: OGT plays an oncogenic role in NAFLD-associated HCC through regulating palmitic acid and inducing ER stress, consequently activating oncogenic JNK/c-jun/AP-1 and NF-κB cascades. LAY SUMMARY: OGT, a unique glycosyltransferase enzyme, was identified to be upregulated in non-alcoholic fatty liver disease-associated hepatocellular carcinoma tissues by transcriptome sequencing. Here, we found that OGT plays a role in cancer by promoting tumor growth and metastasis in both cell models and animal models. This effect is mediated by the induction of palmitic acid.

Octamer 4/microRNA-1246 signaling axis drives Wnt/ß-catenin activation in liver cancer stem cells.

Wnt/ß-catenin signaling is activated in CD133 liver cancer stem cells (CSCs), a subset of cells known to be a root of tumor recurrence and therapy resistance in hepatocellular carcinoma (HCC). However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human microRNA (miRNA), miR-1246, promotes cancer stemness, including self-renewal, drug resistance, tumorigencity, and metastasis, by activation of the Wnt/ß-catenin pathway through suppressing the expression of AXIN2 and glycogen synthase kinase 3ß (GSK3ß), two key members of the ß-catenin destruction complex. Clinically, high endogenous and circulating miR-1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified octamer 4 (Oct4) to be the direct upstream regulator of miR-1246, which cooperatively drive ß-catenin activation in liver CSCs. CONCLUSION: These findings uncover the noncanonical regulation of Wnt/ß-catenin in liver CSCs by the Oct4/miR-1246 signaling axis, and also provide a novel diagnostic marker as well as therapeutic intervention for HCC. (Hepatology 2016;64:2062-2076).

Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

Loss of ATOH8 Increases Stem Cell Features of Hepatocellular Carcinoma Cells.

BACKGROUND & AIMS: Levels of atonal homolog 8 (ATOH8) are reduced in 48% of hepatitis B virus-associated hepatocellular carcinoma cells (HCCs). ATOH8 downregulation is associated with loss of tumor differentiation, indicating an effect mediated by cancer stem cells. We investigated the effects of loss of ATOH8 in human hepatocellular carcinoma (HCC) cells and cell lines. METHODS: HCC and adjacent nontumor tissues were collected, from 2001 through 2012, from 242 patients undergoing hepatectomy at Sun Yat-Sen University Cancer Center in China; 83% of HCCs were associated with hepatitis B virus (HBV) infection. CD133(+) cells were isolated from tumor tissues by flow cytometry. Experiments were performed in HBV-positive and HBV-negative HCC cell lines, the immortalized liver cell line LO2, and 8 other HCC cell lines. ATOH8 was expressed from lentiviral vectors in PLC8024 and Huh7 cells; levels were knocked down with small interfering RNAs in QSG7701 cells. Cells carrying empty vectors were used as controls. Gene regulation by ATOH8 was assessed in mobility shift and luciferase reporter assays. Cells were analyzed in proliferation, foci formation, and colony formation assays. The tumorigenic and chemo-resistant potential of cells were investigated by assessing growth of xenograft tumors in immunocompromised mice. Metastatic features of cells were assessed in Matrigel invasion assays and wound healing analyses. RESULTS: Levels of ATOH8 mRNA were reduced by more than 4-fold, compared to nontumor tissues, in 118 of 242 HCC samples (48.8%). Patients with tumor reductions in ATOH8 had significantly shorter times of disease-free survival (mean, 41.4 months) than patients with normal tissue levels (mean, 52.6 months). ATOH8 expression was reduced in HepG2, Huh7, PLC8024 and CRL8064 HCC cells, as well as CD133(+) cells isolated from human HCC samples. Transgenic expression of ATOH8 in HCC cell lines significantly reduced proliferation and foci colony formation, as well as their invasive and migratory abilities. Transgenic expression of ATOH8 reduced the ability of HBV-positive PLC8024 cells to form tumors in mice, compared to control cells. Cells with ATOH8 knockdown formed xenograft tumors more rapidly, in more mice, than control cells. ATOH8 repressed transcription of stem-cell associated genes including OCT4, NANOG, and CD133. Knockdown of ATOH8 in CD133-negative QSG7701 cells caused them to express CD133; acquire self-renewal, differentiation, chemo-resistance properties; form more xenograft tumors in mice; and generate induced pluripotent stem cells (based on staining for alkaline phosphatase and their ability to form embryoid bodies and teratomas). Alternatively, expression of ATOH8 in PLC8024 and Huh7 cells significantly reduced the numbers of cells expressing CD133, and increased the chemo-sensitivity of Huh7 cells to 5-fluorouracil (5-FU) and cisplatin, in vitro and in mice. CONCLUSIONS: ATOH8 appears to be a tumor suppressor that induces stem-cell features and chemoresistance in HCC cells. Strategies to restore its levels and activities might be developed to treat patients with liver cancer.

Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer.

BACKGROUND: Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. OBJECTIVE: To identify recurrent somatic mutations with prognostic significance in patients with CRC. METHOD: Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. RESULTS: Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6-14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. CONCLUSIONS: The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging.