-
Frontiers in Immunology 2023Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV...
INTRODUCTION
Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV neutralization in patients, their tumor-inhibitory effects have not yet been examined. Hepatitis B immunoglobulin (HBIG) therapy is employed to reduce hepatocellular carcinoma (HCC) recurrence in patients after living donor liver transplantations (LDLT).
HYPOTHESIS
We hypothesized that patient-derived intravenous immunoglobulin (IVIG) binding to HCC associated TICs will reduce self-renewal and cell viability driven by β-CATENIN-downstream pathways. β-CATENIN activity protected TICs from IVIG effects.
METHODS
The effects of HBIG and HCIG binding to TICs were evaluated for cell viability and self-renewal.
RESULTS
Inhibition of β-CATENIN pathway(s) augmented TIC susceptibility to HBIG- and HCIG-immunotherapy. HBV X protein (HBx) upregulates both β-CATENIN and NANOG expression. The co-expression of constitutively active β-CATENIN with NANOG promotes self-renewal ability and tumor-initiating ability of hepatoblasts. HBIG bound to HBV+ cells led to growth inhibition in a TIC subset that expressed hepatitis B surface antigen. The HBx protein transformed cells through β-CATENIN-inducible lncRNAs and . Co-expression of constitutively active β-CATENIN with NANOG promoted self-renewal ability of TICs through EGLN3 induction. β-CATENIN-induced lncRNAs stabilized HIF2 to maintain self-renewal of TICs. Targeting of resulted in destabilization of EZH2-dependent β-CATENIN activity and synergized cell-killing of TICs by HBIG or HCIG immunotherapy.
DISCUSSION
Taken together, WNT and stemness pathways induced HIF2 of TICs via cooperating lncRNAs resulting in resistance to cancer immunotherapy. Therefore, therapeutic use of IVIG may suppress tumor recurrence through inhibition of TICs.
Topics: Humans; beta Catenin; Carcinoma, Hepatocellular; Immunoglobulins, Intravenous; Immunotherapy; Liver Neoplasms; Liver Transplantation; Living Donors; Neoplasm Recurrence, Local; RNA, Long Noncoding
PubMed: 37744383
DOI: 10.3389/fimmu.2023.1204907 -
Hepatology (Baltimore, Md.) Oct 2023Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it...
BACKGROUND AND AIMS
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC.
APPROACH AND RESULTS
Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone.
CONCLUSION
Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth.
Topics: Animals; Humans; Mice; Carcinogenesis; Carcinoma, Hepatocellular; DNA Damage; Doxorubicin; Hepatocytes; Liver Neoplasms; Mice, Knockout; NF-kappa B; Proto-Oncogene Proteins c-rel
PubMed: 36089330
DOI: 10.1002/hep.32781 -
Biomolecules Dec 2023Exosomes are essential for hepatocellular carcinoma (HCC) progression and have garnered significant interest as novel targets for diagnostic, prognostic, and therapeutic...
BACKGROUND
Exosomes are essential for hepatocellular carcinoma (HCC) progression and have garnered significant interest as novel targets for diagnostic, prognostic, and therapeutic approaches. This study aims to identify potential exosome-related biomarkers for the development of useful strategies for HCC diagnosis and therapy.
METHODS
Three datasets obtained from the Gene Expression Omnibus (GEO) were utilized to identify differentially expressed genes (DEGs) in HCC. Through Gene Ontology (GO) analysis and protein-protein interaction (PPI) network, overall survival (OS) analysis, Cox analyses, and diethylnitrosamine (DEN)-induced HCC mouse model detection, exosome-related hub gene was screened out, followed by a prognostic value assessment and immune-correlates analysis based on the Cancer Genome Atlas (TCGA) dataset. The hub gene-containing exosomes derived from Hepa1-6 cells were isolated and characterized using differential ultracentrifugation, transmission electron microscopy scanning, and Western blot. Ultrasound-guided intrahepatic injection, cell co-culture, CCK-8, and flow cytometry were performed to investigate the effects of the hub gene on macrophage infiltration and polarization in HCC.
RESULTS
A total of 83 DEGs enriched in the extracellular exosome term, among which, FTCD, HRA, and C8B showed the strongest association with the progression of HCC. FTCD was independently associated with a protective effect in HCC and selected as the hub gene. The presence of FTCD in exosomes was confirmed. FTCD-stimulated macrophages were polarized towards the M1 type and suppressed HCC cells proliferation.
CONCLUSIONS
FTCD is a potential exosome-related biomarker for HCC diagnosis, prognosis, and treatment. The crosstalk between FTCD-containing exosomes and macrophages in HCC progression deserves further investigation.
Topics: Animals; Mice; Blotting, Western; Carcinoma, Hepatocellular; Exosomes; Liver Neoplasms; Mice, Inbred Strains; Glutamate Formimidoyltransferase
PubMed: 38254641
DOI: 10.3390/biom14010041 -
Hepatology Communications Jul 2023We established a novel diethylnitrosamine (DEN) -induced mouse model that reflected the progression of cholangiocarcinoma (CCA) from atypical cystic hyperplasia.
BACKGROUND
We established a novel diethylnitrosamine (DEN) -induced mouse model that reflected the progression of cholangiocarcinoma (CCA) from atypical cystic hyperplasia.
METHODS
BALB/c mice were administered DEN by oral gavage. Cells isolated from livers were analyzed for expression of CSNK2A1, MAX and MAX-interacting proteins. Human CCA cell lines (MzChA-1, HuCCT1), normal human cholangiocyte (H69), human hepatic stellate cells (LX-2), macrophages (RAW 264.7), and primary hepatic cells were used for cellular and molecular biology assays.
RESULTS
Expression of MAX, CSNK2A1, C-MYC, β-catenin, HMGB1, and IL-6 was upregulated in hepatic cells from CCA liver tissue. The half-life of MAX is higher in CCA cells, and this favors their proliferation. Overexpression of MAX increased growth, migration, and invasion of MzChA-1, whereas silencing of MAX had the opposite effect. MAX positively regulated IL-6 and HMGB1 through paracrine signaling in HepG2, LX2, and RAW cells and autocrine signaling in MzChA-1 cells. CSNK2A1-mediated MAX phosphorylation shifts MAX-MAX homodimer to C-MYC-MAX and β-catenin-MAX heterodimers and increases the HMGB1 and IL-6 promoter activities. Increase of MAX phosphorylation promotes cell proliferation, migration, invasion, and cholangiocarcinogenesis. The casein kinase 2 inhibitor CX-4945 induces cell cycle arrest and inhibits cell proliferation, migration, invasion, and carcinogenesis in MzChA-1 cells through the downregulation of CSNK2A1, MAX, and MAX-interaction proteins.
CONCLUSION
C-MYC-MAX and β-catenin-MAX binding to E-box site or β-catenin-MAX bound to TCFs/LEF1 enhanced HMGB1 or IL-6 promoter activities, respectively. IL-6 and HMGB1 secreted by hepatocytes, HSCs, and KCs exert paracrine effects on cholangiocytes to promote cell growth, migration, and invasion and lead to the progression of cholangiocarcinogenesis. CX-4945 provides perspectives on therapeutic strategies to attenuate progression from atypical cystic hyperplasia to cholangiocarcinogenesis.
Topics: Animals; Mice; Humans; beta Catenin; Interleukin-6; Hyperplasia; Casein Kinase II; HMGB1 Protein; Phosphorylation; Cholangiocarcinoma; Bile Duct Neoplasms; Bile Ducts, Intrahepatic
PubMed: 37347224
DOI: 10.1097/HC9.0000000000000144 -
Gut May 2024Squalene epoxidase (SQLE) promotes metabolic dysfunction-associated steatohepatitis-associated hepatocellular carcinoma (MASH-HCC), but its role in modulating the tumour...
OBJECTIVE
Squalene epoxidase (SQLE) promotes metabolic dysfunction-associated steatohepatitis-associated hepatocellular carcinoma (MASH-HCC), but its role in modulating the tumour immune microenvironment in MASH-HCC remains unclear.
DESIGN
We established hepatocyte-specific transgenic (tg) and knockout mice, which were subjected to a choline-deficient high-fat diet plus diethylnitrosamine to induce MASH-HCC. SQLE function was also determined in orthotopic and humanised mice. Immune landscape alterations of MASH-HCC mediated by SQLE were profiled by single-cell RNA sequencing and flow cytometry.
RESULTS
Hepatocyte-specific tg mice exhibited a marked increase in MASH-HCC burden compared with wild-type littermates, together with decreased tumour-infiltrating functional IFN-γ and Granzyme B CD8 T cells while enriching Arg-1 myeloid-derived suppressor cells (MDSCs). Conversely, hepatocyte-specific knockout suppressed tumour growth with increased cytotoxic CD8 T cells and reduced Arg-1 MDSCs, inferring that SQLE promotes immunosuppression in MASH-HCC. Mechanistically, SQLE-driven cholesterol accumulation in tumour microenvironment underlies its effect on CD8 T cells and MDSCs. SQLE and its metabolite, cholesterol, impaired CD8 T cell activity by inducing mitochondrial dysfunction. Cholesterol depletion in vitro abolished the effect of SQLE-overexpressing MASH-HCC cell supernatant on CD8 T cell suppression and MDSC activation, whereas cholesterol supplementation had contrasting functions on CD8 T cells and MDSCs treated with SQLE-knockout supernatant. Targeting SQLE with genetic ablation or pharmacological inhibitor, terbinafine, rescued the efficacy of anti-PD-1 treatment in MASH-HCC models.
CONCLUSION
SQLE induces an impaired antitumour response in MASH-HCC via attenuating CD8 T cell function and augmenting immunosuppressive MDSCs. SQLE is a promising target in boosting anti-PD-1 immunotherapy for MASH-HCC.
PubMed: 38744443
DOI: 10.1136/gutjnl-2023-331117 -
Clinical and Translational Medicine Dec 2023Sex disparities constitute a significant issue in hepatocellular carcinoma (HCC). However, the mechanism of gender dimorphism in HCC is still not completely understood.
BACKGROUND
Sex disparities constitute a significant issue in hepatocellular carcinoma (HCC). However, the mechanism of gender dimorphism in HCC is still not completely understood.
METHODS
5-Hydroxymethylcytosine (5hmC)-Seal technology was utilised to detect the global 5hmC levels from four female and four male HCC samples. Methylation of XIST was detected by Sequenom MassARRAY methylation profiling between HCC tissues (T) and adjacent normal liver tissues (L). The role of Tet methylcytosine dioxygenase 2 (TET2) was investigated using diethylnitrosamine (DEN)-administered Tet2 female mice, which regulated XIST in hepatocarcinogenesis. All statistical analyses were carried out by GraphPad Prism 9.0 and SPSS version 19.0 software.
RESULTS
The results demonstrated that the numbers of 5hmC reads in the first exon of XIST from female HCC tissues (T) were remarkably lower than that in female adjacent normal liver tissues (L). Correspondingly, DNA methylation level of XIST first exon region was significantly increased in female T than in L. By contrast, no significant change was observed in male HCC patients. Compared to L, the expression of XIST in T was also significantly downregulated. Female patients with higher XIST in HCC had a higher overall survival (OS) and more extended recurrence-free survival (RFS). Moreover, TET2 can interact with YY1 binding to the promoter region of XIST and maintain the hypomethylation state of XIST. In addition, DEN-administered Tet2 mice developed more tumours than controls in female mice.
CONCLUSIONS
Our study provided that YY1 and TET2 could interact to form protein complexes binding to the promoter region of XIST, regulating the methylation level of XIST and then affecting the expression of XIST. This research will provide a new clue for studying sex disparities in hepatocarcinogenesis.
HIGHLIGHTS
XIST was significantly downregulated in HCC tissues and had gender disparity. Methylation levels in the XIST first exon were higher in female HCC tissues, but no significant change in male HCC patients. The TET2-YY1 complex regulate XIST expression in female hepatocytes. Other ways regulate XIST expression in male hepatocytes.
Topics: Animals; Female; Humans; Male; Mice; Carcinoma, Hepatocellular; Chromosomes; DNA Methylation; Liver Neoplasms; Sex Characteristics
PubMed: 38148658
DOI: 10.1002/ctm2.1518 -
JHEP Reports : Innovation in Hepatology Feb 2024The mechanism behind the progressive pathological alteration in metabolic dysfunction-associated steatotic liver disease/steatohepatitis (MASLD/MASH)-associated...
BACKGROUND & AIMS
The mechanism behind the progressive pathological alteration in metabolic dysfunction-associated steatotic liver disease/steatohepatitis (MASLD/MASH)-associated hepatocellular carcinoma (HCC) is poorly understood. In the present study, we investigated the role of the polyol pathway enzyme AKR1B1 in metabolic switching associated with MASLD/MASH and in the progression of HCC.
METHODS
AKR1B1 expression was estimated in the tissue and plasma of patients with MASLD/MASH, HCC, and HCC with diabetes mellitus. The role of AKR1B1 in metabolic switching assessed through media conditioning, lentiviral transfection, and pharmacological probes. A proteomic and metabolomic approach was applied for the in-depth investigation of metabolic pathways. Preclinically, mice were subjected to a high-fructose diet and diethylnitrosamine to investigate the role of AKR1B1 in the hyperglycemia-mediated metabolic switching characteristic of MASLD-HCC.
RESULTS
A significant increase in the expression of AKR1B1 was observed in tissue and plasma samples from patients with MASLD/MASH, HCC, and HCC with diabetes mellitus compared to normal samples. Mechanistically, assays revealed that AKR1B1 modulates the Warburg effect, mitochondrial dynamics, the tricarboxylic acid cycle, and lipogenesis to promote hyperglycemia-mediated MASLD and cancer progression. A pathological increase in the expression of AKR1B1 was observed in experimental MASLD-HCC, and expression was positively correlated with high blood glucose levels. High-fructose diet + diethylnitrosamine-treated animals also exhibited statistically significant elevation of metabolic markers and carcinogenesis markers. AKR1B1 inhibition with epalrestat or NARI-29 inhibited cellular metabolism in and models.
CONCLUSIONS
Pathological AKR1B1 modulates hepatic metabolism to promote MASLD-associated hepatocarcinogenesis. Aldose reductase inhibition modulates the glycolytic pathway to prevent precancerous hepatocyte formation.
IMPACT AND IMPLICATIONS
This research work highlights AKR1B1 as a druggable target in metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC), which could provide the basis for the development of new chemotherapeutic agents. Moreover, our results indicate the potential of plasma AKR1B1 levels as a prognostic marker and diagnostic test for MASLD and associated HCC. Additionally, a major observation in this study was that AKR1B1 is associated with the promotion of the Warburg effect in HCC.
PubMed: 38283757
DOI: 10.1016/j.jhepr.2023.100974 -
Translational Oncology May 2024The "one drug-one target" paradigm has various limitations affecting drug efficacy, such as resistance profiles and adverse effects. Combinational therapies help reduce...
BACKGROUND
The "one drug-one target" paradigm has various limitations affecting drug efficacy, such as resistance profiles and adverse effects. Combinational therapies help reduce unexpected off-target effects and accelerate therapeutic efficacy. Sorafenib- an FDA-approved drug for liver cancer, has multiple limitations. Therefore, it is recommended to identify an agent that increases its effectiveness and reduces toxicity. In this regard, Apigenin, a plant flavone, would be an excellent option to explore.
METHODS
We used in silico, in vitro, and animal models to explore our hypothesis. For the in vitro study, HepG2 and Huh7 cells were exposed to Apigenin (12-96 μM) and Sorafenib (1-10 μM). For the in vivo study, Diethylnitrosamine (DEN) (25 mg/kg) induced tumor-bearing animals were given Apigenin (50 mg/kg) or Sorafenib (10 mg/kg) alone and combined. Apigenin's bioavailability was checked by UPLC. Tumor nodules were studied macroscopically and by Scanning Electron Microscopy (SEM). Biochemical analysis, histopathology, immunohistochemistry, and qRT-PCR were done.
RESULTS
The results revealed Apigenin's good bioavailability. In silico study showed binding affinity of both chemicals with p53, NANOG, ß-Catenin, c-MYC, and TLR4. We consistently observed a better therapeutic efficacy in combination than alone treatment. Combination treatment showed i) better cytotoxicity, apoptosis induction, and cell cycle arrest of tumor cells, ii) tumor growth reduction, iii) increased expression of p53 and decreased Cd10, Nanog, ß-Catenin, c-Myc, Afp, and Tlr4.
CONCLUSIONS
In conclusion, Apigenin could enhance the therapeutic efficacy of Sorafenib against liver cancer and may be a promising therapeutic approach for treating HCC. However, further research is imperative to gain more in-depth mechanistic insights.
PubMed: 38394865
DOI: 10.1016/j.tranon.2024.101920 -
Molecular Therapy. Nucleic Acids Sep 2023We have shown previously that polymorphism of activating transcription factor 6 (ATF6) is associated with susceptibility to hepatocellular carcinoma (HCC). Therefore,...
We have shown previously that polymorphism of activating transcription factor 6 (ATF6) is associated with susceptibility to hepatocellular carcinoma (HCC). Therefore, genes down-regulated by ATF6 might play a tumor-suppressing role. In the present study, we identified that expression of protein phosphatase magnesium- or manganous-dependent 1H (PPM1H) mRNA and protein can be inhibited by ATF6 in hepatoma cells and mice with liver knockdown. Tumor tissues from 134 HCC patients were analyzed by immunohistochemistry, and PPM1H exhibited higher expression levels in adjacent para-cancer tissues than in HCC tissues. Therefore, patients with higher expression of PPM1H had a better prognosis. PPM1H inhibited proliferation, migration, and invasion of hepatoma cells. In addition, PPM1H inhibited induced HCC nodule formation as well as tumor xenograft growth in diethylnitrosamine/CCl-induced HCC mouse model and nude mouse tumorigenicity assay, respectively. A 3D model of PPM1H was obtained by homology multi-template modeling, and ribosomal protein S6 kinase B1 (RPS6KB1) in the bone morphogenetic protein (BMP)/transforming growth factor β (TGF-β) pathway was screened out as the potential substrate of PPM1H by Rosetta. PPM1H could directly dephosphorylate p-RPS6KB1. To conclude, we discovered RPS6KB1 as a new PPM1H dephosphorylation substrate. PPM1H exhibited a suppressive effect on HCC progression by dephosphorylating p-RPS6KB1.
PubMed: 37456776
DOI: 10.1016/j.omtn.2023.06.013 -
Cancer Medicine Sep 2023In liver cancer, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) compartment represents an important tumor-initiating cell (TIC) population and served...
BACKGROUND & AIMS
In liver cancer, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) compartment represents an important tumor-initiating cell (TIC) population and served as a potential therapeutic target. Cancer-associated fibroblasts (CAFs) is a critical part of the tumor microenvironment, heavily influenced TIC function and fate. However, deeply investigations have been hindered by the lack of accurate preclinical models to investigate the interaction between CAFs and TIC. Organoids model have achieved major advancements as a precious research model for recapitulating the morphological aspects of organs, and thus also serving as a candidate model to investigate the mutual interaction between different cell types. Consequently, this study aimed to construct a three-dimensional (3D) co-culture organoid model of primary LGR5-expressing tumor stem cells from primary murine liver tumors with CAFs to investigate the impact of CAFs on LGR5 marked TICs in liver cancer.
MATERIALS AND METHODS
First, both of the transgenic LGR5-diphtheria toxin receptor (DTR)-GFP knock-in mice and transgenic Rosa26-mT mice developed primary liver tumors by diethylnitrosamine (DEN) administration. Tumor organoids and CAFs were generated from those primary liver cancer separately. Second, LGR5-expressing TICs organoid with CAFs were established ex vivo based on cell-cell contact or trans-well co-culture system, and the mutual influence between those two types of cells was further investigated. Subsequently, immunodeficient mouse-based xenograft model was further adopted to evaluate the influence of CAFs to LGR5 tumor stem cell, tumor formation, and metastasis.
RESULTS
The co-culture organoid model composed of murine liver tumor LGR5+ tumor-initiating cells and CAFs in 3D co-culture was successfully established, with the intention to investigate their mutual interaction. The existence of CAFs upon engrafting tumor organoids resulted in dramatic higher number of LGR5+ cells in the neoplasia when compared with engrafting tumor organoids alone. Furthermore, ex vivo culture of isolated LGR5+ cells from tumors of co-engrafted mice formed significantly larger size of organoids than mono-engrafted. Our results also indicated significantly larger size and number of formed organoids, when LGR5+ cells co-cultured with CAF in both cell-cell contact and paracrine signaling in vitro, comparing to LGR5+ cells alone. Furthermore, we found that specific knockout of LGR5 expressing cells suppressed CAF-mediated promotion of tumor formation, growth, and metastasis in the experimental mice model.
CONCLUSIONS
Altogether, in a 3D co-culture type of murine liver LGR5+ cells and cancer-associated fibroblasts, we have demonstrated robust effects of CAFs in the promotion of LGR5 marked liver TICs. We also further revealed the influence of tumor microenvironment on stem cell-related therapy, suggesting the possibility of combing CAF-targeted and tumor stem cell targeted therapy in treating liver cancer.
PubMed: 37578396
DOI: 10.1002/cam4.6408