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Gastroenterology Jul 2024Hepatocellular carcinoma (HCC) is characterized by an immune-suppressive microenvironment, which contributes to tumor progression, metastasis, and immunotherapy...
BACKGROUND & AIMS
Hepatocellular carcinoma (HCC) is characterized by an immune-suppressive microenvironment, which contributes to tumor progression, metastasis, and immunotherapy resistance. Identification of HCC-intrinsic factors regulating the immunosuppressive microenvironment is urgently needed. Here, we aimed to elucidate the role of SYR-Related High-Mobility Group Box 18 (SOX18) in inducing immunosuppression and to validate novel combination strategies for SOX18-mediated HCC progression and metastasis.
METHODS
The role of SOX18 in HCC was investigated in orthotopic allografts and diethylinitrosamine/carbon tetrachloride-induced spontaneous models by using murine cell lines, adeno-associated virus 8, and hepatocyte-specific knockin and knockout mice. The immune cellular composition in the HCC microenvironment was evaluated by flow cytometry and immunofluorescence.
RESULTS
SOX18 overexpression promoted the infiltration of tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) while diminishing cytotoxic T cells to facilitate HCC progression and metastasis in cell-derived allografts and chemically induced HCC models. Mechanistically, transforming growth factor-beta 1 (TGF-β1) upregulated SOX18 expression by activating the Smad2/3 complex. SOX18 transactivated chemokine (C-X-C motif) ligand 12 (CXCL12) and programmed death ligand 1 (PD-L1) to induce the immunosuppressive microenvironment. CXCL12 knockdown significantly attenuated SOX18-induced TAMs and Tregs accumulation and HCC dissemination. Antagonism of chemokine receptor 4 (CXCR4), the cognate receptor of CXCL12, or selective knockout of CXCR4 in TAMs or Tregs likewise abolished SOX18-mediated effects. TGFβR1 inhibitor Vactosertib or CXCR4 inhibitor AMD3100 in combination with anti-PD-L1 dramatically inhibited SOX18-mediated HCC progression and metastasis.
CONCLUSIONS
SOX18 promoted the accumulation of immunosuppressive TAMs and Tregs in the microenvironment by transactivating CXCL12 and PD-L1. CXCR4 inhibitor or TGFβR1 inhibitor in synergy with anti-PD-L1 represented a promising combination strategy to suppress HCC progression and metastasis.
Topics: Animals; Carcinoma, Hepatocellular; Liver Neoplasms; SOXF Transcription Factors; B7-H1 Antigen; Tumor Microenvironment; Humans; Receptors, CXCR4; Transforming Growth Factor beta1; Mice; Disease Progression; Chemokine CXCL12; Up-Regulation; Cyclams; Benzylamines; T-Lymphocytes, Regulatory; Cell Line, Tumor; Tumor-Associated Macrophages; Mice, Knockout; Gene Expression Regulation, Neoplastic; Signal Transduction; Immune Checkpoint Inhibitors; Mice, Inbred C57BL; Diethylnitrosamine; Male
PubMed: 38417530
DOI: 10.1053/j.gastro.2024.02.025 -
Arquivos de Gastroenterologia 2023•In this review, we described different murine models of carcinogenesis: classic models, new transgenic and combined models, that reproduce the key points for HCC and... (Review)
Review
•In this review, we described different murine models of carcinogenesis: classic models, new transgenic and combined models, that reproduce the key points for HCC and CCA genesis allowing a better understanding of its genetic physiopathological, and environmental abnormalities. •Each model has its advantages, disadvantages, similarities, and differences with the corresponding human disease and should be chosen according to the specificity of the study. Ultimately, those models can also be used for testing new anticancer therapeutic approaches. •Cholangiocarcinoma has been highlighted, with an increase in prevalence. This review has an important role in understanding the pathophysiology and the development of new drugs. Background - This manuscript provides an overview of liver carcinogenesis in murine models of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Objective - A review through MEDLINE and EMBASE was performed to assess articles until August 2022.Methods - Search was conducted of the entire electronic databases and the keywords used was HCC, CCA, carcinogenesis, animal models and liver. Articles exclusion was based on the lack of close relation to the subject. Carcinogenesis models of HCC include HCC induced by senescence in transgenic animals, HCC diet-induced, HCC induced by chemotoxicagents, xenograft, oncogenes, and HCC in transgenic animals inoculated with B and C virus. The models of CCA include the use of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), thioacetamide (TAA), and carbon tetrachloride (CCl4). CCA murine models may also be induced by: CCA cells, genetic manipulation, Smad4, PTEN and p53 knockout, xenograft, and DEN-left median bile duct ligation. Results - In this review, we described different murine models of carcinogenesis that reproduce the key points for HCC and CCA genesis allowing a better understanding of its genetic, physiopathological, and environmental abnormalities. Conclusion - Each model has its advantages, disadvantages, similarities, and differences with the corresponding human disease and should be chosen according to the specificity of the study. Ultimately, those models can also be used for testing new anticancer therapeutic approaches.
Topics: Animals; Mice; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinogenesis; Carcinoma, Hepatocellular; Cholangiocarcinoma; Disease Models, Animal; Liver Neoplasms
PubMed: 37792769
DOI: 10.1590/S0004-2803.230302023-58 -
Hepatology (Baltimore, Md.) Sep 2023HCC, the third leading cause of cancer-related death, arises in the context of liver fibrosis. Although HCC is generally poorly fibrogenic, some tumors harbor focal...
BACKGROUND AND AIMS
HCC, the third leading cause of cancer-related death, arises in the context of liver fibrosis. Although HCC is generally poorly fibrogenic, some tumors harbor focal intratumor extracellular matrix (ECM) deposits called "fibrous nests." To date, the molecular composition and clinical relevance of these ECM deposits have not been fully defined.
APPROACH AND RESULTS
We performed quantitative matrisome analysis by tandem mass tags mass spectrometry in 20 human cancer specific matrisome (HCCs) with high or low-grade intratumor fibrosis and matched nontumor tissues, as well as in 12 livers from mice treated with vehicle, carbon tetrachloride, or diethylnitrosamine. We found 94 ECM proteins differentially abundant between high and low-grade fibrous nests, including interstitial and basement membrane components, such as several collagens, glycoproteins, proteoglycans, enzymes involved in ECM stabilization and degradation, and growth factors. Pathway analysis revealed a metabolic switch in high-grade fibrosis, with enhanced glycolysis and decreased oxidative phosphorylation. Integrating the quantitative proteomics with transcriptomics from HCCs and nontumor livers (n = 2,285 samples), we identified a subgroup of fibrous nest HCCs, characterized by cancer-specific ECM remodeling, expression of the WNT/TGFB (S1) subclass signature, and poor patient outcome. Fibrous nest HCCs abundantly expressed an 11-fibrous-nest - protein signature, associated with poor patient outcome, by multivariate Cox analysis, and validated by multiplex immunohistochemistry.
CONCLUSIONS
Matrisome analysis highlighted cancer-specific ECM deposits, typical of the WNT/TGFB HCC subclass, associated with poor patient outcomes. Hence, histologic reporting of intratumor fibrosis in HCC is of clinical relevance.
Topics: Humans; Mice; Animals; Carcinoma, Hepatocellular; Liver Neoplasms; Fibrosis; Extracellular Matrix; Extracellular Matrix Proteins
PubMed: 36999534
DOI: 10.1097/HEP.0000000000000362 -
Cell Reports. Medicine Sep 2023Inhibition of adenosine A2A receptor (A2AR) is a promising approach for cancer immunotherapy currently evaluated in several clinical trials. We here report that...
Inhibition of adenosine A2A receptor (A2AR) is a promising approach for cancer immunotherapy currently evaluated in several clinical trials. We here report that anti-obesogenic and anti-inflammatory functions of A2AR, however, significantly restrain hepatocellular carcinoma (HCC) development. Adora2a deletion in mice triggers obesity, non-alcoholic steatohepatitis (NASH), and systemic inflammation, leading to spontaneous HCC and promoting dimethylbenzyl-anthracene (DMBA)- or diethylnitrosamine (DEN)-induced HCC. Conditional Adora2a deletion reveals critical roles of myeloid and hepatocyte-derived A2AR signaling in restraining HCC by limiting hepatic inflammation and steatosis. Remarkably, the impact of A2AR pharmacological blockade on HCC development is dependent on pre-existing NASH. In support of our animal studies, low ADORA2A gene expression in human HCC is associated with cirrhosis, hepatic inflammation, and poor survival. Together, our study uncovers a previously unappreciated tumor-suppressive function for A2AR in the liver and suggests caution in the use of A2AR antagonists in patients with NASH and NASH-associated HCC.
Topics: Humans; Animals; Mice; Carcinoma, Hepatocellular; Non-alcoholic Fatty Liver Disease; Receptor, Adenosine A2A; Liver Neoplasms; Inflammation
PubMed: 37729873
DOI: 10.1016/j.xcrm.2023.101188 -
EBioMedicine Feb 2024Gut probiotic depletion is associated with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we investigated the prophylactic...
BACKGROUND
Gut probiotic depletion is associated with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we investigated the prophylactic potential of Lactobacillus acidophilus against NAFLD-HCC.
METHODS
NAFLD-HCC conventional and germ-free mice were established by diethylnitrosamine (DEN) injection with feeding of high-fat high-cholesterol (HFHC) or choline-deficient high-fat (CDHF) diet. Orthotopic NAFLD-HCC allografts were established by intrahepatic injection of murine HCC cells with HFHC feeding. Metabolomic profiling was performed using liquid chromatography-mass spectrometry. Biological functions of L. acidophilus conditional medium (L.a CM) and metabolites were determined in NAFLD-HCC human cells and mouse organoids.
FINDINGS
L. acidophilus supplementation suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice. This was confirmed in orthotopic allografts and germ-free tumourigenesis mice. L.a CM inhibited the growth of NAFLD-HCC human cells and mouse organoids. The protective function of L. acidophilus was attributed to its non-protein small molecules. By metabolomic profiling, valeric acid was the top enriched metabolite in L.a CM and its upregulation was verified in liver and portal vein of L. acidophilus-treated mice. The protective function of valeric acid was demonstrated in NAFLD-HCC human cells and mouse organoids. Valeric acid significantly suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice, accompanied by improved intestinal barrier integrity. This was confirmed in another NAFLD-HCC mouse model induced by CDHF diet and DEN. Mechanistically, valeric acid bound to hepatocytic surface receptor GPR41/43 to inhibit Rho-GTPase pathway, thereby ablating NAFLD-HCC.
INTERPRETATION
L. acidophilus exhibits anti-tumourigenic effect in mice by secreting valeric acid. Probiotic supplementation is a potential prophylactic of NAFLD-HCC.
FUNDING
Shown in Acknowledgments.
Topics: Humans; Animals; Mice; Carcinoma, Hepatocellular; Non-alcoholic Fatty Liver Disease; Lactobacillus acidophilus; Liver Neoplasms; Liver; Cell Transformation, Neoplastic; Carcinogenesis; Diet, High-Fat; Choline; Probiotics; Mice, Inbred C57BL; Pentanoic Acids
PubMed: 38176203
DOI: 10.1016/j.ebiom.2023.104952 -
Cancer Research Mar 2024N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology....
UNLABELLED
N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology. As the m6A reader YTHDF1 has been reported to promote progression of hepatocellular carcinoma (HCC), it represents a potential therapeutic target. In this study, we evaluated the clinical significance of YTHDF1 using human HCC samples and found that YTHDF1 was significantly upregulated in HCCs with high stemness scores and was positively associated with recurrence and poor prognosis. Analysis of HCC spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSC). Stem cell-specific conditional Ythdf1 knockin (CKI) mice treated with diethylnitrosamine showed elevated tumor burden as compared with wild-type mice. YTHDF1 promoted CSCs renewal and resistance to the multiple tyrosine kinase inhibitors lenvatinib and sorafenib in patient-derived organoids and HCC cell lines, which could be abolished by catalytically inactive mutant YTHDF1. Multiomic analysis, including RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing identified NOTCH1 as a direct downstream of YTHDF1. YTHDF1 bound to m6A modified NOTCH1 mRNA to enhance its stability and translation, which led to increased NOTCH1 target genes expression. NOTCH1 overexpression rescued HCC stemness in YTHDF1-deficient cells in vitro and in vivo. Lipid nanoparticles targeting YTHDF1 significantly enhanced the efficacy of lenvatinib and sorafenib in HCC in vivo. Taken together, YTHDF1 drives HCC stemness and drug resistance through an YTHDF1-m6A-NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC.
SIGNIFICANCE
Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.
Topics: Humans; Animals; Mice; Carcinoma, Hepatocellular; Sorafenib; Drug Resistance, Neoplasm; Liver Neoplasms; Adenosine; RNA, Messenger; RNA; Receptor, Notch1; RNA-Binding Proteins; Phenylurea Compounds; Quinolines
PubMed: 38241695
DOI: 10.1158/0008-5472.CAN-23-1916 -
Cancer Communications (London, England) Dec 2023Hepatic inflammation is the major risk factor of hepatocellular carcinoma (HCC). However, the underlying mechanism by which hepatic inflammation progresses to HCC is...
BACKGROUND
Hepatic inflammation is the major risk factor of hepatocellular carcinoma (HCC). However, the underlying mechanism by which hepatic inflammation progresses to HCC is poorly understood. This study was designed to investigate the role of ETS translocation variant 4 (ETV4) in linking hepatic inflammation to HCC.
METHODS
Quantitative real-time PCR and immunoblotting were used to detect the expression of ETV4 in HCC tissues and cell lines. RNA sequencing and luciferase reporter assays were performed to identify the target genes of ETV4. Hepatocyte-specific ETV4-knockout (ETV4 ) and transgenic (ETV4 ) mice and diethylnitrosamine-carbon tetrachloride (DEN-CCL ) treatment experiments were applied to investigate the function of ETV4 in vivo. The Cancer Genome Atlas (TCGA) database mining and pathological analysis were carried out to determine the correlation of ETV4 with tumor necrosis factor-alpha (TNF-α) and mitogen-activated protein kinase 11 (MAPK11).
RESULTS
We revealed that ETV4 was highly expressed in HCC. High levels of ETV4 predicted a poor survival rate of HCC patients. Then we identified ETV4 as a transcription activator of TNF-α and MAPK11. ETV4 was positively correlated with TNF-α and MAPK11 in HCC patients. As expected, an increase in hepatic TNF-α secretion and macrophage accumulation were observed in the livers of ETV4 mice. The protein levels of TNF-α, MAPK11, and CD68 were significantly higher in the livers of ETV4 mice compared with wild type mice but lower in ETV4 mice compared with ETV4 mice as treated with DEN-CCL , indicating that ETV4 functioned as a driver of TNF-α/MAPK11 expression and macrophage accumulation during hepatic inflammation. Hepatocyte-specific knockout of ETV4 significantly prevented development of DEN-CCL -induced HCC, while transgenic expression of ETV4 promoted growth of HCC.
CONCLUSIONS
ETV4 promoted hepatic inflammation and HCC by activating transcription of TNF-α and MAPK11. Both the ETV4/TNF-α and ETV4/MAPK11 axes represented two potential therapeutic targets for highly associated hepatic inflammation and HCC. ETV4+TNF-α were potential prognostic markers for HCC patients.
Topics: Humans; Animals; Mice; Carcinoma, Hepatocellular; Tumor Necrosis Factor-alpha; Liver Neoplasms; Transcription Factors; Inflammation; Proto-Oncogene Proteins c-ets
PubMed: 37670477
DOI: 10.1002/cac2.12482 -
Clinical and Translational Medicine Oct 2023Hepatocellular carcinoma (HCC) is one of the major causes of death from cancer and has a very poor prognosis with few effective therapeutic options. Despite the approval...
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the major causes of death from cancer and has a very poor prognosis with few effective therapeutic options. Despite the approval of lenvatinib for the treatment of patients suffering from advanced HCC, only a small number of patients can benefit from this targeted therapy.
METHODS
Diethylnitrosamine (DEN)-CCL4 mouse liver tumour and the xenograft tumour models were used to evaluate the function of KDM6A in HCC progression. The xenograft tumour model and HCC cell lines were used to evaluate the role of KDM6A in HCC drug sensitivity to lenvatinib. RNA-seq and ChIP assays were conducted for mechanical investigation.
RESULTS
We revealed that KDM6A exhibited a significant upregulation in HCC tissues and was associated with an unfavourable prognosis. We further demonstrated that KDM6A knockdown remarkably suppressed HCC cell proliferation and migration in vitro. Moreover, hepatic Kdm6a loss also inhibited liver tumourigenesis in a mouse liver tumour model. Mechanistically, KDM6A loss downregulated the FGFR4 expression to suppress the PI3K-AKT-mTOR signalling pathway, leading to a glucose and lipid metabolism re-programming in HCC. KDM6A and FGFR4 levels were positively correlated in HCC specimens and mouse liver tumour tissues. Notably, KDM6A knockdown significantly inhibited the efficacy of lenvatinib therapy in HCC cells in vitro and in vivo.
CONCLUSIONS
Our findings revealed that KDM6A promoted HCC progression by activating FGFR4 expression and may be an essential molecule for influencing the efficacy of lenvatinib in HCC therapy.
Topics: Humans; Animals; Mice; Carcinoma, Hepatocellular; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Phenylurea Compounds; Receptor, Fibroblast Growth Factor, Type 4
PubMed: 37846441
DOI: 10.1002/ctm2.1452 -
Journal of Hepatology Sep 2023Recurrent somatic mutations of the RPS6KA3 gene encoding for the serine/threonine kinase RSK2 were identified in hepatocellular carcinomas (HCCs), suggesting its...
BACKGROUND & AIMS
Recurrent somatic mutations of the RPS6KA3 gene encoding for the serine/threonine kinase RSK2 were identified in hepatocellular carcinomas (HCCs), suggesting its tumour-suppressive function. Our goal was to demonstrate the tumour suppressor role of RSK2 in the liver and investigate the functional consequences of its inactivation.
METHODS
We analysed a series of 1,151 human HCCs for RSK2 mutations and 20 other driver genetic alterations. We then modelled RSK2 inactivation in mice in various mutational contexts recapitulating or not those naturally found in human HCC, using transgenic mice and liver-specific carcinogens. These models were monitored for liver tumour appearance and subjected to phenotypic and transcriptomic analyses. Functional consequences of RSK2 rescue were also investigated in a human RSK2-deficient HCC cell line.
RESULTS
RSK2-inactivating mutations are specific to human HCC and frequently co-occur with AXIN1-inactivating or β-catenin-activating mutations. Modelling of these co-occurrences in mice showed a cooperative effect in promoting liver tumours with transcriptomic profiles recapitulating those of human HCCs. By contrast, there was no cooperation in liver tumour induction between RSK2 loss and BRAF-activating mutations chemically induced by diethylnitrosamine. In human liver cancer cells, we also showed that RSK2 inactivation confers some dependency to the activation of RAS/MAPK signalling that can be targeted by MEK inhibitors.
CONCLUSIONS
Our study demonstrates the tumour suppressor role of RSK2 and its specific synergistic effect in hepatocarcinogenesis when its loss of function is specifically combined with AXIN1 inactivation or β-catenin activation. Furthermore, we identified the RAS/MAPK pathway as a potential therapeutic target for RSK2-inactivated liver tumours.
IMPACT AND IMPLICATIONS
This study demonstrated the tumour suppressor role of RSK2 in the liver and showed that its inactivation specifically synergises with AXIN1 inactivation or β-catenin activation to promote the development of HCC with similar transcriptomic profiles as found in humans. Furthermore, this study highlights that activation of the RAS/MAPK pathway is one of the key signalling pathways mediating the oncogenic effect of RSK2 inactivation that can be targeted with already available anti-MEK therapies.
Topics: Animals; Humans; Mice; Axin Protein; beta Catenin; Carcinoma, Hepatocellular; Liver Neoplasms; Mutation; Signal Transduction
PubMed: 37201672
DOI: 10.1016/j.jhep.2023.05.004 -
Nature Cell Biology Sep 2023Oxidative stress contributes to tumourigenesis by altering gene expression. One accompanying modification, 8-oxoguanine (oG) can change RNA-RNA interactions via oG•A...
Oxidative stress contributes to tumourigenesis by altering gene expression. One accompanying modification, 8-oxoguanine (oG) can change RNA-RNA interactions via oG•A base pairing, but its regulatory roles remain elusive. Here, on the basis of oG-induced guanine-to-thymine (oG > T) variations featured in sequencing, we discovered widespread position-specific oGs in tumour microRNAs, preferentially oxidized towards 5' end seed regions (positions 2-8) with clustered sequence patterns and clinically associated with patients in lower-grade gliomas and liver hepatocellular carcinoma. We validated that oG at position 4 of miR-124 (4oG-miR-124) and 4oG-let-7 suppress lower-grade gliomas, whereas 3oG-miR-122 and 4oG-let-7 promote malignancy of liver hepatocellular carcinoma by redirecting the target transcriptome to oncogenic regulatory pathways. Stepwise oxidation from tumour-promoting 3oG-miR-122 to tumour-suppressing 2,3oG-miR-122 occurs and its specific modulation in mouse liver effectively attenuates diethylnitrosamine-induced hepatocarcinogenesis. These findings provide resources and insights into epitranscriptional oG regulation of microRNA functions, reprogrammed by redox changes, implicating its control for cancer treatment.
Topics: Animals; Mice; Carcinoma, Hepatocellular; MicroRNAs; Carcinogenesis; Guanine; Glioma; Oxidation-Reduction; Liver Neoplasms
PubMed: 37696949
DOI: 10.1038/s41556-023-01209-6