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Hepatocellular carcinoma (HCC), the predominant primary liver tumor, remains one of the most lethal cancers worldwide, despite the advances in therapy in recent years. In addition to the traditional chemically and dietary-induced HCC models, a broad spectrum of novel preclinical tools have been generated following the advent of transgenic, transposon, organoid, and in silico technologies to overcome this gloomy scenario. These models have become rapidly robust preclinical instruments to unravel the molecular pathogenesis of liver cancer and establish new therapeutic approaches against this deadly disease. The present review article aims to summarize and discuss the commonly used preclinical models for HCC, evaluating their strengths and weaknesses.
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Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods: In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27KIP1 non-phosphorylatable (p27KIP1-T187A) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27KIP1 activators.
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Carcinoma Hepatocelular , Inibidor de Quinase Dependente de Ciclina p27 , Neoplasias Hepáticas , Proteínas Quinases Associadas a Fase S , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Ácido Graxo Sintases/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintase Tipo I/genética , Regulação para Baixo , MasculinoRESUMO
TBX3 behaves as a tumor suppressor or oncoprotein across cancer. However, TBX3 function remains undetermined in intrahepatic cholangiocarcinoma (iCCA), a deadly primary liver malignancy with few systemic treatment options. This study sought to investigate the impact of TBX3 on iCCA. We found that overexpression of TBX3 strongly inhibited human iCCA cell growth. In the Akt/FBXW7ΔF mouse iCCA model, overexpression of Tbx3 reduced cholangiocarcinogenesis in vivo, while inducible genetic knockout of Tbx3 accelerated iCCA growth. RNA-seq identified MAD2L1 as a downregulated gene in TBX3-overexpressing cells, and ChIP confirmed that TBX3 binds to the MAD2L1 promoter. CRISPR-mediated knockdown of Mad2l1 significantly reduced the growth of two iCCA models in vivo. Finally, we found that TBX3 expression is upregulated in ~20% of human iCCA samples, and its high expression is associated with less proliferation and better survival. MAD2L1 expression is upregulated in most human iCCA samples and negatively correlated with TBX3 expression. Altogether, our findings suggest that overexpression of TBX3 suppresses CCA progression via repressing MAD2L1 expression.
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Neoplasias dos Ductos Biliares , Carcinogênese , Colangiocarcinoma , Proteínas com Domínio T , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Humanos , Animais , Camundongos , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de CélulasRESUMO
Altered lipid metabolism is a common hallmark of various cancers, including intrahepatic cholangiocarcinoma (ICC), a highly lethal carcinoma that lacks effective treatment options. To elucidate the lipid metabolism changes in ICC, we coupled the expression of the firefly luciferase gene (FFL) to AKT1 (AKT-FFL) via an IRES linker, and then hydrodynamically injected mice with AKT-FFL and Notch1 intracellular cytoplasmic domain (NICD) to establish a luciferase-positive ICC model. This model not only enabled us to monitor and quantify tumor growth by injecting the mice with luciferin, but also allowed us to assess the fatty acid uptake rate by injecting the mice with free fatty acid luciferin (FFA-Luc). The ICC model exhibited robust uptake of exogenous fatty acids compared with the HCC model induced by AKT-FFL/ neuroblastoma Ras (Ras). Lipidomics analysis showed a dramatically higher level of fatty acid in ICC, further supporting the increased fatty acids uptake. Mechanistic studies identified FATP5 as the predominant mediator of fatty acid uptake required for ICC growth using Fatp5 knockout mice and AAV-based shRNA silencing of Fatp5. Our study discovered a novel therapeutic target for the treatment of ICC and shed light on the contributions of lipid metabolism to ICC development. IMPLICATIONS: This study provides the first in vivo evidence that FATP5 is a potential therapeutic target for treating ICC.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Animais , Camundongos , Humanos , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Camundongos Knockout , Metabolismo dos Lipídeos , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Proto-Oncogênicas c-akt/metabolismoAssuntos
Carcinoma Hepatocelular , Dieta Hiperlipídica , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Carcinoma Hepatocelular/metabolismo , Dieta Hiperlipídica/efeitos adversos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , CamundongosRESUMO
OBJECTIVE: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. DESIGN: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. RESULTS: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. CONCLUSIONS: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Animais , Camundongos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Perfilação da Expressão Gênica , Transcriptoma , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismoRESUMO
BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.
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Neoplasias dos Ductos Biliares , Fibroblastos Associados a Câncer , Colangiocarcinoma , Células Estreladas do Fígado , Proteína-Lisina 6-Oxidase , Microambiente Tumoral , Humanos , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/enzimologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Fibroblastos Associados a Câncer/enzimologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/enzimologia , Regulação Neoplásica da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/enzimologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/enzimologia , Fosforilação Oxidativa , Proteína-Lisina 6-Oxidase/metabolismo , Proteína-Lisina 6-Oxidase/genética , Transdução de SinaisRESUMO
BACKGROUND: Upregulation of the mitogen-activated protein kinase (MAPK) cascade is common in hepatocellular carcinoma (HCC). Neuroblastoma RAS viral oncogene homolog (NRAS) is mutated in a small percentage of HCC and is hitherto considered insufficient for hepatocarcinogenesis. We aimed to characterize the process of N-Ras-dependent carcinogenesis in the liver and to identify potential therapeutic vulnerabilities. METHODS: NRAS V12 plasmid was delivered into the mouse liver via hydrodynamic tail vein injection (HTVI). The resulting tumours, preneoplastic lesions, and normal tissue were characterized by NanoString® gene expression analysis, Western Blot, and Immunohistochemistry (IHC). The results were further confirmed by in vitro analyses of HCC cell lines. RESULTS: HTVI with NRAS V12 plasmid resulted in the gradual formation of preneoplastic and neoplastic lesions in the liver three months post-injection. These lesions mostly showed characteristics of HCC, with some exceptions of spindle cell/ cholangiocellular differentiation. Progressive upregulation of the RAS/RAF/MEK/ERK signalling was detectable in the lesions by Western Blot and IHC. NanoString® gene expression analysis of preneoplastic and tumorous tissue revealed a gradual overexpression of the cancer stem cell marker CD133 and Dual Specificity Phosphatases 4 and 6 (DUSP4/6). In vitro, transfection of HCC cell lines with NRAS V12 plasmid resulted in a coherent upregulation of DUSP4 and DUSP6. Paradoxically, this upregulation in PLC/PRF/5 cells was accompanied by a downregulation of phosphorylated extracellular-signal-regulated kinase (pERK), suggesting an overshooting compensation. Silencing of DUSP4 and DUSP6 increased proliferation in HCC cell lines. CONCLUSIONS: Contrary to prior assumptions, the G12V NRAS mutant form is sufficient to elicit hepatocarcinogenesis in the mouse. Furthermore, the upregulation of the MAPK cascade was paralleled by the overexpression of DUSP4, DUSP6, and CD133 in vivo and in vitro. Therefore, DUSP4 and DUSP6 might fine-tune the excessive MAPK activation, a mechanism that can potentially be harnessed therapeutically.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Hepáticas/patologiaRESUMO
Hepatocellular carcinoma (HCC) is a deadly malignancy with high genetic heterogeneity. TP53 mutation and c-MET activation are frequent events in human HCCs. Here, we discovered that the simultaneous mutations in TP53 and activation of c-MET occur in ~20% of human HCCs, and these patients show a poor prognosis. Importantly, we found that concomitant deletion of Trp53 and overexpression of c-MET (c-MET/sgp53) in the mouse liver led to HCC formation in vivo. Consistent with human HCCs, RNAseq showed that c-MET/sgp53 mouse HCCs were characterized by activated c-MET and Ras/MAPK cascades and increased tumor cell proliferation. Subsequently, a stably passaged cell line derived from a c-MET/sgp53 HCC and corresponding subcutaneous xenografts were generated. Also, in silico analysis suggested that the MEK inhibitor trametinib has a higher inhibition score in TP53 null human HCC cell lines, which was validated experimentally. We consistently found that trametinib effectively inhibited the growth of c-MET/sgp53 HCC cells and xenografts, supporting the possible usefulness of this drug for treating human HCCs with TP53-null mutations. Altogether, our study demonstrates that loss of TP53 cooperates with c-MET to drive hepatocarcinogenesis in vivo. The c-MET/sgp53 mouse model and derived HCC cell lines represent novel and useful preclinical tools to study hepatocarcinogenesis in the TP53 null background.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-met , Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Neoplasias Hepáticas/patologia , Mutação/genética , Proteína Supressora de Tumor p53/genética , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
Many patients with hepatocellular carcinoma (HCC) do not respond to the first-line immune checkpoint inhibitor treatment. Immunization with effective cancer vaccines is an attractive alternative approach to immunotherapy. However, its efficacy remains insufficiently evaluated in preclinical studies. Here, we investigated HCC-associated self/tumor antigen, α-fetoprotein-based (AFP-based) vaccine immunization for treating AFP (+) HCC mouse models. We found that AFP immunization effectively induced AFP-specific CD8+ T cells in vivo. However, these CD8+ T cells expressed exhaustion markers, including PD1, LAG3, and Tim3. Furthermore, the AFP vaccine effectively prevented c-MYC/Mcl1 HCC initiation when administered before tumor formation, while it was ineffective against full-blown c-MYC/Mcl1 tumors. Similarly, anti-PD1 and anti-PD-L1 monotherapy showed no efficacy in this murine HCC model. In striking contrast, AFP immunization combined with anti-PD-L1 treatment triggered significant inhibition of HCC progression in most liver tumor nodules, while in combination with anti-PD1, it induced slower tumor progression. Mechanistically, we demonstrated that HCC-intrinsic PD-L1 expression was the primary target of anti-PD-L1 in this combination therapy. Notably, the combination therapy had a similar therapeutic effect in the cMet/ß-catenin mouse HCC model. These findings suggest that combining the AFP vaccine and immune checkpoint inhibitors may be effective for AFP (+) HCC treatment.
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Vacinas Anticâncer , Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Animais , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Linfócitos T CD8-Positivos , Vacinas Anticâncer/uso terapêuticoRESUMO
Hepatocellular carcinoma (HCC) is a primary liver tumor with high lethality and increasing incidence worldwide. While tumor resection or liver transplantation is effective in the early stages of the disease, the therapeutic options for advanced HCC remain limited and the benefits are temporary. Thus, novel therapeutic targets and more efficacious treatments against this deadly cancer are urgently needed. Here, we investigated the pathogenetic and therapeutic role of eukaryotic initiation factor 4A1 (eIF4A1) in this tumor type. We observed consistent eIF4A1 upregulation in HCC lesions compared with non-tumorous surrounding liver tissues. In addition, eIF4A1 levels were negatively correlated with the prognosis of HCC patients. In HCC lines, the exposure to various eIF4A inhibitors triggered a remarkable decline in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Significantly, anti-growth effects were achieved at nanomolar concentrations of the eIF4A1 inhibitors and were further increased by the simultaneous administration of the pan mTOR inhibitor, Rapalink-1. In conclusion, our results highlight the pathogenetic relevance of eIF4A1 in HCC and recommend further evaluation of the potential usefulness of pharmacological combinations based on eIF4A and mTOR inhibitors in treating this aggressive tumor.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Prognóstico , Apoptose , Proliferação de Células , Linhagem Celular TumoralRESUMO
Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Neoplasias dos Ductos Biliares/terapia , Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/etiologia , Colangiocarcinoma/terapia , Consenso , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologiaRESUMO
BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignancy, with increasing incidence worldwide and limited therapeutic options. Aberrant protein glycosylation is a hallmark of cancer. Here, we thoroughly investigated the possible involvement of fucosylation in cholangiocarcinogenesis. APPROACH AND RESULTS: We discovered that the levels of global fucosylation and members of the fucosylation pathway are ubiquitously upregulated in human iCCA tissues compared to nontumorous surrounding livers and normal biliary cells. In addition, total fucosylation levels correlate with poor patients' prognosis. Furthermore, fucosylation inhibition following 6-alkynylfucose (6AF) administration triggered a dose-dependent decrease in the proliferation and migration of iCCA cell lines. Notably, adding fucose to the cell medium annulled these effects. At the molecular level, 6AF administration or small interfering RNA-mediated silencing of GDP-L-fucose synthetase (FX) and the GDP-fucose transmembrane transporter (SLC35C1), both pivotal players of cellular fucosylation, decreased NOTCH activity, NOTCH1/Jagged1 interaction, NOTCH receptors, and related target genes in iCCA cell lines. In the same cells, EGFR, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and Bcl-xL protein levels diminished, whereas IκBα (a critical cellular NF-κB inhibitor) increased after FX/SLC35C1 knockdown or 6AF administration. In the chick chorioallantoic membrane assay, 6AF treatment profoundly suppresses the growth of iCCA cells. CONCLUSIONS: Elevated global fucosylation characterizes human iCCA, contributing to cell growth and migration through the upregulation of the NOTCH and EGFR/NF-κB pathways. Thus, aberrant fucosylation is a novel pathogenetic player and a potential therapeutic target for human iCCA.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , NF-kappa B/metabolismo , Glicosilação , Prognóstico , Fucose/metabolismo , Colangiocarcinoma/patologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Receptores ErbB/metabolismoRESUMO
Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.
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BACKGROUND AND AIMS: Gain-of-function (GOF) mutations of CTNNB1 and loss-of-function (LOF) mutations of AXIN1 are recurrent genetic alterations in hepatocellular carcinoma (HCC). We aim to investigate the functional contribution of Hippo/YAP/TAZ in GOF CTNNB1 or LOF AXIN1 mutant HCCs. APPROACH AND RESULTS: The requirement of YAP/TAZ in c-Met/ß-Catenin and c-Met/sgAxin1-driven HCC was analyzed using conditional Yap , Taz , and Yap;Taz knockout (KO) mice. Mechanisms of AXIN1 in regulating YAP/TAZ were investigated using AXIN1 mutated HCC cells. Hepatocyte-specific inducible TTR-CreER T2KO system was applied to evaluate the role of Yap;Taz during tumor progression. Cabozantinib and G007-LK combinational treatment were tested in vitro and in vivo . Nuclear YAP/TAZ was strongly induced in c-Met/sgAxin1 mouse HCC cells. Activation of Hippo via overexpression of Lats2 or concomitant deletion of Yap and Taz significantly inhibited c-Met/sgAxin1 driven HCC development, whereas the same approaches had mild effects in c-Met/ß-Catenin HCCs. YAP is the major Hippo effector in c-Met/ß-Catenin HCCs, and both YAP and TAZ are required for c-Met/sgAxin1-dependent hepatocarcinogenesis. Mechanistically, AXIN1 binds to YAP/TAZ in human HCC cells and regulates YAP/TAZ stability. Genetic deletion of YAP/TAZ suppresses already formed c-Met/sgAxin1 liver tumors, supporting the requirement of YAP/TAZ during tumor progression. Importantly, tankyrase inhibitor G007-LK, which targets Hippo and Wnt pathways, synergizes with cabozantinib, a c-MET inhibitor, leading to tumor regression in the c-Met/sgAxin1 HCC model. CONCLUSIONS: Our studies demonstrate that YAP/TAZ are major signaling molecules downstream of LOF AXIN1 mutant HCCs, and targeting YAP/TAZ is an effective treatment against AXIN1 mutant human HCCs.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , beta Catenina/genética , Carcinogênese/genética , Mutação , Via de Sinalização Wnt/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína Axina/genéticaRESUMO
BACKGROUND: Recently, molecular tumour boards (MTBs) have been integrated into the clinical routine. Since their benefit remains debated, we assessed MTB outcomes in the Comprehensive Cancer Center Ostbayern (CCCO) from 2019 to 2021. METHODS AND RESULTS: In total, 251 patients were included. Targeted sequencing was performed with PCR MSI-evaluation and immunohistochemistry for PD-L1, Her2, and mismatch repair enzymes. 125 treatment recommendations were given (49.8%). High-recommendation rates were achieved for intrahepatic cholangiocarcinoma (20/30, 66.7%) and gastric adenocarcinoma (10/16, 62.5%) as opposed to colorectal cancer (9/36, 25.0%) and pancreatic cancer (3/18, 16.7%). MTB therapies were administered in 47 (18.7%) patients, while 53 (21.1%) received alternative treatment regimens. Thus 37.6% of recommended MTB therapies were implemented (47/125 recommendations). The clinical benefit rate (complete + partial + mixed response + stable disease) was 50.0% for MTB and 63.8% for alternative treatments. PFS2/1 ratios were 34.6% and 16.1%, respectively. Significantly improved PFS could be achieved for m1A-tier-evidence-based MTB therapies (median 6.30 months) compared to alternative treatments (median 2.83 months; P = 0.0278). CONCLUSION: The CCCO MTB yielded a considerable recommendation rate, particularly in cholangiocarcinoma patients. The discrepancy between the low-recommendation rates in colorectal and pancreatic cancer suggests the necessity of a weighted prioritisation of entities. High-tier recommendations should be implemented predominantly.