Hepatocellular Carcinoma Model Research Articles

NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

BackgroundNUAK1 is associated with metastasis and drug resistance in hepatocellular carcinoma (HCC). However, little is known about the immune functions of NUAK1 in HCC. Therefore, the aim of this study was to elucidate the novel role of NUAK1 in facilitating immune evasion in HCC and to investigate the mechanisms underpinning this process.MethodThe levels of NUAK1 expression and the infiltration of CD8+ T cells were assessed in tumor tissues from HCC patients and mice xenograft model. HCC cell lines were used to validate the role of NUAK1 in regulating the transcription of PD-L1, the diethylnitrosamine-induced HCC model was established and the expression levels of NUAK1 and PD-L1 proteins in the rat livers were detected. Western blotting, immunofluorescence, real time PCR, and immunohistochemical staining were used to investigate the underlying mechanisms by which NUAK1 regulates PD-L1 expression in hepatocellular carcinoma.ResultsNUAK1 expression was negatively correlated with CD8+ T cell infiltration in tumor tissues from HCC patients and mice xenograft model. Both gain and loss of functions have identified NUAK1 promoted PD-L1 expression at transcriptional level in HCC cells. The increased expression of NUAK1 and PD-L1 proteins were observed in the rat livers of diethylnitrosamine-induced HCC model. Moreover, overexpression of NUAK1 promotes GSK3β Ser9 phosphorylation, β-catenin expression and nuclear accumulation in HCC cells. By contrast, knockdown of NUAK1 has opposite effects. Inhibition of GSK3β activity significantly promoted β-catenin expression and PD-L1 expression in HCC cells. IHC analyses of tumor tissues from HCC patients suggested that the levels of p-GSK3β and β-catenin were positively correlated with NUAK1 expression. Knockdown of β-catenin also reversed NUAK1-mediated PD-L1 expression in HCC cells.ConclusionsThis study revealed a novel role for NUAK1, which promotes the transcriptional expression of PD-L1 by activating GSK3β/β-catenin signaling pathway, leading to immune escape of hepatocellular carcinoma.Registry and the registration no. of the study/trial: Not applicable.

TYRA-430: First reversible FGFR4/3 inhibitor designed to overcome current challenges in FGF19-driven hepatocellular carcinoma treatment.

583 Background: Hepatocellular carcinoma (HCC) is an aggressive malignancy that accounts for approximately 80-90% of all primary liver cancers and is projected to become the third leading cause of cancer-related mortality by 2030. Previous work demonstrated that human Fibroblast Growth Factor 19 (FGF19) was overexpressed in 20-30% of HCC, thereby exerting an oncogenic effect via signaling through its cognate receptors FGFR4, FGFR3, and the co-receptor Klotho b (KLB). Multiple selective covalent FGFR4 inhibitors have been clinically evaluated in FGF19-overexpressing HCC. However, selective inhibition of FGFR4 alone was insufficient and resulted in low response rates and limited duration, likely due to redundant signaling through FGFR3. Here we describe the first report on the preclinical profile of TYRA-430, a first-in-class reversible FGFR4/3 inhibitor. Methods: The preclinical profile of TYRA-430 was assessed in vitro in HCC cellular assays and in vivo in mouse xenograft models. The in vitro potency was assessed by measuring cell viability using Cell Titer-Glo 2.0 Luminescent assay. Percentage of tumor growth inhibition (TGI) was used as a measure to evaluate in vivo efficacy of TYRA-430. Results: TYRA-430 exhibited potency against Ba/F3 cells dependent on wildtype FGFR3 and FGFR4, as well as the known Cys552 and Val550 gatekeeper (GK) resistance mutations in FGFR4. In vitro data showed that TYRA-430 displayed superior potency over the reversible multi-kinase inhibitors sorafenib and lenvatinib, and the covalent FGFR4 inhibitors fisogatinib (BLU-554) and roblitinib (FGF401) in KLB/FGF-19/FGFR3/4 driven models of HCC (Hep3B, HuH-7, and JHH-7 cells). Furthermore, in a human HuH-7 HCC xenograft model in nu/nu mice, TYRA-430 achieved 96% tumor growth inhibition (TGI), compared to 75% TGI for lenvatinib and 86% TGI for FGF401. Additionally, in the GA180 FGF19-driven gastric cancer PDX model, TYRA-430 demonstrated superior efficacy with 93% tumor growth inhibition (TGI), compared to 56% TGI achieved by roblitinib. Conclusions: TYRA-430 was active and potent in multiple KLB/FGF-19/FGFR3/4 models of human hepatocellular carcinoma in vitro and in vivo . Moreover, TYRA-430 displayed potent activity against known FGFR4 resistance mutations compared to covalent FGFR4-specific inhibitors in the clinic. TYRA-430 will be investigated in a Phase 1 clinical trial in hepatocellular carcinoma and other advanced solid tumors.

Phase 1/2 study of FOG-001, a first-in-class direct β-catenin:TCF inhibitor, in patients with colorectal cancer, hepatocellular carcinoma, and other locally advanced or metastatic solid tumors.

TPS322 Background: Wnt/B-catenin pathway activation, which is frequently present in most colorectal cancers and a variety of other solid tumors, is associated with poor prognosis and resistance to immunotherapy. FOG-001 is a Helicon peptide that competitively inhibits the interaction between β-catenin and the T-cell factor (TCF) family of transcription factors. Experiments in patient-derived-xenograft (PDX) models of colorectal cancer (CRC) and hepatocellular carcinoma (HCC) have demonstrated that FOG-001 can inhibit tumor growth and promote tumor regression when administered as monotherapy or in combination with immune checkpoint inhibitors or standard of care therapies, including bevacizumab and 5-FU. Methods: This first-in-human, Phase 1/2, multicenter, open-label, dose escalation (Part 1) and dose-expansion (Part 2) study evaluates the safety/tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor effects of FOG-001 as monotherapy and in combination with other anti-cancer therapies. Eligible patients must have received at least one prior systemic anti-cancer therapy and either progressed on, not responded to, or be unfit for available therapies. In Part 1, FOG-001 is administered intravenously, either weekly or every other week, at escalating dose levels evaluated sequentially in a standard 3+3 design as monotherapy in patients with: (1a) microsatellite stable (MSS) CRC or any solid tumor with a documented Wnt/b-catenin pathway activating mutation (WPAM) and (1c) HCC with WPAM. Part 1b will evaluate PD effects in cohorts of approximately six patients with MSS CRC. Upon selection of the preliminary recommended Phase 2 dose from Part 1a for weekly dosing, combination dose escalation (1d) evaluates the safety/tolerability, PK and anti-tumor effects of FOG-001 in CRC patients combined with FOLFOX+bevacizumab, anti-PD-1/PD-L1 (also includes other solid tumors with known WPAM), or trifluridine/tipiracil+bevacizumab. Part 2 dose expansion evaluates FOG-001 monotherapy in MSS CRC, HCC, and other solid tumors with documented WPAM. Combination dose expansion evaluates the combinations initially evaluated in Part 1d. Primary endpoints are safety/tolerability and preliminary anti-tumor effects (objective response rate). Secondary endpoints are PK, PD, and other anti-tumor responses (e.g., best objective response, duration of response, and progression free survival). Enrollment in Parts 1 and 2 is ongoing in the USA. Clinical trial information: NCT05919264 .

Improving Outcomes in Hepatocellular Carcinoma through Integration of Machine Learning: Development of a Tumor-Associated Macrophage Signature.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors globally. Macrophages, as essential components of the immune system, play crucial roles in immune regulation, inflammation modulation, and antitumor activity. However, it remains unclear whether tumor-associated macrophages can serve as prognostic markers for HCC. First, we identified tumor-associated macrophages based on single-cell data from GSE140228. Then, using a machine learning approach with a combination of 101 module genes, we constructed an optimal prognostic model. Subsequently, we compared our constructed model with other published prognostic models for HCC. Finally, we utilized the generated model score to predict the response to chemotherapy and immune therapy. First, we identified clusters of tumor-associated macrophages using single-cell data. Subsequently, we calculated the tumor-associated macrophage score based on module genes from the previous step. Compared to traditional clinical indicators, tumor-associated macrophage signature (TAMS) exhibits significant advantages. The TAMS C-index not only predicts overall survival, but also recurrence-free survival in HCC patients. Additionally, there was a higher prevalence of TP53 mutations in HCC patients with high TAMS. Furthermore, patients with low TAMS showed greater sensitivity to immunotherapy compared to those with high TAMS. Notably, the number and intensity of interactions between TAM and other T lymphocytes were significantly higher than those involving other cell populations. Interestingly, the high TAMS group exhibited significantly elevated levels of immune checkpoint markers and M2 macrophage markers. TAMS can serve as a novel and potent tool, offering improved treatment options and prognostic assessment for patients with HCC.

The Oncopig hepatocellular carcinoma model: An innovative large animal platform for evaluating treatment effects.

619 Background: Hepatocellular carcinoma (HCC) is an aggressive disease associated with poor prognosis and limited treatment options. Systemic therapies for advanced HCC include sorafenib, lenvatinib, and atezolizumab plus bevacizumab. However, given the limited efficacy of systemic treatment options, novel treatment approaches including combination of systemic and locoregional therapies (LRTs) is required to improve patient outcomes. These approaches require advanced large animal models to prove effectiveness. Pigs represent an ideal platform that provide both regulatory acceptable and clinically relevant large animal cancer models due to their similar physiology, size, genetics, immunity, and metabolism in relation to humans. The Oncopig HCC model is an ideal tool for testing LRT and other oncolytic based treatments. Methods: The Oncopig cancer model develops tumors following induced expression of KRAS G12D and TP53 R167H driver mutations utilizing an adenoviral vector encoding Cre recombinase (AdCre). Oncopig HCC cell lines were developed by isolating hepatocytes from Oncopig liver biopsies and transformed in vitro via exposure to AdCre. Transgene expression and hepatocyte cell origin were validated by RT-PCR and arginase-1 staining, respectively. Comparison of in vitro Oncopig, human, and murine HCC cell line phenotypes (proliferation, migration, and chemotherapeutic response) was performed. Oncopig HCC tumor formation was performed via autologous intrahepatic injection of Oncopig HCC cells. Tumor formation was evaluated using ultrasound and CT imaging. Results: Oncopig and human HCC cell lines displayed similar cell cycle lengths and migration rates. Oncopig HCC cells were consistently more predictive of human HCC responses than murine cells when treated with standard chemotherapeutic agents (doxorubicin, cisplatin, mitomycin C, and sorafenib). Importantly, consistent with human HCC, Oncopig HCC cells were non-responsive to 5-fluorouracil, while murine HCC cells were responsive. Intrahepatic injection of Oncopig HCC cells resulted in tumors visible on ultrasound and CT scan within 2-4 weeks. Resulting tumors were consistent with human tumors on imaging. Histological analysis of tumor biopsies confirmed the identity of the resulting masses as HCC tumors based on positive arginase-1 and KRAS G12D staining. Conclusions: The Oncopig HCC model may be more predictive of human HCC chemotherapeutic responses than currently employed murine models. Tumors develop rapidly (within 2-4 weeks) and are readily identified on ultrasound and CT imaging, making this an ideal model for evaluation of novel therapeutic approaches. Further exploration and development of additional tumor models including further translational characterization will be important for broader utility.

Development and biological evaluation of a novel CEACAM6-targeted PET tracer for distinguishing malignant nodules in early-stage lung adenocarcinoma.

Low-dose CT (LDCT) screening effectively reduces lung adenocarcinoma (LUAD) mortality. However, accurately evaluating the malignant potential of indeterminate lung nodules remains a challenge. Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6), a potential biomarker for distinguishing benign pulmonary nodules from LUAD, may be leveraged for noninvasive positron emission tomography (PET) imaging to aid LUAD diagnosis. This study utilized mRNA, protein, and survival datasets of LUAD patients, along with an animal model of malignant pulmonary nodules, to investigate CEACAM6 expression specificity and its correlation with LUAD. Targeting ligands for CEACAM6 were designed using the Rosetta platform, labeled with [68Ga]Ga, and screened through high-throughput PET imaging to identify the optimal tracer. CEACAM6 was found to be specifically overexpressed in LUAD and was significantly associated with poor prognosis and disease progression. In vivo, [68Ga]Ga-NODA-P3 demonstrated high specificity for delineating CEACAM6-positive A549 xenografts, a LUAD model, via PET imaging, achieving a highest target-to-background ratio of 7.68 ± 0.44. Region of interest (ROI) analysis showed significantly higher tracer uptake in A549 xenografts compared to CEACAM6-negative Huh7 xenografts (a hepatocellular carcinoma model) at 30min post-injection (1.81 ± 0.10%ID/g vs. 0.54 ± 0.06%ID/g). Pre-treatment with an excess of unlabeled NODA-P3 significantly reduced tumor uptake to 0.52 ± 0.07%ID/g. These preclinical findings indicate that [68Ga]Ga-NODA-P3 is a candidate radiotracer for the non-invasive visualization of CEACAM6-positive LUAD, demonstrating favorable imaging contrast. Although the current tumor uptake limits its immediate clinical application, ongoing optimization efforts are expected to improve its efficacy, enabling earlier and more accurate diagnosis of malignant pulmonary nodules in LUAD.

P-467. The clinical prognostic risk stratification system for HIV infected hepatocellular carcinoma

Abstract Background Patients with human immunodeficiency virus (HIV) are more susceptible to liver cancer because of their compromised immune system. There is no specific prognostic model for HIV-infected hepatocellular carcinoma (HCC) patients. Methods Clinical data of 85 patients with HIV-infected HCC was divided into a 7:3 ratio for training and internal validation sets, while the data of 23 patients with HIV-infected HCC was served as the external validation set. Data of 275 HIV-negative HCC patients was considered as external HIV-negative validation set. Variables associated with overall survival (OS) in the training set were used to develop the HIV-infected HCC prognosis (HIHP) model. The model was tested in the internal and external validation sets. The predictive accuracy of the model was assessed with conventional HIV-negative HCC prognostic scoring systems. Results The HIHP model demonstrated a significant association with OS in the training set, with a median OS of 13 months for low risk, 7 months for medium risk, and 3 months for high risk (p < 0.001). The HIHP model showed a significant association with OS, and exhibited greater discriminative abilities compared to conventional HIV-negative HCC prognostic models both in the internal and external validation sets. In the external HIV-negative validation set, the HIHP model did not show better discrimination than conventional HIV-negative HCC scores. Conclusion The new model presented in the work provided a more accurate prognostic prediction of OS in HIV-infected HCC patients. However, the model is not applicable to patients with HIV-negative HCC. Disclosures All Authors: No reported disclosures

A Risk Prediction Model for Hepatocellular Carcinoma in the General Population Without Traditional Risk Factors for Liver Disease.

Existing hepatocellular carcinoma (HCC) prediction models for the general population without traditional risk factors for chronic liver disease are limited. This study aimed to develop an HCC prediction model for individuals lacking these traditional risk factors. The total of 138 452 adult participants without chronic viral hepatitis or significant alcohol intake who underwent regular health checkup at a tertiary hospital in South Korea were followed up for the development of HCC. Risk factors for HCC development were analyzed using Cox regression analysis, and prediction model was developed using the risk factors. Significant predictors of HCC development included older age, male sex, higher body mass index, presence of diabetes mellitus, and levels of aspartate aminotransferase, total cholesterol, and platelet count. A new HCC prediction model using these variables was developed. Harrell's concordance index and Heagerty's integrated area under the receiver operating characteristics (AUROC) curve of the model were 0.88 (95% confidence interval [CI] 0.85-0.91) and 0.89 (95% CI 0.86-0.91), respectively. The 5- and 10-year AUROC were 0.89 (95% CI 0.88-0.89) and 0.87 (95% CI 0.87-0.88), respectively. This model significantly outperformed the FIB-4 scoring model in predicting HCC and effectively stratified individuals into low-, intermediate-, and high-risk groups with significantly different cumulative incidences of HCC. The new model, based on clinical parameters, provides a valuable tool for clinicians to stratify HCC risk in the general population without risk factors for chronic liver disease.

Farnesoid X Receptor Attenuates the Tumorigenicity of Liver Cancer Stem Cells by Inhibiting STAT3 Phosphorylation

The Farnesoid X receptor (FXR) has recently been identified as being closely associated with the progression of primary hepatocellular carcinoma. Cancer stem cells (CSCs) play a crucial role in tumor initiation, progression, invasion, metastasis, recurrence, and drug resistance. The elucidation of the role and regulatory mechanism of FXR in CSCs is therefore deemed significant. Here, bioinformatics analysis has revealed a downregulation of FXR in hepatocellular carcinoma (HCC), which showed a negative correlation with HCC malignancy. This result was further confirmed through clinical sample analysis. Subsequently, CSCs were isolated from HCC cell lines and exhibited a significant decrease in the expression of FXR. The activation of FXR resulted in a remarkable inhibition of the proliferation, invasion, and tumorigenicity of CSCs. Furthermore, activated FXR prominently upregulated the expression of SOCS3 while suppressing STAT3 phosphorylation in CSCs. To further investigate this discovery, we established a DEN-induced HCC model in mice and observed that FXR-deficient mice exhibited heightened susceptibility to HCC. This was accompanied by decreased expression levels of SOCS3 and elevated expression and phosphorylation levels of STAT3, as well as significantly enhanced HCC CSCs markers and stemness-related genes expression in DEN-induced HCC tissues of FXR-deficient mice. Additionally, we also found a significant upregulation of CSCs markers and stemness-related genes within HCC clinical samples. Based on these findings, we postulated that targeted regulation of SOCS3 by FXR inhibits STAT3 phosphorylation, thereby exerting an inhibitory effect on CSCs.

Stromal architecture and fibroblast subpopulations with opposing effects on outcomes in hepatocellular carcinoma

Dissecting the spatial heterogeneity of cancer-associated fibroblasts (CAFs) is vital for understanding tumor biology and therapeutic design. By combining pathological image analysis with spatial proteomics, we revealed two stromal archetypes in hepatocellular carcinoma (HCC) with different biological functions and extracellular matrix compositions. Using paired single-cell RNA and epigenomic sequencing with Stereo-seq, we revealed two fibroblast subsets CAF-FAP and CAF-C7, whose spatial enrichment strongly correlated with the two stromal archetypes and opposing patient prognosis. We discovered two functional units, one is the intratumor inflammatory hub featured by CAF-FAP plus CD8_PDCD1 proximity and the other is the marginal wound-healing hub with CAF-C7 plus Macrophage_SPP1 co-localization. Inhibiting CAF-FAP combined with anti-PD-1 in orthotopic HCC models led to improved tumor regression than either monotherapy. Collectively, our findings suggest stroma-targeted strategies for HCC based on defined stromal archetypes, raising the concept that CAFs change their transcriptional program and intercellular crosstalk according to the spatial context.

CXCR4 antagonist-loaded nanoparticles reprogram the tumor microenvironment and enhance immunotherapy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death that has limited treatment options for advanced stages. Although PD-1 inhibitors such as nivolumab and pembrolizumab have been approved for advanced HCC treatment, their effectiveness is often hampered by the immunosuppressive tumor microenvironment (TME), which is due to hypoxia-driven CXCL12/CXCR4 axis activation. In this study, we developed 807-NPs, lipid-coated tannic acid (TA) nanoparticles that encapsulate BPRCX807, a potent CXCR4 antagonist to target HCC. 807-NPs enhance the pharmacokinetics and improve the tumor availability of BPRCX807 without causing systemic toxicity. Our findings show that 807-NPs block the CXCR4/CXCL12 pathway, inhibiting Akt and mTOR activation in HCC cells and M2 macrophages and promoting their repolarization toward the antitumor M1 phenotype. In orthotopic murine HCC models, systemic administration of 807-NPs significantly remodeled the immunosuppressive TME by reprogramming tumor-associated macrophages (TAMs) toward an immunostimulatory phenotype and promoting cytotoxic T-cell infiltration into tumors. This led to suppressed primary tumor growth and metastasis, while enhancing the efficacy of cancer immunotherapies, including PD-1 blockade and whole-cancer cell vaccines, by promoting T-cell activation. Our work demonstrates the potential of using nanotechnology to deliver CXCR4 antagonists for cancer immunotherapy.

Fibroblast activation protein peptide-targeted NIR-I/II fluorescence imaging for stable and functional detection of hepatocellular carcinoma.

Cancer-associated fibroblasts (CAFs) are the primary stromal component of the tumor microenvironment in hepatocellular carcinoma (HCC), affecting tumor progression and post-resection recurrence. Fibroblast activation protein (FAP) is a key biomarker of CAFs. However, there is limited evidence on using FAP as a target in near-infrared (NIR) fluorescence imaging for HCC. Thus, this study aims to develop a novel NIR fluorescent imaging strategy targeting FAP+ CAFs in HCC. The ICG-FAP-TATA probe was synthesized by conjugating a novel cyclization anti-FAP peptide with an indocyanine green derivative (ICG-NH2) as fluorophore, capable for NIR window I (NIR-I, 700-900nm) and II (NIR-II, 1000-1700nm) imaging. Its efficacy in lesion localization and other potential applications was evaluated. In vivo imaging of subcutaneous HCC models revealed that ICG-FAP-TATA specifically targeted FAP+ CAFs in the stroma and detected differences in CAFs loading within lesions. The fluorescence intensity/tumor-to-background ratio (TBR) positively correlated with FAP expression (R2 > 0.8, p < 0.05). Ex vivo incubation of tumor tissues with ICG-FAP-TATA provided stable fluorescence imaging of tumors in subcutaneous and orthotopic HCC models, including different cell line co-culture systems (LM3-luc, MHCC97H-luc, HepG2-luc + LX2), and various liver backgrounds (healthy/fibrotic) (n = 5 per group). TBR of the tumor mice models was higher for NIR-II than NIR-I imaging (3.89 ± 1.27 vs. 2.64 ± 0.64, p < 0.05). Moreover, NIR-I/II imaging of fresh tissues from seven patients with HCC undergoing surgery incubated with ICG-FAP-TATA visually provided the spatial distribution heterogeneity of CAFs. The targeted fluorescence was relatively enriched more in the blood flow direction and at the tumor edge, both of which were associated with tumor metastasis (all p < 0.05). This study presents a rapid and effective method for detecting HCC lesions, locating FAP+ CAFs, and visualizing high-risk areas for tumor metastasis at the macroscopic level. It offers a new promising approach with translational potential for imaging HCC.

TREM2 promotes the formation of a tumor-supportive microenvironment in hepatocellular carcinoma

BackgroundTriggering receptor expressed on myeloid cells 2 (TREM2), a surface receptor predominantly expressed on myeloid cells, is a major hub gene in pathology-induced immune signaling. However, its function in hepatocellular carcinoma (HCC) remains controversial. This study aimed to evaluate the role of TREM2 in the tumor microenvironment in the context of HCC progression.MethodsHCC was experimentally induced in wild-type (WT) and Trem2-deficient (Trem2−/−) mice, and clinical sample analysis and in vitro studies on macrophages were conducted. HCC cells were treated with conditioned medium from WT or Trem2−/− macrophages, and their malignant phenotypes and underlying mechanisms were analyzed.ResultsTREM2 deficiency reduced liver tumor burden in orthotopic and subcutaneous HCC models by altering CD8+ T cell infiltration. Trem2-deficient macrophages presented increased chemokine secretion. TGF-β1 was found to be positively correlated with TREM2 expression in HCC, and TGF-β blockade reversed TREM2 induction. On the other hand, TREM2+ macrophages were found to be associated with glycolysis and PKM2 expression in HCC cells; this association may be related to the secretion of IL-1β, which enhances the malignant phenotypes of HCC cells.ConclusionsThese results reveal that TREM2+ macrophages play a driving role in HCC progression by suppressing CD8+ T cell infiltration and promoting tumor cell glycolysis, providing a new therapeutic target for HCC.

Isoxazole-based molecules restore NK cell immune surveillance in hepatocarcinogenesis by targeting TM4SF5 and SLAMF7 linkage

Dynamic communication between hepatocytes and the environment is critical in hepatocellular carcinoma (HCC) development. Clinical immunotherapy against HCC is currently unsatisfactory and needs more systemic considerations, including the identification of new biomarkers and immune checkpoints. Transmembrane 4 L six family member 5 (TM4SF5) is known to promote HCC, but it remains unclear how cancerous hepatocytes avoid immune surveillance and whether avoidance can be blocked. We investigated how TM4SF5-mediated hepatic tumorigenesis avoids surveillance by natural killer (NK) cells, which are prevalent in the liver, and whether the avoidance can be blocked by anti-TM4SF5 agents. We used comprehensive structure activity relationship analysis to identify TM4SF5-specific isoxazole (TSI)-based small molecules that inhibit TM4SF5-mediated effects. TM4SF5 expressed by hepatocytes reduced NK cell cytotoxicity by downregulating stimulatory ligands/receptors, including signaling lymphocytic activation molecule family member 7 (SLAMF7). TM4SF5 bound SLAMF7 depending on N-glycosylation and caused intracellular trafficking of SLAMF7 from the plasma membrane to lysosomes for degradation. TSI treatments in cell lines and animal models of HCC blocked this binding, intracellular trafficking, and downregulation, resulting in higher levels of stimulatory NK cell ligands. In mouse xenograft models, TSI treatment abrogated HCC development by increasing the abundance and dispersion of Slamf7-positive cells in liver tissues, recapitulating the phenotype of Tm4sf5-knockout mice and indicating TSI-mediated restoration of NK cell surveillance. These findings suggest that TSIs can inhibit TM4SF5-mediated liver carcinogenesis by increasing NK cell surveillance.

Targeting aldolase A in hepatocellular carcinoma leads to imbalanced glycolysis and energy stress due to uncontrolled FBP accumulation.

Increased glycolytic flux is a hallmark of cancer; however, an increasing body of evidence indicates that glycolytic ATP production may be dispensable in cancer, as metabolic plasticity allows cancer cells to readily adapt to disruption of glycolysis by increasing ATP production via oxidative phosphorylation. Using functional genomic screening, we show here that liver cancer cells show a unique sensitivity toward aldolase A (ALDOA) depletion. Targeting glycolysis by disrupting the catalytic activity of ALDOA led to severe energy stress and cell cycle arrest in murine and human hepatocellular carcinoma cell lines. With a combination of metabolic flux analysis, metabolomics, stable-isotope tracing and mathematical modelling, we demonstrate that inhibiting ALDOA induced a state of imbalanced glycolysis in which the investment phase outpaced the payoff phase. Targeting ALDOA effectively converted glycolysis from an energy producing into an energy-consuming process. Moreover, we found that depletion of ALDOA extended survival and reduced cancer cell proliferation in an animal model of hepatocellular carcinoma. Thus, our findings indicate that induction of imbalanced glycolysis by targeting ALDOA presents a unique opportunity to overcome the inherent metabolic plasticity of cancer cells.

ERG mediates the differentiation of hepatic progenitor cells towards immunosuppressive PDGFRα+ cancer-associated fibroblasts during hepatocarcinogenesis

Cancer-associated fibroblasts (CAFs) play important roles in the occurrence and development of hepatocellular carcinoma (HCC) and are a key component of the immunosuppressive microenvironment. However, the origin of CAFs has not been fully elucidated. We employed single-cell sequencing technology to identify the dynamic changes in different subsets of fibroblasts at different time points in rat primary HCC model. Inflammation-associated CAFs (Pdgfrα+ CAFs) were subsequently identified, which demonstrated a significant correlation with the survival duration of HCC patients and a dual role in the tumour microenvironment (TME). On the one hand, they secrete the chemokines CCL3 and CXCL12, which recruit macrophages to the tumour site. On the other hand, they produce TGFβ, inducing the polarization of these macrophages towards an immunosuppressive phenotype. According to the in vitro and in vivo results, hepatic progenitor cells (HPCs) can aberrantly differentiate into PDGFRα+ CAFs upon stimulation with inflammatory cytokine. This differentiation is mediated by the activation of the MAPK signaling pathway and the downstream transcription factor ERG via the TLR4 receptor. Downregulating the expression of ERG in HPCs significantly reduces the number of PDGFRα+ CAFs and the infiltration of tumour-associated macrophages in HCC, thereby suppressing hepatocarcinogenesis. Collectively, our findings elucidate the distinct biological functions of PDGFRα+ cancer-associated fibroblasts (PDGFRα+ CAFs) within the TME. These insights contribute to our understanding of the mechanisms underlying the establishment of an immunosuppressive microenvironment in HCC, paving the way for the exploration of novel immunotherapeutic strategies tailored for HCC treatment.

The expression landscape and clinical significance of methyltransferase-like 17 in human cancer and hepatocellular carcinoma: a pan-cancer analysis using multiple databases

BackgroundMethyltransferase-like (METTL) family protein plays a crucial role in the progression of malignancies. However, the function of METTL17 across pan-cancers, especially in hepatocellular carcinoma (HCC) is still poorly understood.MethodsAll original data were downloaded from TCGA, GTEx, HPA, UCSC databases and various data portals. First, we comprehensively analyzed RNA-seq data from the HPA database of 25 human tissues. An array of bioinformatics methods was employed to explore the potential oncogenic roles of METTL17, including analyzing its related prognosis, mutation, landscapes, tumor stemness index, immune cell infiltration, and other factors among different tumors. Additionally, gene set enrichment analysis (GSEA) was used to analyze pathways associated with METTL17 in HCC. Immunohistochemistry (IHC) was performed on clinical samples to validate the differential expression of METTL17 in HCC and normal tissues. Ultimately, we constructed a METTL17-related risk-score model of HCC and validated its prognostic classification efficiency. Survival rates were calculated using the Kaplan-Meier method. Statistical significance was defined as P < 0.05.ResultsMETTL17 was differentially expressed in various cancers. METTL17 maintained strong correlations with the cancer patient’s prognosis, genetic alterations, tumor stemness index, and immune-infiltrated cells, etc. In addition, IHC experiments verified that METTL expression was significantly decreased in liver tissues of HCC patients compared to normal liver tissue. GESA analysis indicated METTL17 mainly involves oncogenic and immune-related pathways among HCC. MRPS5, CHCHD2, NCBP1, LRPPRC, DAP3, and BMS1 were included in a prognostic model based on METTL17’s interaction networks. Kaplan–Meier survival analysis of the prognostic model showed that the overall survival (OS) of the low-risk group was significantly better than that of the high-risk group (P < 0.001). The area under the receiver operating characteristic (ROC) curve (AUC) of the 1-year, 3-year, and 5-year OS were 0.747, 0.671, and 0.631, respectively.ConclusionsMETTL17 may serve as a novel prognostic marker and therapeutic target for human tumors, offering a theoretical foundation for formulating more effective and tailored clinical treatment options for cancers, particularly HCC.

THSWD upregulates the LTF/AMPK/mTOR/Becn1 axis and promotes lysosomal autophagy in hepatocellular carcinoma cells by regulating gut flora and metabolic reprogramming.

THSWD has the effect of reducing inflammation, improving microcirculation, and regulating immune status in patients with hepatocellular carcinoma. Regardless of its clear therapeutic effect, the underlying mechanism of action against hepatocellular carcinoma is not clear. To identify critical gut microbiota and its associated metabolites related to THSWD inhibition against hepatocellular carcinoma progression, we assessed the microbe-dependent anti-hepatocellular carcinoma effects of THSWD through 16s rRNA gene sequencing, fecal microbial transplantation and antibiotic treatment. Metabolic analyses, transcriptomic analyses, and molecular experiments were performed to explore how THSWD modulates the gut microbiota against hepatocellular carcinoma progression. As confirmed by in vivo and in vitro assays, THSWD reduced tumour growth rate and promoted apoptosis in hepatocellular carcinoma cells in hepatocellular carcinoma model mice, and liver and kidney indexes were detected and confirmed the safety of THSWD. Transcriptomic analysis revealed that the targets of THSWD were significantly enriched in multiple lysosomal autophagy signalling pathways, suggesting that lysosomal autophagy is probably associated with THSWD's therapeutic effect. Based on the integrated data analysis, THSWD delays hepatocellular carcinoma progression by increasing the intestinal microbiota Duncaniella and augmenting the metabolite glabrol, and the joint analysis of metabolic and genomic data suggests that this metabolite is associated with lysosomal autophagy, and cellular experiments confirmed that the The differential metabolite glabrol induces apoptosis in hepatocellular carcinoma cells by triggering the lysosomal autophagy-mediated apoptosis signalling pathway. Supplementation with glabrol metabolites up regulates the LTF/AMPK/mTOR/Beclin1 axis and promotes hepatocellular carcinoma cells with lysosomal autophagy and induced apoptosis in hepatocellular carcinoma cells.

Transcriptomic landscape of Hras12V oncogene-induced hepatocarcinogenesis with gender disparity

The genesis of hepatocellular carcinoma (HCC) is closely related to male factors and hyper-activated Ras signals. A transcriptomic database was established via RNA-Seq of HCC (T) and the adjacent precancerous liver tissue (P) of Hras12V transgenic mice (Ras-Tg, HCC model) and the normal liver tissue of wild-type mice (W) of both sexes. Comparative analysis within W, P, and T and correlation expression pattern analysis revealed common/unique cluster-enriched items towards HCC between the sexes. Specifically, the numbers of differentially expressed genes (DEGs) were much higher in females than in males, and tumor suppressor genes, such as p21Waf1/Cip1 and C6, were significantly higher in the female P. This finding denotes the higher sensitivity of female hepatocytes to the Ras oncogene and, therefore, the difficulty in developing HCC. Moreover, convergence in HCC between the sexes suggests the underlying mechanisms for the ineffectiveness of sex hormone therapies. Additionally, expression pattern analysis revealed that the DEGs and their relevant pathways were either positively or negatively associated with the HCC/Ras oncogene. Among them, the vital role of glutathione metabolism in HCC was established. This work provides a basis for future research on elucidating the underlying mechanisms, selecting the diagnostic biomarker, and planning the clinical therapy in HCC.

Iron metabolism in a mouse model of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains the most prevalent type of primary liver cancer worldwide. p53 is one of the most frequently mutated tumor-suppressor genes in HCC and its deficiency in hepatocytes triggers tumor formation in mice. To investigate iron metabolism during liver carcinogenesis, we employed a model of chronic carbon tetrachloride injections in liver-specific p53-deficient mice to induce liver fibrosis, cirrhosis and subsequent carcinogenesis. A transcriptome analysis of liver carcinoma was employed to identify p53-dependent gene expression signatures with subsequent in-depth analysis of iron metabolic parameters being conducted locally within liver cancers and at systemic levels. We show that all mutant mice developed liver cancer by 36-weeks of age in contrast to 3.4% tumors identified in control mice. All liver cancers with a p53-deficient background exhibited a local iron-poor phenotype with a “high transferrin receptor 1 (Tfr1) and low hepcidin (Hamp)” signature. At systemic levels, iron deficiency was restricted to female mice. Additionally, liver tumorigenesis correlated with selective deficits of selenium, zinc and manganese. Our data show that iron deficiency is a prevalent phenomenon in p53-deficient liver cancers, which is associated with alterations in Hamp and Tfr1 and a poor prognosis in mice and patients.