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Nucleotide-binding Oligomerization Domain 1 (NOD1) is a cytosolic pattern recognition receptor that senses specific bacterial peptidoglycan moieties, leading to the induction of inflammatory response. Besides, sensing peptidoglycan, NOD1 has been reported to sense metabolic disturbances including the ER stress-induced unfolded protein response (UPR). However, the underpinning crosstalk between the NOD1 activating microbial ligands and the metabolic cues to alter metabolic response is not yet comprehensively defined. Here, we show that underlying ER stress aggravated peptidoglycan-induced NOD1-mediated inflammatory response in hepatoma cells. The HepG2 cells, undergoing ER stress induced by thapsigargin exhibited an amplified inflammatory response induced by peptidoglycan ligand of NOD1 (i.e. iE-DAP). This aggravated inflammatory response disrupted lipid and glucose metabolism, characterized by de novo lipogenic response, and increased gluconeogenesis in HepG2 cells. Further, we characterized that the aggravation of NOD1-induced inflammatory response was dependent on inositol-requiring enzyme 1-α (IRE1-α) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) activation, in conjunction with calcium flux. Altogether, our findings suggest that differential UPR activation makes liver cells more sensitive towards bacterial-derived ligands to pronounce inflammatory response in a NOD1-dependent manner that impairs hepatic nutrient metabolism., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare that are relevant to the content of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hypoxia is one of the main hallmarks of hepatocellular carcinoma (HCC) resulting from improper oxygenation and insufficient nourishment of the HCC microenvironment. The effect of hypoxia is mediated by hypoxia-inducible factor-1A (HIF-1A) via targeting various downstream pathways, including glycolysis, angiogenesis, and survival signaling. However, HCC cell lines in a 2-dimensional (2D) setting do not resemble the metabolic signature of HCC. Here we aim to overcome these limitations by developing an HCC organoid that recapitulates the HIF-1A metabolic shift. The enrichment analysis of the RNA-Seq data revealed that HIF-1A-driven glycolytic shift is of the significant pathways. The established organoid model, using xeno-free plasma-derived extracellular matrix (ECM) as a scaffold and nutritive biomatrix, maintained its structural integrity and viability for up to 14 days; the comparative analysis of the cobalt (II) chloride (CoCl 2 )-treated organoids to the untreated ones unveiled reduced size and proliferative capacity. Interestingly, our organoid model showed an elevated expression of HIF-1A and glycolysis enzymes compared to their counterparts in the CoCl 2 -treated organoids. HIF-1A molecular expression-translated biochemical signature is further assessed in our spontaneously growing organoids showing an increase in glucose uptake, intracellular pyruvate, extracellular lactate dehydrogenase expression, and extracellular lactate production, while hydrogen peroxide (H 2 O 2 ), a marker for oxidative metabolism, is reduced. Our data confirmed the potency of the established organoid model to mimic the molecular and biochemical HIF-1A-driven metabolism, which validates its potential use as an in vitro HCC model. Our model naturally simulates hypoxic conditions and simultaneous HIF-1A-dependent glycolysis within HCC rather than using of CoCl 2 -induced hypoxic conditions., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the Institution Review Board (IRB) of the National Liver Institute (NLI), Menoufiya University (date: September 1st, 2018, number: 00140/2018). Consent to participate: Not applicable. Consent to publish: Not applicable., (© 2024. The Author(s).)

Hepatocellular carcinoma (HCC) ranks among the most lethal malignancies around the world. However, the current management strategies for predicting prognosis in HCC patients remain unreliable. Our study developed a robust prognostic model based on glutamine metabolism associated-genes (GMAGs), utilizing data from The Cancer Genome Atlas database. The prognostic values of model were validated through the databases of the Gene Expression Omnibus and International Cancer Genome Consortium via Kaplan‒Meier curves and receiver operating characteristic (ROC). The potential biological pathways associated with prognostic risk were investigated through different enrichment analysis, and Gene variation analysis. The correlation between prognostic model and therapeutic responses were analyzed. Quantitative real-time PCR (qRT-PCR) and cellular experiments were measured to analyze the GMAGs. Consequently, a prognostic model was constructed of 4 GMAGs (RRM1, RRM2, G6PD, and GPX7) through least absolute shrinkage and selection operator (LASSO) regression analysis. The Kaplan‒Meier curves and ROC curves showed a reliable predictive capacity of prognosis for HCC patients (p < 0.05). The enrichment analyses revealed a multitude of biological pathways that are significantly associated with cancer. Patients with high prognostic risk might be sensitive to immunotherapy (p < 0.05). The results of qRT-PCR revealed that all 4 GMAGs exhibited significantly higher expression levels in HCC samples compared to normal samples (p < 0.05). Moreover, the knockdown of RRM1 suppresses the progression of HCC cells. In this study, we developed a robust prognostic model for predicting the prognosis of HCC patients based on GMAGs, and identified RRM1 as a potential therapeutic target for HCC., Competing Interests: Declarations Ethical approval This research was approved by the Ethics Committee of Dalian medical university. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Curcumin (CUR) as one of the natural edible pigments is approved by the World Health Organization due to its nontoxic and anticancer effect. However, the utility of CUR is restricted due to its low oral bioavailability. Nanoparticle drug delivery systems like mesoporous silica nanoparticles (MSNs) have been extensively used due to their high specific surface area, high loading rate, and ease of modification. This study developed lactobionic acid (LA)-modified carboxymethyl chitosan (CMCS)-coated MSNs to deliver CUR specifically targeting hepatocellular carcinoma. Among these nanoparticles, LA targets liver cancer cells. CMCS utilizes pH-responsive release of CUR. The LA-CMCS-MSN@CUR (MSN@CUR) were evaluated using several methods, including Fourier transform infrared spectroscopy, transmission electron microscopy, and zeta potential measurements. Liver cellular uptake of MSN@CUR depends on a specific LA receptor-mediated endocytosis mechanism. Additionally, MSN@CUR performed with a better antitumor effect than Cur in H22 orthotopic transplantation of liver cancer and H22 solid tumor mouse models. Treatment with MSN@CUR significantly reduced the protein of VEGF, p-PI3K, and AKT, increased the protein of caspases 3 and 8, ultimately inhibited tumor migration, and promoted apoptosis. This study provides a new path for delivery of natural active ingredients with excellent bioavailability in the antitumor field.

Background: The response of hepatocellular carcinoma (HCC) to transarterial chemoembolization (TACE) treatment and its underlying mechanisms remain elusive. This study investigates the role of enzymes involved in fatty acid activation, specifically Acyl-CoA synthetase long chain 4 (ACSL4), in HCC patients treated with postoperative adjuvant TACE (PA-TACE) and in nutrient-deprived HCC cells., Methods: We examined the expression of ACSL4 and its family members in HCC clinical samples and cell lines. The clinical significance of ACSL4, particularly regarding the prognosis of patients treated with PA-TACE, was assessed using two independent HCC cohorts. We further explored the role of ACSL4 in glucose starvation-induced cell death in HCC cells and xenograft mouse models., Results: Among the family members, ACSL4 is the most up-regulated enzyme, associated with poor survival in HCC patients, particularly in post-recurrent TACE-treated patients in a Singapore cohort. ACSL4 is essential for HCC cell survival in response to glucose starvation, rather than to hypoxia or to the combination of hypoxia with doxorubicin or cisplatin. ACSL4-mediated arachidonic acid (AA) metabolism supports mitochondrial β-oxidation and energy production. CCAAT/enhancer binding protein α (CEBPA) transcriptionally regulates ACSL4 by binding 3 motifs (-623 to -613, -1197 to -1187 and -1745 to -1735) of ACSL4 upstream promoter region, enhancing its pro-survival effects. Furthermore, canagliflozin (Cana), a clinical-approved drug for type 2 diabetes, mimics glucose starvation and inhibits the growth of ACSL4-low xenograft tumors. Moreover, high ACSL4 or CEBPA expressions correlate with increased recurrence susceptibility after PA-TACE in the China-Guangxi HCC cohort., Conclusions: The CEBPA-ACSL4 pathway is critical in protecting HCC cells from glucose starvation-induced cell death, suggesting that ACSL4 and CEBPA could serve as valuable prognostic indicators and potential therapeutic targets in the context of PA-TACE treatment for HCC., Competing Interests: Declarations Ethics approval and consent to participate The studies involving two HCC cohorts were conducted with approval from the National Healthcare Group Domain Specific Review Board (NHG DSRB Ref: 2011/01580) and the Guangxi Medical University Institutional Review Board (GXMU #20160302–10). Informed consent was obtained from all patients participating in the study. All animal experiments were approved by the Guangxi Medical University Institutional Animal Care and Use Committees (#201910029) and performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Consent for publication The consents for publication from all authors were obtained. Competing interests The authors declare that they have no conflicts of interest., (© 2024. The Author(s).)

Radiofrequency ablation (RFA) is a commonly used minimally invasive treatment for hepatocellular carcinoma (HCC). However, incomplete radiofrequency ablation (iRFA) promotes tumor progression and metastasis. There is an urgent need to develop innovative strategies to enhance the efficacy of iRFA. The upregulation of heat shock proteins (HSPs) and activation of protective autophagy in tumor cells upon exposure to sublethal heat enhance the thermotolerance, thereby promoting tumor cell survival. Here, 3-methyladenine (3-MA) and lonidamine (LND) co-encapsulated liposomes (Lip@LND/3-MA) are designed to enhance the efficacy of iRFA by simultaneous inhibition of glycolysis and autophagy. On one hand, LND inhibits hexokinase, a key enzyme in glycolysis, and thus reduces ATP production and consequently suppresses the expression of HSPs. On the other hand, 3-MA, as an autophagy inhibitor, can inhibit protective autophagy after iRFA. Lip@LND/3-MA is confirmed to suppress the expression of HSPs and reduce the autophagy level during RFA. Therefore, the thermotolerance of tumor cells is significantly weakened, leading to remarkably enhanced therapeutic efficacy of iRFA. It is believed that simultaneous inhibition of HSPs and autophagy is a promising therapeutic strategy in clinical practice of RFA.

Lenvatinib resistance presents a significant challenge in the clinical management of advanced hepatocellular carcinoma (HCC). To address this issue, we established lenvatinib resistant HCC cells and performed high-throughput screening using FDA-approved anti-cancer drug library. Through quantitative selective drug sensitivity scoring (sDSS), pacritinib, a well-known JAK inhibitor, emerged as the top candidate, displaying the highest sDSS score among 219 compounds. We further demonstrated that pacritinib reduced the viability of a panel of HCC cell lines in a dose-dependent manner, while exhibiting minimal effects on normal liver cells. Interestingly, pacritinib, but not other JAK inhibitors such as ruxolitinib, upadacitinib, or filgotinib, acted synergistically with lenvatinib in HCC cells. In lenvatinib-resistant HCC cells, pacritinib significantly decreased proliferation and induced apoptosis. Rescue studies using IL-1 receptor-associated kinase 1 (IRAK1) overexpression and knockdown revealed that pacritinib's effects are mediated through IRAK1, with minimal impact on normal liver cells. In a xenograft model of lenvatinib-resistant HCC, oral administration of pacritinib significantly reduced tumor size without affecting body weight. Immunohistochemical analysis of tumor sections revealed that pacritinib reduced Ki67 staining and phosphorylated IRAK1. Our findings suggest that pacritinib may be a promising therapeutic option for the treatment of advanced HCC, particularly in patients who have developed resistance to lenvatinib., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) is a common malignancy with a poor prognosis. The recommended treatment of unresectable HCC involves targeted therapy, for example sorafenib, combined with immunotherapy. A recent article reported that sorafenib could induce ferroptosis escape in HCC. Brusatol is a novel Nrf2 inhibitor that takes effects in various diseases. In our study, we aimed to identify whether the addition of Brusatol to sorafenib could reverse ferroptosis escape in Huh7 cells., Methods: The cultured Huh7 cells treated by sorafenib with or without Brusatol addition were harvested for ferroptotic phenotype experiments and ferroptosis-related markers such as GPX4 and SLC7A11 were detected. In vivo experiments were conducted to discover the effect of Brusatol in combination with sorafenib in liver tumor bearing mice. Mechanism signaling pathways were detected by RNA-sequencing., Results: Brusatol alone could induce Huh7 cell death and sorafenib could moderately mediate Huh7 cell ferroptosis by paradoxically inhibiting GPX4. However, sorafenib simultaneously upregulates Nrf2 signaling in Huh7 cells fighting against ferroptosis to result in sorafenib resistance. The addition of Brusatol could potentiate ferroptosis in Huh7 cells through downregulating Nrf2 and the downstream HO-1 and NQO1, thus enhancing the efficacy of sorafenib, which could be reversed by ferrostatin-1 treatment., Conclusion: In conclusion, Brusatol improves the efficacy of sorafenib by inducing ferroptosis via hindering Nrf2 signaling activation in HCC., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The molecular profile of paracancerous tissue has been reported to be prognostic for recurrence in patients with different cancers. This study investigated the clinical significance of the protein expression of gamma-butyrobetaine hydroxylase 1 (BBOX1) in tumor and paracancerous tissues of hepatocellular carcinoma (HCC) patients. This study assessed the role of BBOX1 mRNA in tumor and paracancerous tissues in HCC via bioinformatics analysis. The protein levels of BBOX1 in paired tumor and paracancerous tissues from 83 HCC patients were determined by immunohistochemistry. The associations among BBOX1 protein levels, clinicopathological characteristics and the survival probability of HCC patients were also analyzed. The results revealed that BBOX1 mRNA expression was significantly lower in HCC tissues than in noncancerous tissues. HCC patients with low BBOX1 mRNA expression had a significantly poorer overall survival than those with high BBOX1 expression did. Immunohistochemical analysis of BBOX1 protein expression further confirmed that its levels were markedly lower in HCC tissues than in paracancerous tissues. High BBOX1 expression in paracancerous tissues correlated with poor overall survival and disease-free survival in HCC patients. These findings suggest that the paracancerous BBOX1 expression might be a credible indicator for overall survival or disease-free survival in HCC patients., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: The relationship between hepatocellular carcinoma (HCC) metastasis and cancer metabolism reprogramming is becoming increasingly evident. Ubiquitin-specific protease 11 (USP11), a member of the deubiquitinating enzyme family, has been linked to various cancer-related processes. While USP11 is known to promote HCC metastasis and proliferation, the precise mechanisms, especially those related to cancer metabolism, remain unclear., Methods: Through mass spectrometry, co-immunoprecipitation, immunofluorescence, and ubiquitination assays, we identified USP11 as the key deubiquitinase for SREBF1.Lipogenesis was evaluated using Oil Red O and Nile Red staining, along with the detection of triglycerides and cholesterol. To assess HCC cell proliferation, migration, and invasion in vitro, Transwell assays, EDU, colony formation, and CCK-8 were conducted. Xenograft models in nude mice were developed to verify the role of the USP11/SREBF1 axis in lipogenesis and tumor growth in vivo., Results: USP11 directly interacts with SREBF1, and its silencing leads to the disruption of SREBF1 stabilization through K48-linked deubiquitination and degradation. Importantly, the truncated mutant USP11 (503-938 aa) interacts with the truncated mutant SREBF1 (569-1147aa), with K1151 playing a crucial role in this interaction. Higher levels of USP11 enhance lipogenesis, proliferation, and metastasis in HCC cells. Importantly, the knockdown of SREBF1 weakened the effects of USP11 in enhancing lipogenesis and tumorigenesis. Futhermore, the elevated expression of USP11 and SREBF1 in HCC tissue serves as an indicator of poor prognosis in HCC patients., Conclusions: In summary, our study reveals that USP11 promotes HCC proliferation and metastasis through SREBF1-induced lipogenesis. These findings provide a foundation for novel therapies targeting lipid metabolism in HCC., Competing Interests: Declarations Ethics approval and consent to participate The Ethics Committee of the Second Affiliated Hospital of Nanchang University approved the inclusion of human participants and the use of human data and tissues in this study, and conducted them in accordance with the principles of the Declaration of Helsinki. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co., Ltd. (Ethics ID: RYE2023120301) Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide, with death rates increasing by approximately 2-3% per year. The high mortality and poor prognosis of HCC are primarily due to inaccurate early diagnosis and lack of monitoring when liver transplantation is not feasible. Fatty acid (FA) metabolism is a critical metabolic pathway that provides energy and signaling factors in cancer, particularly in HCC, and promotes malignancy. Therefore, it is essential to explore specific FA metabolism-related diagnostic and prognostic signatures that can enable the effective early diagnosis and monitoring of HCC., Methods: In this study, we used genes associated with FA metabolism pathway and weighted gene co-expression network analysis (WGCNA) to establish a gene co-expression network and identify hub genes related to HCC (disease WGCNA) and FA clusters (cluster WGCNA). A diagnostic model was constructed using data downloaded from the Gene Expression Omnibus database (GSE25097), and a prognostic model was established using The Cancer Genome Atlas cohort, in which Univariate Cox regression analysis, multivariate Cox risk model, and LASSO Cox regression analysis were applied. The immune infiltration of HCC cells was evaluated using CIBERSORT. The function of the key SLC22A1 gene was experimentally verified in vitro and in vivo., Results: Twelve overlapping genes (CPEB3, ASPDH, DEPDC7, ETFDH, UGT2B7, GYS2, F11, ANXA10, CYP2C8, GLYATL1, C6, and SLC22A1) from disease and cluster WGCNA were identified as key genes and used in the construction of the diagnostic and prognostic models. The RF model had the highest area under the ROC curve (AUC) of 0.994 was identified as the most effective for distinguishing patients with HCC with different features. The top five important genes (C6, UGT2B7, SLC22A1, F11, and CYP2C8) from the RF model were selected as diagnostic genes for further analysis (ROC curves: AUC value = 0.986, 95% confidence interval [95% CI] = 0.967-0.999). Moreover, a risk score formula consisting of four genes (GYS2, F11, ANXA10 and SLC22A1) was established and its independent prognostic ability was further demonstrated (univariate Cox regression analysis: hazard ratio [HR] = 3.664%, 95% CI = 2.033-6.605, P < 0.001; multivariate Cox regression analysis: HR = 2.801%, 95% CI = 1.553-5.049, P < 0.001). Additionally, in vitro and in vivo experiments demonstrated that SLC22A1 inhibits HCC tumor development, suggesting it may be a potential therapeutic target for HCC., Conclusions: These findings indicate a considerable value of specific FA metabolism-related genes in the diagnostic and prognostic evaluation of HCC, which provide novel insights into the disease's management, as well as has potential implications for personalized treatment strategies. However, further investigation of the effects of these model genes on HCC is required., Competing Interests: Declarations Ethics approval and consent to participate All participants signed informed consent forms. This study was reviewed and approved by the Institutional Review Board (Approval Number: 21K196) of Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai. The animal study also approved under declaration with the approval number of SHDSYY-2021-4709. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Niemann-Pick Type C1 (NPC1) plays a significant role in the development of liver diseases and liver cancer. Our objective was to investigate the involvement of NPC1 in regulating liver cancer development. We observed that high levels of NPC1 expression in tumor tissues from patients with liver cancer were associated with a poor prognosis. Through in vitro experiments, we found that inhibiting NPC1 expression reduced the proliferation, invasion, and migration of liver cancer cells, while also inducing apoptosis in these cells. Additionally, the inhibition of NPC1 led to decreased activation of Wnt/β-catenin signaling. In vivo studies further supported our findings by demonstrating that the suppression of liver cancer cell growth was effectively achieved through the inhibition of NPC1. Overall, our results strongly indicate that the inhibition of NPC1 suppresses liver cancer cell proliferation. Targeting NPC1 is a promising potential therapeutic strategy for liver cancer., Competing Interests: Declarations Ethics approval and consent to participate Clinical SamplesThe study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the ethics committee of the West China Hospital of Sichuan University (No. 2016-91). All subjects provided informed consent for inclusion before participating in the study, and the tissue was used for future studies.Animal StudyThis animal study was approved by the Ethics Committee of West China Hospital, Sichuan University, and conformed to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Consent for publication Not Applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Aims: Ficolin 3 (FCN3) has the highest complement-activating capacity through the lectin pathway and is synthesized mainly in the liver and lung. Yet, its potential molecular mechanism in hepatocarcinogenesis is not fully understood., Materials and Methods: The expression of FCN3 in hepatocellular carcinoma (HCC) tumor and non-tumor tissues was analyzed by RT-qPCR, Western blotting and immunofluorescence staining assays. Lentivector-mediated ectopic overexpression was performed to explore the role of FCN3 in vitro and in vivo. Whether FCN3 inhibited HCC cell growth and survival via complement pathway was determined with immunocytochemical staining for C3b, membrane attack complex (MAC) formation and complement killing assay using recombinant FCN3 (rFCN3) in combination with human serum with or without heat inactivation, and with C6 blocking antibody., Key Findings: The transcript and protein of FCN3 were found to be remarkably down-regulated in HCC tumor tissues. FCN3 expression was found to be associated with better survival of HCC patients. Restoration of FCN3 expression significantly inhibited proliferation, migration and anchorage independent growth of HCC cell lines, and xenograft tumor growth. FCN3 expression induced apoptosis of HCC cells. C3 and MAC formation was stimulated by FCN3 overexpression or rFCN3 treatment. rFCN3 enhanced human serum-induced complement activation and cell death. C6 blocking antibody significantly attenuated complement-mediated cell death and restored the growth of FCN3-overexpressing HCC cells., Significance: FCN3 has a malignant suppressor role in HCC cells. Our study provides new insights into the molecular mechanisms that drive HCC progression and potential therapeutic targets for treating HCC., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is a prevalent and lethal malignancy with significant global impact, necessitating the development of novel therapeutic strategies and drugs. Ferroptosis, a newly identified form of iron-dependent programmed cell death, has emerged as a promising strategy to combat HCC. Sappanone A, an isoflavone compound derived from the heartwood of Biancaea sappan (L.) Tod., is known for its anti-inflammatory and antioxidant properties. However, its anti-HCC effects and underlying mechanisms remain unclear. This study is the first time to demonstrate the anti-tumor effect of Sappanone A on HCC both in vitro and in vivo, through the assessment of cell viability and apoptosis following Sappanone A treatment. Flow cytometry and confocal microscopy revealed that Sappanone A induced ferroptosis in HCC cells by increasing Fe 2+ accumulation, reactive oxygen (ROS) level, and lipid peroxidation, specifically targeting inosine monophosphate dehydrogenase-2 (IMPDH2). Additionally, Western blot analysis suggested that the anti-HCC effects of Sappanone A were mediated through the regulation of the NRF2/xCT/GPX4 axis, highlighting its potential to enhance ferroptosis in HCC cells and underscoring the critical role of IMPDH2 in HCC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: This study aimed to explore novel targets for hepatocellular carcinoma (HCC) treatment by investigating the role of fatty acid metabolism., Methods: RNA-seq and clinical data of HCC were obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatic analyses were employed to identify differentially expressed genes (DEGs) related to prognosis. A signature was then constructed using the Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression to classify HCC patients from the TCGA database into low-risk and high-risk groups. The predictive performance of the signature was evaluated through principal components analysis (PCA), Kaplan Meier (KM) survival analysis, receiver operating characteristics (ROC) curves, nomogram, genetic mutations, drug sensitivity analysis, immunological correlation analysis, and enrichment analysis. Single-cell maps were constructed to illustrate the distribution of core genes. Immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR), and western blot were employed to verify the expression of core genes. The function of one core gene was validated through a series of in vitro assays, including cell viability, colony formation, wound healing, trans-well migration, and invasion assays. The results were analyzed in the context of relevant signaling pathways., Results: Bioinformatic analyses identified 15 FAMGs that were related to prognosis. A 4-gene signature was constructed, and patients were divided into high- and low-risk groups according to the signature. The high-risk group exhibited a poorer prognosis compared to the low-risk group in both the training (P < 0.001) and validation (P = 0.020) sets. Furthermore, the risk score was identified as an independent predictor of OS (P < 0.001, HR = 8.005). The incorporation of the risk score and clinicopathologic features into a nomogram enabled the effective prediction of patient prognosis. The model was able to effectively predict the immune microenvironment, drug sensitivity to chemotherapy, and gene mutation for each group. Single-cell maps demonstrated that FAMGs in the model were distributed in tumor cells. Enrichment analyses revealed that the cell cycle, fatty acid β oxidation and PPAR signaling pathways were the most significant pathways. Among the four key prognostically related FAMGs, Spermine Synthase (SMS) was selected and validated as a potential oncogene affecting cell cycle, PPAR-γ signaling pathway and fatty acid β oxidation in HCC., Conclusions: The risk characteristics based on FAMGs could serve as independent prognostic indicators for predicting HCC prognosis and could also serve as evaluation criteria for gene mutations, immunity, and chemotherapy drug therapy in HCC patients. Meanwhile, targeted fatty acid metabolism could be used to treat HCC through related signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Liuwei dihuang pills is a famous Traditional Chinese Medicine with various anti-cancer properties. Over 50 pharmaceutical manufacturers produce Liuwei dihuang pills in China and an estimated millions of people around the world orally take it every day. D-glucaro-1,4-lactone (1,4-GL) was quantified to be about 12.0 mg/g in Liuwei dihuang pills and a primary bioactive component of it inhibiting the activity of β-glucuronidase in vivo. 1,4-GL can prevent and effectively inhibit various types of cancer. However, its exact mechanism of action remains unknown. The study would justify the traditional usage of Liuwei dihuang pills against cancers., Aim of the Study: 1,4-GL, a bioactive ingredient derived from Liuwei dihuang pills, a famous Traditional Chinese Medicine, could delay the progression of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The mechanism underpinning the effect, however, remains poorly understood., Materials and Methods: Healthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and 1 HNMR-based metabonomic analysis was employed. 10 metabolites in uric acid pathway were quantitatively determined by UPLC-MS/MS. The expression of xanthine dehydrogenase (XDH), SLC2A9 mRNA, and SLC2A9 protein was determined using RT-qPCR and Western Blot. The effect of 1,4-GL on HCC-LM3 cells was verified in vitro. The alterations of ROS activity, SLC2A9 and XDH gene levels were observed in NCTC-1469 cells induced by lipopolysaccharide (LPS) after 1,4-GL treatment., Results: After the intervention of 1,4-GL, improved pathological morphology, liver lesions in HCC rats was observed with restored serum levels of AFP, AST, ALP, γ-GGT and Fisher's ratio. Hepatic metabonomics revealed that puring metabolism were significantly regulated by 1,4-GL in HCC rats. Uric acid, xanthine and hypoxanthine levels were quantified by UPLC-MS/MS and found to be nearly restored to control levels after 1,4-GL treatment in HCC rats. Changes in xanthine oxidase activity, XDH mRNA expression, and SLC2A9 mRNA and protein expression were also reversed. 1,4-GL treatment in LM3 HCC cells were consistent with the results in vivo. Furthermore, oxidative stress indicators such as T-SOD, GSH, CAT and MDA in serum and liver were improved after HCC rats treated with 1,4-GL. In vitro, 1,4-GL was observed to reduce lipopolysaccharide-induced ROS levels in NCTC-1469 cells with enhanced mRNA and protein expression of SLC2A9 and decreased mRNA level of XDH., Conclusion: The protective effects of 1,4-GL against DEN-induced HCC by reducing uric acid and ROS levels due to down-regulation of uric acid production and up-regulation of SLC2A9 expressions. 1,4-GL may represent a novel treatment that improves recovery from HCC by targeting uric acid-ROS pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) is a common cancer that is fatal and has a dismal prognosis. Obovatol (Ob), a novel lignan derived from the leaf and stem bark of Magnolia obovata Thunb, has exhibited anti-tumor effect on diverse tumors. However, its effect and mechanisms on HCC remain to be further explored., Methods: Huh7 and Hep3B cells, as well as BALB/c nude mice were used to determine the function and mechanisms of Ob on growth, invasion and immune escape by cell counting kit-8, transwell, enzyme-linked immunosorbent assay (ELISA) and western blot experiments., Results: Ob reduced the cell viability of Huh7 and Hep3B cells, with a IC50 value of 57.41 µM and 62.86 µM, respectively. Ob declined the invasion ability, the protein expression of N-cadherin and the concentrations of IL-10 and TGF-β, whereas increased the E-cadherin expression and the contents of IFN-γ and IL-2 in Hep3B and Huh7 cells. Mechanically, Ob decreased the protein level of p-JAK/JAK, p-STAT3/STAT3 and PD-L1, which was partly restored with the treatment of RO8191, an activator of JAK/STAT3 axis. The effect of Ob on the cell viability, the invasion ability, the protein level of N-cadherin and E-cadherin, and the concentrations of IL-10, TGF-β, IFN-γ and IL-2 in both Hep3B and Huh7 cells was reversed with the management of RO8191. In vivo, Ob reduced tumor volume and weight, the level of N-cadherin, PD-L1, p-JAK/JAK, and p-STAT3/STAT3, with an elevated expression of E-cadherin and IFN-γ., Conclusion: Ob downregulated the JAK/STST3/PD-L1 pathway to attenuate the growth, invasion and immune escape of HCC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: CircRNAs have been demonstrated to play a crucial role in regulating the growth and progression of various cancers, including hepatocellular carcinoma (HCC). Nevertheless, the circRNA's expression pattern and function in HCC need more investigation., Methods: Bioinformatics techniques were used to identify differentially expressed circRNAs in HCC. CircASH2L expression in HCC tissues was assessed through qRT-PCR and ISH analysis. To assess circASH2L's impact on HCC progression, a variety of experiments were carried out both in vitro and in vivo, such as CCK8, colony formation, EdU assay, flow cytometry, transwell assay, and xenograft mouse model. Various experimental techniques including qRT-PCR, dual luciferase reporter assay, FISH, RNA pull-down, and RIP experiments were utilized to evaluate the relationship between circASH2L, miR-525-3p, and MTUS2. Additionally, experiments were conducted to explore the impact of m 6 A modification on circASH2L expression, including RNA stability assay, m 6 A RNA immunoprecipitation assay (MeRIP), and Co-IP experiments., Results: We found that circASH2L was downregulated in HCC tissues and the downregulation of circASH2L was significantly correlated with malignant characteristics as well as poor overall survival of patients with HCC. CircASH2L was found to inhibit cells growth, migration and invasion as well as tumorigenesis and metastasis in vivo. Mechanistically, we established that circASH2L directly interacted with miR-525-3p to enhance MTUS2 expression, subsequently leading to tumor suppression. Moreover, the influence of circASH2L on tumor suppression was attenuated by increasing miR-525-3p levels, and MTUS2 was recognized as an essential intermediary in circASH2L-induced tumor suppression. Additionally, N 6- methyladenosine (m6A) modification was identified in circASH2L. Our data suggested that METTL3 was responsible for mediating m 6 A methylation of circASH2L, ultimately regulating circASH2L expression through the promotion of its degradation. These findings collectively highlight the role of circASH2L as a tumor suppressor through a unique circASH2L/miR-525-3p/MTUS2 axis, shedding light on the significance of m 6 A modification in regulating circASH2L function., Conclusion: The work emphasizes circASH2L as a promising therapeutic target for treating HCC, offering new insights into the role of circRNAs in HCC development., Competing Interests: Declarations Ethics approval and consent to participate The procedure of human sample collection was approved by the Institutional Review Board of Tongji Medical College of Huazhong University of Science and Technology on 16 November 2021 (NO. TJ-IRB20211163) and all procedures were meet the criteria of the Declaration of Helsinki. Written informed consent for specimens was obtained from all patients. All of the animal experiments met the National Institutes of Health guidelines and were approved by the Committee on the Ethics of Animal Experiments of the Tongji Medical College, HUST on 20 September 2021 (approval NO. TJH-202109032, IACUC Number: 2750). All animal experiments were performed in accordance with the Declaration of Helsinki and “Guide for The Care and Use of Laboratory Animals” (NIH publication 86-23 revised 1985). Consent for publication All authors had read and agreed to publish the paper. Competing interests All authors declare that they have no conflict of interests., (© 2024. The Author(s).)

RNA-dependent protein kinase (PKR) may have a positive regulatory role in controlling tumor growth and progression in hepatocellular carcinoma (HCC). However, the downstream substrates and the molecular mechanism of PKR in the growth and progression of HCC have not been clarified. In this study, mass spectrometry analysis was performed with immunoprecipitated samples, and 4.1R was identified as a protein that binds to PKR. In transfected COS7 cells, an immunoprecipitation experiment showed that 4.1R binds to wild-type PKR, but not to a kinase-deficient mutant PKR, suggesting that PKR binds to 4.1R in a kinase activity-dependent manner. In HCC cell lines, HuH7 and HepG2, the expression level of 4.1R protein was shown to be regulated by protein expression and activation of PKR. Interestingly, high expression of 4.1R, as well as PKR, is associated with a worse prognosis in HCC. PKR increased HCC cell growth in both anchorage-dependent and anchorage-independent manners, whereas 4.1R was involved in HCC cell growth only in an anchorage-independent manner, not in an anchorage-dependent manner. The rescue experiment indicated that increased anchorage-independent growth of HCC cells by PKR might be caused by 4.1R. In conclusion, PKR associates with 4.1R and promotes anchorage-independent growth of HCC. The PKR-4.1R axis might be a new therapeutic target in HCC., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The PI3K/Akt pathway is overexpressed in nearly 50% of hepatocellular carcinomas and inhibits apoptosis by promoting the expression of antiapoptotic genes. Serine protease inhibitors have been shown to induce apoptosis in hepatoma cells by downregulating SPINK13 in the PI3K/Akt pathway. In this study, SPINK13 was expressed in lentiviral vectors. Changes in signaling pathway adapter proteins, apoptosis regulatory proteins, cell cycle regulatory proteins, and the biological behavior of hepatocellular carcinoma were observed in cell and nude mouse xenograft models. The underlying mechanism of endogenous SPINK13-induced apoptosis in hepatocellular carcinoma cells was explored via transcriptomics. As a result, endogenous SPINK13 might inhibit the activity of Furin protease, downregulate the Notch1/Hes1 pathway in a binding manner, activate the direct effector PTEN, inhibit Akt phosphorylation, inactivate the downstream PI3K/Akt pathway, and ultimately lead to mitochondrial apoptosis and cell cycle arrest in hepatoma cells. Therefore, the Notch1/Hes1/PTEN pathway may act upstream of SPINK13 to downregulate the PI3K/Akt signaling pathway. Our study helps elucidate the underlying mechanism of SPINK13 in anti-hepatocellular carcinoma and lays a theoretical foundation for the development of novel therapeutic serine protease inhibitors., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Despite public health measures, type 2 diabetes (T2D) is still a significant concern worldwide, given its associated complications, including hepatic alterations. The role of epithelial-to-mesenchymal transition (EMT) in liver fibrosis is well established. However, its effects on the progression of diabetic liver diseases are not well understood. Therefore, this study aims to investigate the mRNA/miRNA axes involved in this process. Bioinformatic analysis revealed new EMT-associated genes (CDH2, ITGB1, COL4A1, COL1A1, TNC, CCN2, and JUN), which showed higher expression in obese T2D and hepatocellular carcinoma (HCC) patients. In addition, six miRNAs (miR-21-5p, miR-26a-5p, miR-34a-5p, miR-106a-5p, miR-320a-3p and miR-424-5p) have been found as potential targets. Among them, miR-26a-5p and miR-424-5p were significantly downregulated in nonalcoholic steatohepatitis (NASH) and HCC (p < 0.05), being considered potential negative regulators of the EMT process. In our hepatic mesenchymal culture model, miR-26a-5p negatively regulated cadherin 2 (also known as N-cadherin, CDH2) and promoted the cadherin 1 (also known as E-cadherin, CDH1) expression. Our results reveal potential molecules involved in liver tumor development. Moreover, we observe that miR-26a-5p impairs EMT in the initial stages of diabetic liver disease by inhibiting CDH2 and could be a valuable target in this pathology., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval All research was conducted in accordance with the Declarations of Helsinki. The research experimental procedure was approved by the Ethics Committee of the Fundeni Clinical Institute (51715/27.09.2022). Informed consent All enrolled patients gave written informed consent., (© 2024. The Author(s).)

The long noncoding RNA (lncRNA) HOTAIR has been shown to act as an oncogene in a variety of cancers, including hepatocellular carcinoma (HCC). MicroRNA-122 (miR-122) is a key liver-specific miRNA that is frequently inhibited in HCC and is associated with poor prognosis. However, a potential relationship between HOTAIR and miR-122 in individual hepatocytes has not been explored. To this end, we propose here an intracellular catalytic hairpin self-assembly-CRISPR/Cas12a tandem multiplexed signal amplification strategy for the simultaneous quantification of HOTAIR and miRNA-122 in a single hepatocyte. We applied this method to analyze both normal HL-7702 liver cells and HepG2 HCC cells, and found that HL-7702 cells contained large amounts of miRNA-122, while the content of miRNA-122 in HepG2 cells was low. However, the level of HOTAIR in HepG2 cells was much higher than that in HL-7702 cells, confirming the overexpression of HOTAIR in HCC cells. We achieved the simultaneous absolute quantification of HOTAIR and miRNA-122 in single cells, providing an important method to study the relationships between these two RNA molecules in individual cells.

SLC16A3/monocarboxylate transporter 4 (MCT4) regulates intracellular lactate transport and is highly expressed in many tumors, indicating poor prognosis. It may be related to inducing hypoxia, apoptosis and other mechanisms, but the study of MCT4 in HCC is far from complete. In this study, we first analyzed the expression of SLC16A3 in HCC tumor and non-tumor tissue samples based on TCGA data and immunohistochemistry. Subsequently, the effects of SLC16A3 expression on cell proliferation and invasion were analyzed using hepatocellular carcinoma (HCC) lines, and Western blot (WB) analysis was performed to explore the changes in pathway proteins and ferroptosis proteins. Finally, the drug sensitivity was tested by CCK8 kit. We found that SLC16A3 was significantly upregulated in tumor tissues, and was significantly correlated with TNM stage, histological grade, and macrovascular invasion. TCGA data and WB analysis showed that the high expression of SLC16A3 induced hypoxia, and knockdown could reverse hypoxia and inhibit ERK phosphorylation, thus limiting the malignant behavior of HCC cells. Moreover, knockdown of SLC16A3 significantly increased the level of lipid peroxidation and reactive oxygen species (ROS), while the expressions of GPX4, DHODH and SLC7A11 were inhibited. The expression of SLC16A3 affected the sensitivity of HCC cells to chemotherapy and targeted drugs, and RNA sequencing data suggested that the expression level influenced tumor microenvironment and response to immunotherapy. So, we draw a conclude that SLC16A3 is associated with poor prognosis of HCC. Inhibition of SLC16A3 expression is a potential therapeutic target for HCC., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The mitochondrial malic enzyme 2 (ME2), which is frequently elevated during carcinogenesis and may be a target for cancer therapy, catalyzes the conversion of malate to pyruvate. The processes controlling ME2 activity, however, remain largely unclear. In this work, we show that human hepatocellular carcinoma (HCC) tissues contain high levels of ME2 and that the methylation of ME2 stimulates the growth and migration of HCC cells. Furthermore, we observed that ME2 interacts with protein arginine methyltransferase 1 (PRMT1) and that ME2 enzymatic activity is activated by mutation of ME2 at lysine 67. Mitochondrial respiration was markedly increased by activated ME2, which promoted cell division and carcinogenesis. Furthermore, a negative prognosis for patients was strongly linked with the expression levels of PRMT1 and ME2 R67K in HCC tissues. These findings imply that hepatocellular carcinoma growth is aided by PRMT1-mediated ME2 methylation, that is an essential signaling event that cancer cells need to continue mitochondrial respiration., Competing Interests: Competing interests The authors declare no competing interests. Ethics approval and consent to participate The study was approved by the Human Research Ethics Committee of Guizhou Medical University. All procedures involving humans were in accordance with the ethical standards of the national responsible committee on human experimentation and with the Helsinki Declaration of 1964 and later versions. Informed consent was obtained from all patients. All animal studies were approved by the Guizhou Medical University Committee on Ethics of Animal Experiments (IRB). All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Research Council’s Guide for the Care and Use of Laboratory Animals. Consent for publication All authors have agreed to publish this manuscript., (© 2024. The Author(s).)

Objective: To compare the performance of [ 18 F]FDG and [ 18 F]FAPI-04 in PET/CT evaluation for liver cancer lesions, with a further exploration of the associations between PET semiquantitative data and immunohistochemical markers to liver cancer., Methods: Patients with suspected malignant liver lesions (MLL) underwent [ 18 F]FDG and [ 18 F]FAPI-04 PET/CT scanning. Liver lesions were visually classified as positive or negative based on their uptake level exceeding that of adjacent normal liver tissue. SUVmax and tumor-to-background ratio (TBR) were recorded for semi-quantitative analysis. Sensitivity, specificity and accuracy of each tracer were determined using pathological findings as the gold standard. Furthermore, immunohistochemical analysis provided the molecular characteristics of all MLLs. Comprehensive analysis explored correlations between these molecular markers and PET semiquantitative parameters (SUVmax andTBR) to identify potential associations., Results: The study enrolled 44 patients, with 39 confirmed cases of MLL, comprising 28 hepatocellular carcinomas (HCC) and 11 intrahepatic cholangiocarcinomas (ICC). For MLL detection, [ 18 F]FAPI-04 and [ 18 F]FDG exhibited sensitivities of 84.6% (33/39) and 76.9% (30/39), specificitiesy of 60% (3/5) and 100%(5/5), and accuracy of 81.8% (36/44) and 79.5%(35/44). Across all liver lesions, [ 18 F]FAPI-04 significantly surpassed [ 18 F]FDG in SUVmax(10.54 ± 6.72 VS. 7.68 ± 6.79) and TBR(4.35 ± 3.78 Vs. 3.17 ± 3.05). Notably, [ 18 F]FAPI-04 displayed markebly elevated SUVmax in benign liver lesions (BLLs) (P = 0.032), HCCs (P = 0.005), and ICCs (P = 0.011). Lesions with hepatocyte negativity (P = 0.023), CD34 negativity(P = 0.044), and high Ki67 expression (> 30%) (P = 0.001) had higher SUVmax on [ 18 F]FAPI-04. Additionally, ARG-1-negative lesions demonstrated higher TBR on [ 18 F]FAPI-04 than ARG-1-positive lesions(P = 0.018). No significant SUVmax/TBR differences were observed with [ 18 F]FDG based on these markers. A linear relationship was identified between Ki67 scores and SUVmax of [ 18 F]FAPI-04 (R = 0.603, P < 0.001)., Conclusion: [ 18 F]FAPI-04 exhibits superior performance over [ 18 F]FDG in PET/CT evaluation of liver cancer, characterized by increased sensitivity and SUVmax/TBR. Significant correlations with molecular markers, including Ki67, suggest [ 18 F]FAPI-04's potential for characterizing liver cancer subtypes and assessing tumor proliferation. However, further research is required to validate these findings and their clinical significance., Trial Registration: NCT05485792, Registered 01 August 2022., Competing Interests: Declarations Ethics approval and consent to participate This study was approved by the institutional review board of Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital, Guangzhou Medical University, and written informed consent for publication was obtained from the patient. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Cancer-associated fibroblasts (CAFs) and hepatocellular carcinoma (HCC) cells interact to promote HCC progression, but the underlying mechanisms remain unclear. Serpin family E member 1 (SERPINE1) has conflicting roles in HCC, and microRNAs (miRNAs) are known to regulate tumor progression through intercellular communication. Therefore, we investigated the potential involvement of miRNA/SERPINE1 axis in crosstalk between CAFs and HCC cells., Methods: In this study, candidate miRNAs targeting SERPINE1 3' UTR were predicted using multiple miRNA databases. The miRNAs and SERPINE1 mRNA expression in Huh7 cells was assessed after co-culture with CAFs using RT-qPCR. Huh7 cell proliferation and invasion were detected after SERPINE1 siRNA. The functions of the CAF-derived miR-642a-3p/SERPINE1 axis in HCC cells were examined using CCK-8, wound healing, transwell assays, western blot, and dual-luciferase reporter assays. Moreover, a orthotopic xenograft model was used to investigate the contribution of miR-642a-3p knockdown in HCC., Results: SERPINE1 mRNA expression decreased, while miR-642a-3p expression increased in Huh7 cells co-cultured with CAFs. SERPINE1 knockdown enhanced Huh7 cell proliferation and invasion as well as miR-642a-3p expression. miR-642a-3p overexpression promoted migration, invasion, and epithelial-mesenchymal transition (EMT) in Huh7 cells by targeting SERPINE1, while miR-642a-3p knockdown yielded the opposite effect. Rescue experiments confirmed that SERPINE1 knockdown attenuated the inhibitory effects of miR-642a-3p knockdown on migration, invasion, and EMT in Huh7 cells. Importantly, miR-642a-3p knockdown suppressed growth and EMT in orthotopic liver tumors., Conclusion: CAF-derived miR-642a-3p/SERPINE1 axis facilitated migration, invasion, and EMT in the HCC cells, suggesting miR-642a-3p/SERPINE1 axis can be a potential therapeutic target for HCC., Competing Interests: The authors declare there are no competing interests., (©2024 Zhang et al.)

Background: Hepatocellular carcinoma (HCC) is one of the most common as well as leading causes of mortality worldwide, and sorafenib is the first-line treatment in HCC patients. Unfortunately, drug resistance to sorafenib often develops. However, the underlying mechanism remains unclear. Here, we reveal the important role of macrophage extracellular traps (METs)-mediated crosstalk between macrophages and tumor cells in sorafenib resistance., Methods: METs in HCC tumor tissues were detected using immunofluorescence. The concentrations of MPO-DNA, elastase and cytokines were measured using ELISA. The mRNA expression levels of genes were confirmed by qRT-PCR. The siRNAs were conducted to knock ARHGDIG in Hepa1-6 and Hep3B cells. Western Blot assay was performed to determine protein expression of Rho GDP dissociation inhibitor gamma (ARHGDIG, or RHOGDI-3), PADI2, and PADI4. Cell viability and migration were evaluated by CCK-8 assay and transwell assay, respectively. Cell ferroptosis was assessed by measurement of Fe 2+ concentration, flow cytometry assay of lipid ROS, and western blot assay of GPX4. The functions of sorafenib, DNase I, IL4 neutralization antibody and GPX4 in tumor growth were explored through in vivo experiments., Results: Sorafenib induced MET formation in M2 macrophages rather than M1 macrophages derived from both human and mice. In Hepa1-6 HCC mice, METs clearance by DNase I improved response to sorafenib therapy, detected by tumor weight, tumor growth curve, tumor volume, and survival. By screening candidate cytokines that affect macrophage function, we found that sorafenib-promoting IL4 secretion by HCC cells plays a crucial role in sorafenib-induced MET formation. Understanding the critical role of IL4 in sorafenib-induced MET formation led us to find that IL4 neutralization significantly improved the efficiency of sorafenib in HCC models. Mechanistically, we discovered that sorafenib increased the expression of ARHGDIG in HCC cells, which led to the release of IL4. In M2 macrophages, IL4 triggered MET formation by elevating the mRNA and protein expression of peptidyl arginine deiminase 4 (PADI4) rather than PADI2. In HCC models, GSK484 inhibition of PADI4 could consistently weaken sorafenib resistance and improve sorafenib efficiency. Importantly, we discovered that METs contribute to sorafenib resistance by inhibiting the ferroptosis of HCC cells. Meanwhile, PADI4 inhibition or DNase I could reverse the sorafenib resistance caused by METs-inhibiting ferroptosis of HCC cells., Conclusion: Our study concludes that sorafenib-induced METs inhibit the ferroptosis of tumor cells, suggesting that targeting the IL4/PADI4/METs axis in HCC could reduce or prevent sorafenib resistance., Competing Interests: Declarations Ethics approval and consent to participate The animal experiment in this study was approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The widespread use of oxidative hair colouring cosmetics threatens public health. Phenylenediamine derivatives serve as the main pigment in permanent hair colours. They interact with biological macromolecules, altering their functional and structural physiology. The study aimed to investigate the effect of a typical synthetic hair dye pigment, 4-Chloro-orthophenylenediamine (4-Cl-OPD), under a nitrating environment of DEANO on the calf thymus DNA molecule. The results showed single-stranded regions, base/sugar-phosphate backbone alterations, molecular changes, and nitro-oxidative lesions. These modifications are referred to as neo-epitopes on the DNA molecule. IgGs from cancer patients with a history of permanent hair dye use were screened for the recognition of neo-epitopes on DNA molecules. Hepatocellular carcinoma IgG showed the highest binding with 56% inhibition in the competition ELISA. The immune complex formation was observed through electrophoretic mobility shift assay. In conclusion, synthetic hair dye users are likely to present with heightened immunological triggers under elevated nitric oxide levels. The study reports chronic hair dye exposure as one of the factors responsible for altering the intricacies of the DNA's microarchitectural structure and inducing neo-epitopes on the molecule. The physiological status of NO may define the susceptibility towards 4-Cl-OPD and humoral response in hair dye users. Persistent nitro-oxidative stress due to 4-Cl-OPD and NO may induce a heightened immune response against neoepitopes in the nitro-oxidatively modified DNA. Therefore, chronic hair dye exposure may be identified as a risk to human health. These findings may contribute to a better understanding and reinforcement of hair dye as one of the modifiable risk factors responsible for the pro-inflammatory carcinogenic environment., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with increasing global prevalence and is one of the leading causes of cancer-related mortality in the human population. Developing robust clinical prediction models and prognostic stratification strategies is crucial for developing individualized treatment plans. A range of novel forms of programmed cell death (PCD) plays a role in the pathological progression and advancement of HCC, and in-depth study of PCD is expected to further improve the prognosis of HCC patients. Sixteen patterns (apoptosis, autophagy, anoikis, lysosome-dependent cell death, immunogenic cell death, necroptosis, ferroptosis, netosis, pyroptosis, disulfidptosis, entotic cell death, cuproptosis, parthanatos, netotic cell death, alkaliptosis, and oxeiptosis) related to PCD were collected from the literatures and used for subsequent analysis. Supervised (Elastic net, Random Forest, XgBoost, and Boruta) and unsupervised (Nonnegative Matrix Factorization, NMF) clustering algorithms were applied to develop and validate a novel classifier for the individualized management of HCC patients at the transcriptomic, proteomic and single-cell levels. Multiple machine learning algorithms developed a programmed cell death index (PCDI) comprising five robust signatures (FTL, G6PD, SLC2A1, HTRA2, and DLAT) in four independent HCC cohorts, and a higher PCDI was predictive of higher pathological grades and worse prognoses. Furthermore, a higher PCDI was found to be correlated with the presence of a repressive tumor immune microenvironment (TME), as determined through an integrated examination of bulk and single-cell transcriptome data. In addition, patients with TP53 mutation had higher PCDI in comparison with TP53 WT patients. Three HCC subtypes were identified through unsupervised clustering (NMF), exhibiting distinct prognoses and significant biological processes, among the three subtypes, PCDcluster 3 was of particular interest as it contained a large proportion of patients with high risk and low metabolic activity. Construction and evaluation of the Nomogram model was drawn based on the multivariate logistic regression analysis, and highlighted the robustness of the Nomogram model in other independent HCC cohorts. Finally, to explore the prognostic value, we also validated the frequent upregulation of DLAT in a real-world cohort of human HCC specimens by qPCR, western blot, and immunohistochemical staining (IHC). Together, our work herein comprehensively emphasized PCD-related patterns and key regulators, such as DLAT, contributed to the evolution and prognosis of tumor foci in HCC patients, and strengthened our understanding of PCD characteristics and promoted more effective risk stratification strategies., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Objective: Squalene epoxidase (SQLE) promotes metabolic dysfunction-associated steatohepatitis-associated hepatocellular carcinoma (MASH-HCC), but its role in modulating the tumour immune microenvironment in MASH-HCC remains unclear., Design: We established hepatocyte-specific Sqle transgenic (tg) and knockout mice, which were subjected to a choline-deficient high-fat diet plus diethylnitrosamine to induce MASH-HCC. SQLE function was also determined in orthotopic and humanised mice. Immune landscape alterations of MASH-HCC mediated by SQLE were profiled by single-cell RNA sequencing and flow cytometry., Results: Hepatocyte-specific Sqle tg mice exhibited a marked increase in MASH-HCC burden compared with wild-type littermates, together with decreased tumour-infiltrating functional IFN-γ + and Granzyme B + CD8 + T cells while enriching Arg-1 + myeloid-derived suppressor cells (MDSCs). Conversely, hepatocyte-specific Sqle knockout suppressed tumour growth with increased cytotoxic CD8 + T cells and reduced Arg-1 + MDSCs, inferring that SQLE promotes immunosuppression in MASH-HCC. Mechanistically, SQLE-driven cholesterol accumulation in tumour microenvironment underlies its effect on CD8 + T cells and MDSCs. SQLE and its metabolite, cholesterol, impaired CD8 + T cell activity by inducing mitochondrial dysfunction. Cholesterol depletion in vitro abolished the effect of SQLE-overexpressing MASH-HCC cell supernatant on CD8 + T cell suppression and MDSC activation, whereas cholesterol supplementation had contrasting functions on CD8 + T cells and MDSCs treated with SQLE-knockout supernatant. Targeting SQLE with genetic ablation or pharmacological inhibitor, terbinafine, rescued the efficacy of anti-PD-1 treatment in MASH-HCC models., Conclusion: SQLE induces an impaired antitumour response in MASH-HCC via attenuating CD8 + T cell function and augmenting immunosuppressive MDSCs. SQLE is a promising target in boosting anti-PD-1 immunotherapy for MASH-HCC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Metabolic dysfunction-associated steatohepatitis (MASH) is a form of chronic liver inflammation associated with metabolic syndrome, such as obesity and a major cause of hepatocellular carcinoma (HCC). Multi-biotics, a soymilk fermented with lactic acid bacteria, are known to alleviate obesity by lowering lipid profile. This study aimed to establish and characterize mouse organoids derived from MASH-related HCC models to evaluate drug responses, particularly focusing on Lenvatinib resistance. Organoids were developed using mouse liver tissues subjected to a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) to mimic MASH-related HCC. The study evaluated the effect of multi-biotics, a fermented product, on tumor regression and drug sensitivity. While multi-biotics did not reduce tumor burden, they enhanced the response to Lenvatinib. Additionally, repeated treatment with Lenvatinib led to the development of drug-resistant organoids. Transcriptomic analysis of these resistant organoids identified key pathways related to KRAS signaling, inflammation, and epithelial-mesenchymal transition (EMT), revealing potential targets for overcoming Lenvatinib resistance. This study provides valuable insights into MASH-related HCC progression and drug resistance, offering a model for further therapeutic research., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Enhancer of zeste homolog 2 (EZH2), a key protein implicated in various cancers including hepatocellular carcinoma (HCC), is recognized for its association with epigenetic dysregulation and pathogenesis. Despite clinical explorations into EZH2-targeting therapies, the mechanisms underlying its role in gene suppression in HCC have remained largely unexplored. Here, we integrate epigenomic and transcriptomic analyses to uncover the transcriptional landscape modulated by selective EZH2 inhibition in HCC. By reanalyzing transcriptomic data of HCC patients, we demonstrate that EZH2 overexpression correlates with poor patient survival. Treatment with the EZH2 inhibitor tazemetostat restored expression of genes involved in cysteine-methionine metabolism and lipid homeostasis, while suppressing angiogenesis and oxidative stress-related genes. Mechanistically, we demonstrate EZH2-mediated H3K27me3 enrichment at cis-regulatory elements of transsulfuration pathway genes, which is reversed upon inhibition, leading to increased chromatin accessibility. Among 16 EZH2-targeted candidate genes, BHMT and CDO1 were notably correlated with poor HCC prognosis. Tazemetostat treatment of HCC cells increased BHMT and CDO1 expression while reducing levels of ferroptosis markers FSP1, NFS1, and SLC7A11. Functionally, EZH2 inhibition dose-dependently reduced cell viability and increased lipid peroxidation in HCC cells. Our findings reveal a novel epigenetic mechanism controlling lipid peroxidation and ferroptosis susceptibility in HCC, providing a rationale for exploring EZH2-targeted therapies in this malignancy., (© 2024. The Author(s).)

End-stage liver disease is marked by portal hypertension, systemic elevations in ammonia, and development of hepatocellular carcinoma (HCC). While these clinical consequences of cirrhosis are well described, it remains poorly understood whether hepatic insufficiency and the accompanying elevations in ammonia contribute to HCC carcinogenesis. Using preclinical models, we discovered that ammonia entered the cell through the transporter SLC4A11 and served as a nitrogen source for amino acid and nucleotide biosynthesis. Elevated ammonia promoted cancer stem cell properties in vitro and tumor initiation in vivo. Enhancing ammonia clearance reduced HCC stemness and tumor growth. In patients, elevations in serum ammonia were associated with an increased incidence of HCC. Taken together, this study forms the foundation for clinical investigations using ammonia-lowering agents as potential therapies to mitigate HCC incidence and aggressiveness.

In this manuscript, we comment on the article, which explores the anti-cancer effects of Calculus bovis (CB) in tumor biology. We highlight its potential, particularly in hepatocellular carcinoma (HCC), where it inhibits the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways and induces apoptosis. CB contains compounds such as oleanolic acid and ursolic acid that target interleukin-6, mitogen-activated protein kinase 8, vascular endothelial growth factor, and caspase-3, offering anti-inflammatory and hepatoprotective benefits. The manuscript also discusses CB sativus (CBS), an artificial substitute, which has shown efficacy in reducing hepatic inflammation and oxidative stress in animal models. We emphasize the need for further research on the effects of CBS on the gut-liver axis and gut microbiota, and on targeting Wnt signaling and M2 tumor-associated macrophage as potential therapeutic strategies against HCC., Competing Interests: Conflict-of-interest statement: All authors declare no conflict of interest in publishing the manuscript., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

In this article, we comment on the article by Huang et al . The urgent development of new therapeutic strategies targeting macrophage polarization is critical in the fight against liver cancer. Tumor-associated macrophages (TAMs), primarily of the M2 subtype, are instrumental in cellular communication within the tumor microenvironment and are influenced by various signaling pathways, including the wingless/integrated (Wnt) pathway. Activation of the Wnt signaling pathway is pivotal in promoting M2 TAMs polarization, which in turn can exacerbate hepatocarcinoma cell proliferation and migration. This manuscript emphasizes the burgeoning significance of the Wnt signaling pathway and M2 TAMs polarization in the pathogenesis and progression of liver cancer, highlighting the potential therapeutic benefits of inhibiting the Wnt pathway. Lastly, we point out areas in Huang et al 's study that require further research, providing guidance and new directions for similar studies., Competing Interests: Conflict-of-interest statement: Tam PKH and Chen Y have received research funding from Macau Science and Technology Development Fund., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Objective: Isoscopoletin is one of the primary metabolites of natural product scoparone, which was reported to against tumor proliferation. The aim of this study was to explore the mechanism of isoscopoletin against hepatocellular carcinoma (HCC)., Methods: Transcriptomics was used to reveal the possible pathways of isoscopoletin against HCC in vitro. The potential targets of isoscopoletin against HCC through affecting glycolysis were analyzed by network pharmacology, then the potential binding abilities of isoscopoletin to glycolysis-related proteins were initially verified by high throughput virtual molecular docking. The affinities of isoscopoletin for glycolysis-related proteins were assayed using microscale thermophoresis (MST), which was reverse-validated by inhibiting the binding ability of isoscopoletin to GPD2. Glucose consumption and lactate production were examined to evaluate the effects of isoscopoletin on intracellular glycolysis, and the regulation of glycolysis-related targets by isoscopoletin was detected using RT-qPCR and ELISA kits., Results: The results of transcriptomics showed that the differentially expressed genes (DEGs) were mainly enriched in glycolysis and other metabolic-related pathways. Network pharmacology and molecular docking revealed that GPD2, GPI, HSP90AA1 and PGK2 were the core targets in the glycolysis process of isoscopoletin against HCC. MST results showed that there was a strong affinity between isoscopoletin and GPD2, GPI, Hsp90α and PGK2. In vitro results showed that isoscopoletin inhibited glucose consumption and lactate production, while regulating the levels of glycolysis-related proteins., Conclusion: This study suggests that isoscopoletin may exist an anti-tumor effect by regulating the glycolysis-related proteins GPD2, GPI, Hsp90α and PGK2, inhibiting the glycolysis process in HCC cells, then blocking the energy supply of tumor cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ruan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Accurately identifying effective biomarkers and translating them into clinical practice have significant implications for improving clinical outcomes in hepatocellular carcinoma (HCC). In this study, our objective is to explore appropriate methods to improve the accuracy of biomarker identification and investigate their clinical value., Methods: Concentrating on the N6-methyladenosine (m6A) modification regulators, we utilized dozens of multi-omics HCC datasets to analyze the expression patterns and genetic features of m6A regulators. Through the integration of big data analysis with function experiments, we have redefined the biological roles of m6A regulators in HCC. Based on the key regulators, we constructed m6A risk models and explored their clinical value in estimating prognosis and guiding personalized therapy for HCC., Results: Most m6A regulators exhibit abnormal expression in HCC, and their expression is influenced by copy number variations (CNV) and DNA methylation. Large-scale data analysis has revealed the biological roles of many key m6A regulators, and these findings are well consistent with experimental results. The m6A risk models offer significant prognostic value. Moreover, they assist in reassessing the therapeutic potential of drugs such as sorafenib, gemcitabine, CTLA4 and PD1 blockers in HCC., Conclusions: Our findings suggest that the mutual validation of big data analysis and functional experiments may facilitate the precise identification and definition of biomarkers, and our m6A risk models may have the potential to guide personalized chemotherapy, targeted treatment, and immunotherapy decisions in HCC., (© 2024. The Author(s).)

Nonalcoholic fatty liver disease (NAFLD) has become a common health care burden worldwide. The high heterogeneity of NAFLD remains elusive and impairs outcomes of clinical diagnosis and pharmacotherapy. Several NAFLD classifications have been proposed on the basis of clinical, genetic, alcoholic, or serum metabolic analyses. Yet, accurately predicting the progression of NAFLD to cirrhosis or hepatocellular carcinoma (HCC) in patients remains a challenge. Here, on the basis of a Chinese cohort of patients, we classified NAFLD into three distinct molecular subtypes (NAFLD-mSI, NAFLD-mSII, and NAFLD-mSIII) using integrative multiomics including whole-genome sequencing (WGS), proteomics, phosphoproteomics, lipidomics, and metabolomics across a broad range of liver, blood, and urine specimens. We found that NAFLD-mSI had higher expression of CYP1A2 and CYP3A4, which alleviate hepatic steatosis through mediating free fatty acid/bile acid-mTOR-FXR/PPARα signaling. NAFLD-mSII displayed an elevated risk of liver cirrhosis along with increased hepatic infiltration of M1 and M2 macrophages because of lipid-triggered hepatic CCL2 and CRP production. NAFLD-mSIII exhibited a potential risk for HCC development by increased transcription of CEBPB- and ERCC3-regulated oncogenes because of activation of the EGF-EGFR/CHKA/PI3K-PDK1-AKT cascade. Next, we validated the existence of these three NAFLD molecular subtypes in an external cohort comprising 92 patients with NAFLD across three different Chinese hospitals. These findings may aid in understanding the molecular features underlying NAFLD heterogeneity, thereby facilitating clinical diagnosis and treatment strategies with the aim of preventing the development of liver cirrhosis and HCC.

FKBP4, a member of the FK506-binding protein (FKBP) family, is a promising target for a variety of disorders, including cancer. However, its underlying molecular mechanism and potential function in hepatocellular carcinoma (HCC) are largely elusive. Therefore, we aimed to investigate the expression status, functional implications and underlying mechanisms of FKBP4 in HCC. Our bioinformatics analysis of TCGA LIHC datasets, ICGC LIRI-JP datasets and GEO datasets results showed FKBP4 was upregulated in HCC tissues. We also confirmed the elevated FKBP4 in clinical HCC samples. Additionally, quantitative RT-PCR results revealed FKBP4 was highly expressed in all five tested HCC cell lines. We also observed a correlation between elevated FKBP4 expression and poor prognosis in HCC patients. Loss of FKBP4 can inhibit the proliferation and migration in HCC cells. Furthermore, we found that silencing FKBP4 suppressed glucose uptake, lactic acid production and  18 F-FDG uptake compared with the control group. Mechanistically, our funding indicated that FKBP4 participates in glycolysis through p53 mediated HK2 signaling pathway, specially, FKBP4 knockdown promotes the expression and stability of p53 protein rather than affecting the transcription level. Finally, rescue experiments revealed that simultaneous knockdown of both FKBP4 and p53 partially reversed the inhibitory effects on HK2 protein levels and  18 F-FDG uptake. Our study elucidates a novel role of FKBP4 in promoting HCC development and glycolysis by modulating the p53/HK2 signaling pathway. Given the critical role of aerobic glycolysis in the progression of HCC, targeting FKBP4 may offer a new therapeutic strategy for treating this malignancy., (© 2024. The Author(s).)

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) asscociated hepatocellular carcinoma (HCC) is becoming a growing concern in global healthcare. The early-life gut microbiota plays a crucial role in maintaining healthy. However, the impact of early-life gut microbiota dysbiosis on the advancement of MASLD-HCC remains inadequately understood., Methods: In the present study, we investigated the role of early-life gut microbiota in the development of MASLD-HCC in streptozotocin and high-fat diet (STZ-HFD) induced mouse model. We recorded the body weight and lifespan, and dynamically monitored the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (T-CHO) and blood glucose in the serum monthly. In addition, we examined various immune cells present in the liver, such as T cells, B cells, NK cells, NKT cells, αβT cells, γδT cells, macrophage and MDSC cells by flow cytometry and conducted liquid chromatography mass spectrometry (LC-MS) based analysis on liver tissue from control and early-life antibiotic exposure mice (early-Abx) MASLD-HCC mice., Results: We found that early-Abx mice suffered from more severe tumor burden and further confirmed that hepatocytes and immune cells were all disturbed. Importantly, early-life antibiotic exposure alters the liver metabolic profiling especially glycerophospholipids and lipid accumulation. Furthermore, mice exposed to antibiotics in early-life showed disturbances in glucose metabolism and developed insulin resistance., Conclusions: Collectively, our findings revealed that early-life antibiotic exposure accelerated the progression of MASLD-HCC by impairing the hepatocytes, immune homeostasis and metabolites persistently, highlighting the importance of the early-life microbiota in the development of MASLD-HCC., (© 2024. The Author(s).)

Purpose: Highly complex tumor microenvironment makes hepatocellular carcinoma (HCC) as one of the most malignant tumors worldwide. The role of cellular senescence in HCC has been gradually recognized. The present study aimed to comprehensively elucidate the senescence-related features of HCC in single-cell and spatial dimension., Methods: Single-cell RNA sequencing (scRNA-Seq) data was used to clarify the heterogeneity of senescence-related genes (SRGs) among multiple cell types within HCC. Spatial transcriptome RNA sequencing (stRNA-Seq) data was used for depicting SRGs features in spatial dimension. A prognostic model based on SRGs was constructed by using of bulk sequencing (bulk-Seq) data of HCC. The cell-cell interaction of senescent endothelial cells (ECs) in tumor microenvironment was analyzed. Then, the role of senescent ECs was verified through in vitro and in vivo experiments., Results: The level of senescence demonstrated substantial heterogeneity among different cell types within tumor microenvironment of HCC, where ECs exhibited the most prominent senescent phenotype. Senescent ECs activated specific regulatory pathways through communicating with other cell types, with a potential impact on tumor progression. Spatial analysis revealed senescent ECs mainly located in the core region of HCC. The interaction of senescent ECs and immune cells implicated their role in tumor progression and immunotherapeutic response. In addition, CDKN2A was identified as an independent risk factor for HCC prognosis by constructing a prognostic model. Patients with high risk displayed an even worse outcome. The experimental verification indicated senescence of ECs determined by CDKN2A exhibited a secretory phenotype. Furthermore, senescent ECs with CDKN2A overexpression promote the proliferation and migration of HCC., Conclusion: The present study recognizes the critical effect of senescent ECs defined by CDKN2A in the promotion of tumor progression, which sheds new light on the investigation of ECs senescence in HCC., (© 2024. The Author(s).)

A pan-cancer analysis summarizing the overall changes in mRNA and protein stability of ADM9, as well as its oncogenic function on immune cell line modulation and checkpoints within the tumor microenvironment (TME), is lacking, despite the fact that ADM9 up-regulation is correlated with the progression of many cancers. Therefore, in this study, we comprehensively analyzed the role of ADAM9 expression and its prognostic value in different cancers to fill this gap. Multiple bioinformatics databases such as Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) were used to evaluate the ADAM9 genetic alternation, phosphorylation, and methylation, and indicated highly positive correlated genes that might play a critical interaction with ADAM9 and their molecular function with GO analysis. We also evaluate the effect of higher ADAM9 with prominent immune modulatory genes and immune infiltration especially in liver cancer pathogenesis stimulates lower NK cell effector functions based on its role in MICA shedding and increasing the Tregs infiltration. Immunohistochemistry (IHC) staining from 90 pathologically verified samples proved the positive correlation between ADAM9 and tumor stages and proved the higher expression of ADAM9 correlated genes (SNX9, APP, TNF, CDH1, ITGAV, MAD2L2) in HCC pathogenesis. In conclusion, this pan-cancer study provides a comprehensive understanding of the prognostic value of ADAM9 in various tumors emphasizing its importance to be considered as an innovative treatment approach, especially in tumor immunity shortly., (© 2024. The Author(s).)

Multitarget tyrosine kinase inhibitors (TKIs) serve as first-line therapeutics in the systemic treatment of hepatocellular carcinoma (HCC), yet their clinical effectiveness is hampered by suboptimal pharmacokinetics and bioavailability. There is a critical need to enhance the circulation, tumor targeting, and infiltration of TKIs. In this context, we developed a silk fibroin (SF)-based nanomedicine that exploits the chemical versatility and conformation tunability of SF. Folic acid (FA) with affinity toward HCC cells is utilized to functionalize SF, simultaneously aiding in the pH-sensitive β-sheet transitions of SF. This dynamic conformation behavior is key to improving the nanomedicine's circulation, biological adhesion, and tumor localization. By encapsulating Lenvatinib (Leva) TKI, the nanomedicine exhibits tumor-targeted accumulation and potent inhibition on HCC cell survival and angiogenesis, thereby amplifying Leva's bioavailability and therapeutic impact. Owing to SF's low immunogenicity and high reproducibility, this SF-based approach for TKI delivery holds substantial promise for advancing HCC systemic therapy.

Background: Immune checkpoint inhibitors (ICIs) are therapeutically effective for hepatocellular carcinoma (HCC) but are individually selective. This study examined the role of specific common fragile sites (CFSs) related gene in HCC immunotherapy., Methods: We analyzed HCC tissues using next-generation sequencing and flow cytometry via time-of-flight technology. A humanized orthotopic HCC mouse model, an in vitro co-culture system, untargeted metabolomics and a DNA pulldown assay were used to examine the function and mechanism of WWOX in the tumor immune response., Results: WWOX was the most upregulated CFS-related gene in HCC patients responsive to ICIs. WWOX deficiency renders HCC resistant to PD-1 treatment in humanized orthotopic HCC mouse model. Macrophage infiltration is increased and CD8 T-cell subset infiltration is decreased in WWOX-deficient HCC patients. HCC-derived oleic acid (OA) promotes macrophage conversion to an immunosuppressive phenotype. Mechanistically, WWOX deficiency promoted OA synthesis primarily via competitive binding of NME2 with KAT1, which promoted acetylation of NME2 at site 31 and inhibited NME2 binding to the SCD5 promoter region. Pharmacological blockade of SCD5 enhanced the antitumor effects of anti-PD-1 therapy., Conclusions: WWOX is a key factor for immune escape in HCC patients, which suggests its use as a biomarker for stratified treatment with ICIs in clinical HCC patients., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The efficacy of immune checkpoint inhibitors in the treatment of hepatocellular carcinoma (HCC) remains limited, highlighting the need for further investigation into the mechanisms underlying treatment resistance. Accumulating evidence indicates that tumor-associated macrophages (TAM) within the tumor microenvironment demonstrate a key role in immune evasion and treatment resistance. This study explored the role of TAMs in the HCC tumor microenvironment. Our findings reveal that TAMs expressing CX3C motif chemokine receptor 1 (CX3CR1) induced T-cell exhaustion through IL27 secretion in orthotopic models of HCC following treatment with anti-PD1. Moreover, we identified prostaglandin E2 (PGE2), released by immune-attacked tumor cells, as a key regulator of TAM transition to a CX3CR1+ phenotype. To augment the therapeutic response to anti-PD1 therapy, we propose targeting CX3CR1+ TAMs in addition to anti-PD1 therapy. Our study contributes to the understanding of the role of TAMs in cancer immunotherapy and highlights potential clinical implications for HCC treatment. The combination of targeting CX3CR1+ TAMs with anti-PD1 therapy holds promise for enhancing the efficacy of immunotherapeutic interventions in patients with HCC., (©2024 American Association for Cancer Research.)

Background Sorafenib is a standard therapeutic agent for advanced hepatocellular carcinoma (HCC). However, its efficacy is moderate, as the survival of patients is prolonged for only a few months, and the response rate is low. The mechanism of low efficacy remains unclear. In this study, we investigated the effect of Toll-like receptor 3 (TLR3) on the effects of sorafenib on HCC. Methods Polyinosinic-polycytidylic acid [poly(I: C)] was used as a double-stranded RNA analog and TLR3 agonist in subsequent experiments. After orthotopic implantation of HCC tumors in BALBc nu/nu or C57BL/6 mice, survival time, tumor growth, and metastasis in the abdomen and lungs were analyzed. Flow cytometry and cytotoxicity assays were used to analyze NK cells isolated from the spleen or peripheral blood. ELISA was used to detect the expression of plasma interferon (IFN)-γ and monocyte chemoattractant protein (MCP)-1. In addition, the expression of phosphorylated-extracellular regulated kinase 1/2 (pERK1/2), phosphorylated-protein kinase B (pAKT), ERK1/2 and AKT was analyzed by Western blotting. Results Sorafenib reduced the number and activity of NK cells in tumor-bearing mice and simultaneously decreased the levels of MCP-1 and IFN-γ in the plasma. The combination of sorafenib and poly(I: C) synergistically inhibited tumor growth and metastasis in tumor xenograft mice and prolonged survival. Poly(I: C) not only exerts a direct inhibitory effect on tumor growth and metastasis by targeting the TLR3 receptor on tumor cells but also facilitates the proliferation and activation of NK cells, indirectly impeding tumor progression. Mechanistically, poly(I: C) decreased the sorafenib-induced inhibition of ERK phosphorylation and increased the phosphorylation of IκB in NK cells, thereby enhancing NK cell function. Conclusion Activation of TLR3 can enhance the antitumor effect of sorafenib on HCC. The combination of a TLR3 activator and sorafenib may be a new strategy for the treatment of HCC., (© 2024. The Author(s).)

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. This study investigates the role and mechanisms of ADAM9 as a biomarker and potential therapeutic target in HCC. Utilizing RNA-sequencing data and clinicopathological characteristics from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we conducted survival and meta-analyses, functional enrichment, and immune infiltration studies. Additionally, we evaluated the effects of ADAM9 silencing on HCC cell proliferation, migration, and invasion through in vitro experiments. Our results demonstrate that high ADAM9 expression is associated with poor prognosis and increased immune infiltration in HCC patients. Furthermore, ADAM9 knockdown significantly inhibited tumor cell proliferation and migration. These findings indicate that ADAM9 is a promising prognostic biomarker and potential therapeutic target in HCC. In conclusion, ADAM9 could offer avenues for developing strategies to inhibit tumor progression and improve patient outcomes., (© 2024. The Author(s).)

Background: The occult onset and rapid progression of hepatocellular carcinoma (HCC) lead to an unsatisfactory overall survival (OS) rate. Established prognostic predictive models based on tumor-node-metastasis staging and predictive factors do not report satisfactory predictive efficacy. Arachidonic acid plays pivotal roles in biological processes including inflammation, regeneration, immune modulation, and tumorigenesis. We, therefore, constructed a prognostic predictive model based on seven genes linked to arachidonic acid metabolism, using samples of HCC patients from databases to analyze the genomic profiles. We also assessed the predictive stability of the constructed model., Methods: Sample data of 365 patients diagnosed with HCC were extracted from The Cancer Genome Atlas (TCGA, training set) and HCCDB18, GSE14520, and GSE76427 databases (validation sets). Patient samples were clustered using ConsensusClusterPlus analysis based on the expression levels of 12 genes involved in arachidonic acid metabolism that were significantly associated with HCC prognosis. Differentially expressed genes (DEGs) within different clusters were distinguished and compared using WebGestaltR. Immunohistochemistry (IHC) analysis was performed using a human HCC tissue microarray (TMA). Tumor immune microenvironment assessment was performed using ESTIMATE, ssGSEA, and TIDE., Results: Samples of patients with HCC were classified into three clusters, with significant differences in OS. Cluster 2 showed the best prognosis, whereas cluster 1 presented the worst. The three clusters showed significant differences in immune infiltration. We then performed Cox and LASSO regression analyses, which revealed CYP2C9, G6PD, CDC20, SPP1, PON1, TRNP1, and ADH4 as prognosis-related hub genes, making it a simplified prognostic model. TMA analysis for the seven target genes showed similar results of regression analyses. The high-risk group showed a significantly worse prognosis and reduced immunotherapy efficacy. Our model showed stable prognostic predictive efficacy., Conclusions: This seven-gene-based model showed stable outcomes in predicting HCC prognosis as well as responses to immunotherapy., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Background and Objectives: Hepatocellular carcinoma (HCC) is a major global health concern, and emerging evidence suggests that TIGIT and NKG2A are potential immune checkpoints with implications for HCC progression. This study aimed to evaluate the prognostic significance of TIGIT and NKG2A expression in HCC patients who underwent radical liver resection., Methods: We conducted a retrospective analysis of 144 HCC patients who underwent radical liver resection. TIGIT and NKG2A expression levels were assessed using the immunoreactive score. Cox proportional hazards models were utilized to analyze the association between TIGIT/NKG2A expression and clinical characteristics, relapse-free survival (RFS), and overall survival (OS). Prognostic models for OS and RFS was developed and validated using concordance index and calibration curves. Additionally, the random forest algorithm was employed to identify independent risk factors for OS and RFS and their correlation with predicted survival., Results: TIGIT and NKG2A expression were identified as independent risk factors for RFS, while TIGIT expression alone significantly impacted OS. The prognostic models showed good discriminative ability, with concordance indices exceeding 0.7 for predicting 1-, 3-, and 5-year OS or RFS. Calibration curves confirmed the reliability of the nomograms for OS and RFS prediction. The areas under the ROC curve consistently exceeded 0.7 for predicting OS and RFS. Elevated levels of TIGIT and NKG2A expression were associated with diminished RFS, highlighting their importance as prognostic factors., Conclusions: Our study establishes the prognostic significance of TIGIT and NKG2A expression in predicting OS and RFS following radical liver resection for HCC patients. The developed prognostic models incorporating TIGIT and NKG2A expression hold promise for improving risk stratification and clinical management of HCC patients., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Purpose: Ferroptosis is a regulated form of cell death characterized by iron-dependent accumulation of associated lipid peroxides (LPO), which can induce cell death when a certain level is reached. However, the extremely complex tumor microenvironment (TME) has the characteristics of antioxidant, even if it induces ferroptosis of tumor cells, its killing effect on tumor cells is still very limited. To solve this problem, we constructed a novel nanomaterials (GOx/EC@Fe 3 O 4 @CCM). We evaluated the anticancer effect of this nanomaterial in inhibiting tumor growth through comprehensive in vitro and in vivo experiments., Methods: We successfully synthesized GOx/EC@Fe 3 O 4 by one-pan synthesis method, then coated the Hepatocellular carcinoma cell membrane on its surface by co-extrusion technology, and finally synthesized the GOx/EC@Fe 3 O 4 @CCM nanoplatforms. We characterized the compounds in terms of morphology, particle size, and Zeta potential. In addition, we also studied the anti-tumor effect of GOx/EC@Fe 3 O 4 @CCM nanoplatforms from the following aspects, including the performance test of the nanoplatform, the intracellular effect of the nanoplatform, the anti-tumor effect in vitro, the intracellular ROS analysis, the intracellular effect of EC, and the anti-tumor effect in vivo., Results: The iron-based carriers in GOx/EC@Fe 3 O 4 @CCM nanoplatforms are released and produce ferrous ions (Fe 2+ ) in an acidic environment. Due to the limitation of the endogenous level of hydrogen peroxide (H 2 O 2 ), we introduced GOx into the TME or tumor cells. Under the catalysis of GOx, glucose reacted rapidly to produce a large amount of H 2 O 2 , which then combined with Fe 2+ to produce a large number of Hydroxyl radical (·OH). Its toxicity leads to dysfunction of cell membrane and organelles, and then causes cell damage. EC inhibits Nuclear factor erythroid 2-related factor 2 (Nrf2) in cancer cells, which effectively down-regulates downstream gene products, including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HMOX1). A series of chain reactions reduce the escape effect of oxidative stress (OS) and effectively maintain a high level of intracellular oxidation. Furthermore, it induces sustained and intense ferroptosis in tumor cells. Finally, the use of cancer cell membrane modified nanoplatforms due to the homology of membrane protein components improves the tumor cell targeting of the nanoplatforms, showing significant tumor cell inhibition and killing effect in vivo., Conclusion: The results showed that the GOx/EC@Fe 3 O 4 @CCM nanoplatforms successfully induced significant ferroptosis of Hepatocellular carcinoma cells through a cascade effect, and finally effectively promoted cancer cell regression., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Huang et al.)