Multiple Hepatocellular Carcinoma Research Articles

Hypoxia-induced TRPM7 promotes glycolytic metabolism and progression in hepatocellular carcinoma

BackgroundHypoxia disrupts glucose metabolism in hepatocellular carcinoma (HCC). Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) plays an ontogenetic role. Thus, we aimed to explore the regulation of TRPM7 by hypoxia-induced factor (HIF) and its underlying mechanisms in HCC. Methodshypoxia was induced in multiple HCC cells using 1% O2 or CoCl2 treatment, and subsequently blocked using siRNAs targeting HIF-1α or HIF-2α as well as a HIF-1α protein synthesis inhibitor. The levels of HIF-1α and TRPM7 were assessed using quantitative PCR (qPCR) and Western blot analysis. Chromatin immunoprecipitation (ChIP) and luciferase assays were performed to observe the regulation of TRPM7 promoter regions by HIF-1α. A PCR array was utilized to screen glucose metabolism-related enzymes in HEK293 cells overexpressing TRPM7 induced by tetracycline, and then verified in TRPM7-overexpressed huh7 cells. Finally, CCK-8, transwell, scratch and tumor formation experiments in nude mice were conducted to examine the effect of TRPM7 on proliferation and metastasis in HCC. ResultsExposure to hypoxia led to increase the levels of TRPM7 and HIF-1α in HCC cells, which were inhibited by HIF-1α siRNA or enhanced by HIF-1α overexpression. HIF-1α directly bound to two hypoxia response elements (HREs) in the TRPM7 promoter. Several glycolytic metabolism-related enzymes, were simultaneously upregulated in HEK293 and huh7 cells overexpressing TRPM7 during hypoxia. In vitro and in vivo experiments demonstrated that TRPM7 promoted the proliferation and metastasis of HCC cells. ConclusionsTRPM7 was directly transcriptionally regulated by HIF-1α, leading to glycolytic metabolic reprogramming and the promotion of HCC proliferation and metastasis in vitro and in vivo. Our findings suggest that TRPM7 might be a potential diagnostic indicator and therapeutic target for HCC.

Comprehensive molecular classification predicted microenvironment profiles and therapy response for hepatocellular carcinoma.

Tumor microenvironment (TME) heterogeneity leads to a discrepancy in survival prognosis and clinical treatment response for hepatocellular carcinoma (HCC) patients. The clinical applications of documented molecular subtypes are constrained by several issues. We integrated three single-cell datasets to describe the TME landscape and identified six prognosis-related cell subclusters. Unsupervised clustering of subcluster-specific markers was performed to generate transcriptomic subtypes. The predictive value of these molecular subtypes for prognosis and treatment response was explored in multiple external HCC cohorts and the Xiangya HCC cohort. TME features were estimated using single-cell immune repertoire sequencing, mass cytometry and multiplex immunofluorescence. The prognosis-related score (PRS) was constructed based on machine learning algorithm. Comprehensive single-cell analysis described TME heterogeneity in HCC. The five transcriptomic subtypes possessed different clinical prognoses, stemness characteristics, immune landscapes and therapeutic responses. Class 1 exhibited an inflamed phenotype with better clinical outcomes, while Classes 2 and 4 were characterized by a lack of T cell infiltration. Classes 5 and 3 indicated an inhibitory tumor immune microenvironment. Analysis of multiple therapeutic cohorts suggested that Classes 5 and 3 were sensitive to ICB and targeted therapy, whereas Classes 1 and 2 were more responsive to transcatheter arterial chemoembolization treatment. Class 4 displayed resistant to all conventional HCC therapies. Three potential therapeutic agents and four targets were further identified for high-PRS HCC patients. Our study generated a clinically valid molecular classification to guide precision medicine in patients with HCC.

Abstract 4547: A novel drug combination with improved therapeutic efficacy over sorafenib in multiple preclinical models of hepatocellular carcinoma

Abstract Introduction: Due to the heterogeneity of hepatocellular carcinoma (HCC), the incidence of resistance and toxicity to first-line therapy sorafenib is high. Combinations of drugs can be used to exploit synergistic effects to reduce adverse events, while improving efficacy and survival. In-vitro studies performed by our team identified irinotecan and sonidegib to have potential for synergistic therapeutic effects in combination with sorafenib. Follow up in-vivo dose optimization studies in a murine NASH induced HCC found this 3-drug combination to be more effective than sorafenib-alone at arresting tumor development and progression. In this study, we tested the in-vivo efficacy of the combination in two separate preclinical cell-line derived xenograft (CDX) models. Experimental Procedure: HCC CDX models were generated by injecting two distinct HCC cell lines, Huh7 and HepG2, subcutaneously in the flank of male NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ (NSG) mice. Mice were administered either vehicle, sorafenib, or a 3-drug combination, and mouse tumor volumes and weights were tracked for duration of study. Mice were all euthanized at study endpoint and tissues collected for analysis, including the assessment of tumor weights, differentiation, grade, necrosis, and proliferation status. Serum collected from endpoint blood was measured for human alpha fetoprotein (AFP) and alanine transaminase (ALT) levels. Changes in protein expression of tumors were assessed by western blot analysis. Results: Huh7 and HepG2 tumors were both sensitive to sorafenib-alone treatment. However, Huh7 tumors treated with the 3-drug combination had a significant decrease in tumor volume and weight at endpoint compared to the vehicle and sorafenib-alone. HepG2 tumors treated with the combination also had a significant decrease in tumor volume and weight at endpoint compared to the vehicle. Though the difference in tumor volume and weight was not statistically significant for the combination compared to sorafenib, a trend towards decrease was observed. This was further confirmed by significantly reduced AFP levels in the combination compared to the vehicle and sorafenib in HepG2 and Huh7. Drug toxicities, measured by mouse weights and ALT levels, were not significantly different. Histological and immunohistochemical assessment of tumors showed necrosis and Ki67 staining to be significantly elevated in vehicle and sorafenib-alone compared to the 3-drug group. The targets for all three drugs present in the combination were downregulated in the combination compared to the vehicle and sorafenib-alone. Conclusion: This study provides strong evidence that a novel 3-drug combination of irinotecan, sonidegib, and low dose sorafenib can significantly reduce tumor proliferation and burden in multiple independent pre-clinical in-vivo HCC models compared to high dose sorafenib monotherapy. Citation Format: Anastasia Chambers, Sunaina Shrestha, Alexandra Ladd, Sergio Duarte, Ali Zarrinpar. A novel drug combination with improved therapeutic efficacy over sorafenib in multiple preclinical models of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4547.

Abstract 3124: MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most challenging cancers with high unmet medical need. Glypican-3 (GPC3) is an oncofetal glycoprotein anchored to the cell surface via GPI (glycosylphosphatidylinositol) and frequently over-expressed in HCC with limited expression in normal adult tissues, which makes it an appealing antibody-drug conjugate (ADC) target for HCC. MRG006A is a potentially first-in-class GPC3-targeting ADC composed of a novel humanized IgG1 antibody conjugated to a proprietary linker-payload platform utilizing a potent topoisomerase 1 inhibitor via a peptide-based cleavable linker. Materials and Methods: As a GPC3-targeting ADC intended for clinical therapy of GPC3-expressing cancers, the mechanism of action and therapeutic potential of MRG006A were investigated in a series of preclinical studies, including (1) MRG006A and its antibody moiety binding to human GPC3 expressed on cells assessed by flow cytometry; (2) binding affinity to human and cynomolgus monkey GPC3 measured by SPR (surface plasmon resonance); (3) internalization in Huh7 cells assessed by pHAb Reactive Dye and confocal microscopy; (4) in vitro cytotoxicity against a panel of HCC cell lines (Huh7, HepG2, Hep3B and GPC3-negative SK-HEP-1); (5) anti-tumor activity of MRG006A evaluated in Huh7 and HepG2 cell line-derived xenograft (CDX) as well as a panel of HCC patient-derived xenograft (PDX) mouse models with a different expression level of GPC3; (6) plasma stability of MRG006A evaluated in human, cynomolgus monkey, rat and mouse plasma; and (7) an exploratory pharmacokinetic (PK) and toxicology study of MRG006A in cynomolgus monkey. Results: The GPC3-targeting ADC, MRG006A, and its antibody moiety exhibited superior binding activity to GPC3-expressing cancer cell lines than a clinical antibody benchmark and similar nanomolar binding affinity to both human and cynomolgus monkey GPC3, which makes cynomolgus monkey suitable for toxicological studies. Rapid internalization of MRG006A antibody was observed in GPC3-expressing Huh7 cells, and MRG006A maintained comparable internalization capability to the naked antibody. More importantly, MRG006A exhibited potent GPC3-dependent cytotoxic activity in a panel of HCC cells expressing varying levels of GPC3. Administrations of MRG006A resulted in a robust and dose-dependent tumor growth inhibition of multiple CDX models and HCC PDX models. Remarkable stability of MRG006A was shown in the plasma of various species tested. MRG006A demonstrated a favorable PK and safety profile with good tolerability in the exploratory toxicology study. Conclusion: Overall, the preclinical study results suggest that MRG006A is a feasible ADC drug candidate for treating GPC3-expressing cancers in clinical studies. MRG006A was aimed to submit IND in China and the U.S in 2024. Citation Format: Yanchun Wang, Hongfeng Li, Hao Shen, Wenchao Liu, Shoujia Liu, Kequan Yin, Haili Xu, Xueyuan Cui, Wei Li, Wei Liu, Xiangyu Wu, Liu Yang, Tian Ma, Zhongrun Zhao, Jun Wang, Feifei Cui, Lei Fang, Minmin Qin, Chaohong Hu. MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3124.

Abstract 3599: Recombinant chimeric antibody-based gamma-delta TCR targeting GPC3 induces potent antitumor response against hepatocellular carcinoma

Abstract Background and Significance: Hepatocellular carcinoma (HCC), a major type of primary liver cancer, is the third leading cause of cancer-related death worldwide. While Glypican-3 (GPC3) is highly upregulated in HCC, it is minimally expressed in normal tissues. The research in our laboratory has been focused on the development of GPC3 as an immunotherapeutic target for liver cancer. Despite many clinical trials, there has not been any approved therapy targeting GPC3 largely due to the combination of antigen heterogeneity, poor tumor penetration, and the inability to induce a durable, potent immune response in the tumor microenvironment (TME). Here, we made a recombinant chimeric antibody-based T cell receptors (named AbTCR) targeting GPC3 and explored its anti-tumor efficacy in xenograft mouse models. Methods: The AbTCR constructs were built by fusing an antigen recognition domain (hYP7 scFv or HN3 VH) specific for GPC3 to the gamma-delta TCR constant region along with a secondary T cell signaling activation motif. The NFAT-tdTomato reporter cell line was established and used to measure the AbTCR-triggered T cell signaling upon stimulation with tumor cells. Anti-tumor efficacy of AbTCR-T cells was determined via luciferase-based cell killing assay as well as multiple HCC xenograft mouse models. Results: The hYP7 GPC3 AbTCR-T cell not only enhances antigen-binding capacity and T cell activity, but also induces infiltration and expansion of T cells in the TME, thereby causing robust liver tumor regression in mice. Conclusion: Our data show that new hYP7-directed GPC3 AbTCR is a promising strategy for treating liver cancer. This work provides a rationale for the use of hYP7 GPC3 AbTCR in clinical studies for treating HCC and other GPC3-positive solid tumors. Citation Format: Dan Li, Tianyuzhou Liang, Zhijian Duan, Hongbing Zhang, Cheng Liu, Mitchell Ho. Recombinant chimeric antibody-based gamma-delta TCR targeting GPC3 induces potent antitumor response against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3599.

Abstract 1637: EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma

Abstract Effective therapeutic regimens against advanced hepatocellular carcinoma (HCCs) have recently improved but it remains very limited. Targeting receptor tyrosine kinases (RTKs) has proven to one of the main ways of achieving better survival outcome in HCC patients, yet the precise mechanism for this success remains to be elusive. We have systematically investigated the expression profiles of RTKs transcriptomic datasets i.e. The Cancer Genome Atlas - Liver hepatocellular carcinoma (TCGA-LIHC) (n=371) and our in-house cohort of paired i.e. HKU-QMH (n=41), and identified EPHB4 as one of the most abundantly expressed and frequently mutated RTKs in human HCCs. EPHB4 expression was found to be significantly correlated with Alpha Fetoprotein (AFP) expression, a factor commonly used in the diagnosis of HCCs. Furthermore, EphB4 has been demonstrated to play a regulatory role in VEGF/VEGFR-mediated angiogenesis, critical targets for current successful drugs. We confirmed abundant membrane EphB4 expression in HCC clinical specimens and human HCC cell line models using immunohistochemistry. Clinicopathological correlation analysis of EPHB4 mRNA expression revealed that its overexpression is associated with aggressive tumor features including presence of tumor microsatellite, liver invasion, venous invasion, and absence of tumor encapsulation. Functionally, stably knocking down EPHB4 (shEPHB4) resulted in a strong reduction of proliferation and self-renewal rates of multiple HCC cell lines in vitro. It significantly reduced tumor growth rates in subcutaneous and liver orthotopic models in vivo. Furthermore, limiting dilution assay showed EPHB4 plays a role in tumor initiation property of HCC cells. Mechanistically, we performed Gene Set Enrichment Analysis (GSEA) of transcriptome profiles for shEPHB4 with respective controls and found that knocking down EPHB4 causes HCC cells undergo drastic changes in several cell cycle checkpoints including G1/S transition. Using thymidine blockade, we further confirmed that knockdown of significantly hinders the transition of HCC cells from G1 to S phase of cell cycle. Our data suggests a strong link between EphB4 and cell cycle. Conclusion: We found EphB4 plays a functional role in regulating G1/S transition in human HCCs and understanding regulatory role of EphB4 on cell cycle progression may provide mechanistic insights into the how to effectively target HCC in the clinic. Citation Format: Abdullah Husain, Elley Y. Chiu, Hoi-Tang Ma, Daniel W. Ho, Karen M. Sze, Lo-Kong Chan, Irene O. Ng. EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1637.

Abstract 6019: Biodistribution, molecular imaging and efficacy evaluation of a novel GPC3-targeted radiopharmaceutical therapy for hepatocellular carcinoma

Abstract Introduction: Glypican-3 (GPC3) is a membrane-anchored oncofetal protein with minimal expression in normal tissues. Significant upregulation of GPC3 protein has been observed in the majority of hepatocellular carcinomas (HCC), and is associated with poor prognosis. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy (RPT) to treat HCC, a leading cause for cancer-related deaths worldwide. Methods: The novel RPT agent RYZ-GPC3 comprises a small macrocyclic peptide binder with high affinity to GPC3 linked with a tetraxetan moiety capable of chelating a variety of radioisotopes. The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR) and radioligand binding assays. Cellular internalization was radiometrically measured at multiple time points. Non-human primate (NHP) PET imaging was performed with 64Cu. In vivo biodistribution, monotherapy and combination treatments with 177Lu or 225Ac were performed in HCC xenografts. Human imaging was conducted in HCC patients with 68Ga PET/CT. Results: The novel agent is a highly potent (KD=0.7 nM) and selective peptide binder to GPC3 of human, mouse, canine and NHP origins, and is compatible with multiple radiometal isotopes. In vivo biodistribution study of [177Lu]Lu-RYZ-GPC3 in HCC xenografts showed sustained tumor uptake of 16.6, 16.4, and 8.8 %ID/g at 24, 48, and 96 hours with fast renal clearance, resulting in favorable tumor/kidney ratios of 3.6, 10.2, and 13.0, respectively. Minimal or no uptake was observed in other normal tissues. PET imaging in NHP confirmed absence of on-target uptake by liver or other organs, and fast renal clearance. As monotherapy, durable (>60 days) tumor regression (TGI>100%) was achieved with 177Lu (111 MBq)- and 225Ac (0.111 MBq)-labeled RYZ-GPC3. At 10x lower injected activity, 177Lu- and 225Ac-labeled RYZ-GPC3 significantly improved anti-tumor effect of lenvatinib in multiple HCC models. Furthermore, PET/CT imaging in a 70-year-old patient with histologically proven HCC demonstrated distinct and avid [68Ga]Ga-RYZ-GPC3 tumor uptake. Conclusion: Preclinical data and preliminary human imaging demonstrate the potential of the novel binder as a theranostic agent for the treatment of patients with GPC3+ HCC. Citation Format: Fanching Lin, Renee Clift, Steven Horton, Matt Guest, Alain Noncovich, Abhijit Bhat, Sanjana Ballal, Chandrasekhar Bal, Kathryn Shah, Anna Karmann, Gary Li. Biodistribution, molecular imaging and efficacy evaluation of a novel GPC3-targeted radiopharmaceutical therapy for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6019.

The cell death-related genes machine learning model for precise therapy and clinical drug selection in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the prevailing subtype of hepatocellular malignancy. While previous investigations have evidenced a robust link with programmed cell death (PCD) and tumorigenesis, a comprehensive inquiry targeting the relationship between multiple PCDs and HCC remains scant. Our aim was to develop a predictive model for different PCD patterns in order to investigate their impact on survival rates, prognosis and drug response rates in HCC patients. We performed functional annotation and pathway analysis on identified PCD-related genes (PCDRGs) using multiple bioinformatics tools. The prognostic value of these PCDRGs was verified through a dataset obtained from GEO. Consensus clustering analysis was utilized to elucidate the correlation between diverse PCD clusters and pertinent clinical characteristics. To comprehensively uncover the distinct PCD regulatory patterns, our analysis integrated gene expression profiling, immune cell infiltration and enrichment analysis. To predict survival differences in HCC patients, we established a PCD model. To enhance the clinical applicability for the model, we developed a highly accurate nomogram. To address the treatment of HCC, we identified several promising chemotherapeutic agents and novel targeted drugs. These drugs may be effective in treating HCC and could improve patient outcomes. To develop a cell death feature for HCC patients, we conducted an analysis of 12 different PCD mechanisms using eligible data obtained from public databases. Through this analysis, we were able to identify 1254 PCDRGs likely to contribute to cell death on HCC. Further analysis of 1254 PCDRGs identified 37 genes with prognostic value in HCC patients. These genes were then categorized into two PCD clusters A and B. The categorization was based on the expression patterns of the genes in the different clusters. Patients in PCD cluster B had better survival probabilities. This suggests that PCD mechanisms, as represented by the genes in cluster B, may have a protective effect against HCC progression. Furthermore, the expression of PCDRGs was significantly higher in PCD cluster A, indicating that this cluster may be more closely associated with PCD mechanisms. Furthermore, our observations indicate that patients exhibiting elevated tumour mutation burden (TMB) are at an augmented risk of mortality, in comparison to those displaying low TMB and low-risk statuses, who are more likely to experience prolonged survival. In addition, we have investigated the potential distinctions in the susceptibility of diverse risk cohorts towards emerging targeted therapies, designed for the treatment of HCC. Moreover, our investigation has shown that AZD2014, SB505124, LJI308 and OSI-207 show a greater efficacy in patients in the low-risk category. Conversely, for the high-risk group patients, PD173074, ZM447439 and CZC24832 exhibit a stronger response. Our findings suggest that the identification of risk groups and personalized treatment selection could lead to better clinical outcomes for patients with HCC. Furthermore, significant heterogeneity in clinical response to ICI therapy was observed among HCC patients with varying PCD expression patterns. This novel discovery underscores the prospective usefulness of these expression patterns as prognostic indicators for HCC patients and may aid in tailoring targeted treatment for those of distinct risk strata. Our investigation introduces a novel prognostic model for HCC that integrates diverse PCD expression patterns. This innovative model provides a novel approach for forecasting prognosis and assessing drug sensitivity in HCC patients, driving a more personalized and efficacious treatment paradigm, elevating clinical outcomes. Nonetheless, additional research endeavours are required to confirm the model's precision and assess its potential to inform clinical decision-making for HCC patients.

Targeting RORγ inhibits the growth and metastasis of hepatocellular carcinoma

Approximately 80%-90% of hepatocellular carcinomas (HCC) occur in a premalignant environment of fibrosis and abnormal extracellular matrix (ECM), highlighting an essential role of ECM in the tumorigenesis and progress of HCC. However, the determinants of ECM in HCC are poorly defined. Here, we show that nuclear receptor RORγ is highly expressed and amplified in HCC tumors. RORγ functions as an essential activator of the matrisome program via directly driving the expression of major ECM genes in HCC cells. Elevated RORγ increases fibronectin-1 deposition, cell-matrix adhesion, and collagen production, creating a favorable microenvironment to boost liver cancer metastasis. Moreover, RORγ antagonists effectively inhibit tumor growth and metastasis in multiple HCC xenografts and immune-intact models, and they effectively sensitize HCC tumors to sorafenib therapy in mice. Notably, elevated RORγ expression is associated with ECM remodeling and metastasis in patients with HCC. Taken together, we identify RORγ as a key player of ECM remodeling in HCC and as an attractive therapeutic target for advanced HCC.

A Mouse Model of Hepatocellular Carcinoma Induced by Streptozotocin and High-Fat Diet.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the second most common cause of cancer-related death. HCC is associated to chronic diseases such as viral hepatitis, alcoholic, and non-alcoholic fatty liver disease (NAFLD), diabetes mellitus, and obesity, among others. Although pre-clinical models have been investigated to mimic the transition from NAFLD to HCC, they do not accurately reproduce the phenotypic evolution from simple steatosis to steatohepatitis, fibrosis/cirrhosis, and HCC. Hence, these models have failed to demonstrate the influence of diabetes on hepatic carcinogenesis. Here, we report a novel mouse model of HCC triggered by fast-developing diabetes and NAFLD. The first step consists in a single intraperitoneal injection of a low dose of streptozotocin into neonatal C57BL/6J mice to induce type 2 diabetes. In a second step, mice are fed with high-fat diet to accelerate the development of simple steatosis. Continuous high-fat diet exacerbates hepatic fat deposition with increased lobular inflammation (by activation of foam cell-like macrophages) and fibrosis (by activating hepatic stellate cells), two representative pathological traits of steatohepatitis/fibrosis. After 20weeks, all mice developed multiple HCCs. This model of hepatic carcinogenesis triggered by diabetes mellitus and NAFLD offers the advantage of being rapid and accurately recapitulates the pathogenesis of human HCC without the need of administering hepatic carcinogens.

NIR-II fluorescence-guided liver cancer surgery by a small molecular HDAC6 targeting probe

Hepatocellular carcinoma (HCC) is the sixth most common malignancy globally and ranks third in terms of both mortality and incidence rates. Surgical resection holds potential as a curative approach for HCC. However, the residual disease contributes to a high 5-year recurrence rate of 70%. Due to their excellent specificity and optical properties, fluorescence-targeted probes are deemed effective auxiliary tools for addressing residual lesions, enabling precise surgical diagnosis and treatment. Research indicates histone deacetylase 6 (HDAC6) overexpression in HCC cells, making it a potential imaging biomarker. This study designed a targeted small-molecule fluorescent probe, SeCF3-IRDye800cw (SeCF3-IRD800), operating within the Second near-infrared window (NIR-II, 1000-1700nm). The study confirms the biocompatibility of SeCF3-IRD800 and proceeds to demonstrate its applications in imaging invivo, fluorescence-guided surgery (FGS) for liver cancer, liver fibrosis imaging, and clinical samples incubation, thereby preliminarily validating its utility in liver cancer. SeCF3-IRD800 was synthesized by combining the near-infrared fluorescent dye IRDye800cw-NHS with an improved HDAC6 inhibitor. Initially, a HepG2-Luc subcutaneous tumor model (n=12) was constructed to investigate the metabolic differences between SeCF3-IRD800 and ICG invivo. Subsequently, HepG2-Luc (n=12) and HCCLM3-Luc (n=6) subcutaneous xenograft mouse models were used to assess invivo targeting by SeCF3-IRD800. The HepG2-Luc orthotopic liver cancer model (n=6) was employed to showcase the application of SeCF3-IRD800 in FGS. Liver fibrosis (n=6) and HepG2-Luc orthotopic (n=6) model imaging results were used to evaluate the impact of different pathological backgrounds on SeCF3-IRD800 imaging. Three groups of fresh HCC and normal liver samples from patients with liver cancer were utilized for SeCF3-IRD800 incubation exvivo, while preclinical experiments illustrated its potential for clinical application. The HDAC6 inhibitor 6 (SeCF3) modified with trifluoromethyl was labeled with IRDy800CW-NHS to synthesize the small-molecule targeted probe SeCF3-IRD800, with NIR-II fluorescence signals. SeCF3-IRD800 was rapidly metabolized by the kidneys and exhibited excellent biocompatibility. Invivo validation demonstrated that SeCF3-IRD800 achieved optimal imaging within 8h, displaying high tumor fluorescence intensity (7658.41±933.34) and high tumor-to-background ratio (5.20±1.04). Imaging experiments with various expression levels revealed its capacity for HDAC6-specific targeting across multiple HCC tumor models, suitable for NIR-II intraoperative imaging. Fluorescence-guided surgery experiments were found feasible and capable of detecting sub-visible 2mm tumor lesions under white light, aiding surgical decision-making. Further imaging of liver fibrosis mice showed that SeCF3-IRD800's imaging efficacy remained unaffected by liver pathological conditions. Correlations were observed between HDAC6 expression levels and corresponding fluorescence intensity (R2=0.8124) among normal liver, liver fibrosis, and HCC tissues. SeCF3-IRD800 identified HDAC6-positive samples from patients with HCC, holding advantages for perspective intraoperative identification in liver cancer. Thus, the rapidly metabolized HDAC6-targeted small-molecule NIR-II fluorescence probe SeCF3-IRD800 holds significant clinical translational value. The successful application of NIR-II fluorescence-guided surgery in liver cancer indicates that SeCF3-IRD800 has great potential to improve the clinical diagnosis and treatment of liver cancer, and could be used as an auxiliary tool for surgical treatment of liver cancer without being affected by liver pathology. This paper is supported by the National Natural Science Foundation of China (NSFC) (92,059,207, 62,027,901, 81,930,053, 81,227,901, 82,272,105, U21A20386 and 81,971,773), CAS Youth Interdisciplinary Team (JCTD-2021-08), the Zhuhai High-level Health Personnel Team Project (Zhuhai HLHPTP201703), and Guangdong Basic and Applied Basic Research Foundation under Grant No. 2022A1515011244.

Identification of 13 Novel Loci in a Genome-Wide Association Study on Taiwanese with Hepatocellular Carcinoma.

Liver cancer is caused by complex interactions among genetic factors, viral infection, alcohol abuse, and metabolic diseases. We conducted a genome-wide association study and polygenic risk score (PRS) model in Taiwan, employing a nonspecific etiology approach, to identify genetic risk factors for hepatocellular carcinoma (HCC). Our analysis of 2836 HCC cases and 134,549 controls revealed 13 novel associated loci such as the FAM66C gene, noncoding genes, liver-fibrosis-related genes, metabolism-related genes, and HCC-related pathway genes. We incorporated the results from the UK Biobank and Japanese database into our study for meta-analysis to validate our findings. We also identified specific subtypes of the major histocompatibility complex that influence both viral infection and HCC progression. Using this data, we developed a PRS to predict HCC risk in the general population, patients with HCC, and HCC-affected families. The PRS demonstrated higher risk scores in families with multiple HCCs and other cancer cases. This study presents a novel approach to HCC risk analysis, identifies seven new genes associated with HCC development, and introduces a reproducible PRS model for risk assessment.

Identification of metabolism terms significantly affecting hepatocellular carcinoma immune microenvironment and immunotherapy response.

Metabolic pathways exert a significant influence on the onset and progression of cancer. Public data on hepatocellular carcinoma (HCC) patients were obtained from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases. Analysis was performed in R software using different R packages. Here, we integrated the data from multiple independent HCC cohorts, including TCGA-LIHC, ICGC-FR and ICGC-JP. Then, the enrichment score of 21 metabolism-related pathways was quantified using the ssGSEA algorithm. Next, univariate Cox regression analysis was applied to identify the metabolic terms with significant correlation to patient survival. Finally, a prognosis model based on linoleic acid metabolism, sphingolipid metabolism and regulation of lipolysis in adipocytes was established, which showed good performance in predicting patients' survival. Furthermore, we conducted a biological enrichment analysis to delineate the biological disparities between high- and low-risk patients. Notably, we discerned differences in the microenvironments between these two patient groups. We also found that low-risk patients could potentially respond better to immunotherapy. Drug sensitivity analysis suggested that low-risk patients are more susceptible to bexarotene and erlotinib, yet exhibit resistance to ATRA and bleomycin. Furthermore, through the use of LASSO logistic regression analysis, we identified 19 characteristic genes, which could robustly indicate the risk groups. Our research underscores the role of linoleic acid metabolism, sphingolipid metabolism and the regulation of lipolysis in adipocytes in HCC, pointing towards potential avenues for future research.

Laparoscopic versus open liver resection for multiple hepatocellular carcinoma within and beyond the Milan criteria: An Eastern-Western propensity score-matched analysis.

There is still little knowledge about the outcomes of laparoscopic liver resection (LLR) for multiple hepatocellular carcinomas (HCC). The aim of this study was to assess the short- and long- term outcomes of LLR versus open liver resection (OLR) for patients with multiple HCC within and beyond the Milan criteria, and in both BCLC-A and -B stage. Data regarding all consecutive patients undergoing liver resection for multiple HCC were retrospectively collected from Asian (South Korean) and European (Italian) referral HPB centers. The cases were propensity-score matched for age, BMI, center, extent of the resection, postero-superior location of the lesion, underlying liver condition, BCLB staging and the Milan criteria. A total of 203 patients were included in the study: 27% of patients had undergone hemi-hepatectomy, 26.6% atypical resections, 20.6% sectionectomy and 16.2% segmentectomy. After PSM two cohorts of 57 patients were obtained, with no significant differences in all preoperative characteristics. The length of hospital stay was significantly lower after LLR (median 7 vs. 9 days, p < .01), with no statistically significant differences in estimated blood loss, operation time, transfusions, postoperative bile leak, ascites, severe complications and R1 resection rates. After a median follow-up of 61 (±7) months, there were no significant differences between OLR and LLR in both median OS (69 vs. 59 months, p = .74, respectively) and median DFS (12 vs. 10 months, p = .48, respectively). LLR for multiple HCC can be safe and effective in selected cases and is able to shorten median hospital stay without affecting perioperative and long-term oncological outcomes.

Outcomes and Prognostic Analysis of Surgical Resection for Oligometastasis from Hepatocellular Carcinoma.

This study aimed to evaluate the outcomes of patients who underwent resection for oligometastasis from hepatocellular carcinoma (HCC) and identify the prognostic factors associated with poor survival. Patients who underwent resection for oligometastasis from HCC between January 2000 and April 2021 were retrospectively investigated. Oligometastasis was defined as 1-5 single organ metastases that were detected preoperatively in this study. Clinical characteristics and treatment outcomes were analyzed, and independent risk factors for poor prognosis were identified using cox proportional hazards model. A total of 33 patients were included in this study. Eleven oligometastases were located in the intraabdominal lymph node, 8 in the adrenal gland, 5 in the lung, 4 in the peritoneum, 3 in the pleura, and 1 each in the supraclavicular lymph node and abdominal wall. No re-operation or operative death occurred in this study. The median OS was 44.6 months (range=5.1-150.6 months), and the median survival after primary HCC diagnosis was 116.5 months (range=7.1-253.6 months). The median cumulative incidence of recurrent HCC was 7.2 months (range=0.3-94.7 months). The multivariate analysis showed that an alpha-fetoprotein level ≥20 ng/ml and multiple primary HCC tumors were independent poor prognostic factors. Clinical characteristics and treatment outcomes of patients who underwent resection for oligometastasis from HCC were demonstrated. A high alpha-fetoprotein level and multiple primary HCC tumors were independent poor prognostic factors. Surgical resection can be one of the treatment options for oligometastasis from HCC.

E2F1-mediated Up-regulation of NCAPG Promotes Hepatocellular Carcinoma Development by Inhibiting Pyroptosis.

As a subunit of the condensin complex, NCAPG has an important role in maintaining chromosome condensation, but its biological function and regulatory mechanism in hepatocellular carcinoma (HCC) remains undefined. The prognostic ability of NCAPG in HCC patients was examined by univariate and multivariate Cox regression analysis. ROC curves were plotted to compare the predictive ability of NCAPG and AFP. Double luciferase reporter system, and ChIP were used to investigate transcriptional potential of E2F1 to NCAPG. Pyroptosis was observed by scanning electron microscopy. Protein expression of NCAPG, E2F1, and major proteins constituting NLRP3 inflammasome was determined by western blotting and ELISA. An in vivo tumor formation assay was conducted to verify the in vitro results. Up-regulated NCAPG was identified in HCC tissues compared with adjacent tissue and high NCAPG was positively correlated with poor prognosis. Serum NCAPG mRNA level was a prognostic factor in HCC patients and also a diagnostic factor with higher predictive ability compared with AFP [AUROC 0.766 (95% CI: 0.650-0.881) vs. 0.649 (95% CI 0.506-0.793)]. HBx transfection resulted in concomitant up-regulation of E2F1 and NCAPG. E2F1 significantly increased the activity of luciferase reporter fused with NCAPG reporter, and the interaction of E2F1 and NCAPG gene was confirmed by ChIP. Silencing of E2F1 resulted in significant down-regulation of NCAPG. Knockdown of NCAPG promote pyroptosis mediated by NLRP3 inflammasome activation in multiple HCC cell lines and also suppressed tumorigenesis in vitro. We identified a novel role of NCAPG in the regulation of NLRP3 inflammasome-mediated pyroptosis, which was regulated by its upstream transactivator, E2F1. The role of E2F1-NCAPG-NLRP3 regulation of pyroptosis network may be a potential target in HCC treatment.

Clinical outcome of surgical resection for multifocal T2-T3 hepatocellular carcinoma up to 3 nodules: a comparative analysis with a single nodule.

Although the Barcelona Clinic Liver Cancer staging system seems to underestimate the impact of curative-intent surgical resection for multifocal hepatocellular carcinoma (HCC), recent studies have indicated favorable results for the surgical resection of multiple HCC. This study aimed to assess clinical outcomes and feasibility of surgical resection for multifocal HCC with up to three nodules compared with single tumor cases. Patients who underwent surgical resection for HCC with up to three nodules between 2009 and 2020 were included, and those with the American Joint Committee on Cancer (AJCC) 8th edition, T1 and T4 stages were excluded to reduce differences in disease distribution and severity. Finally, 81 and 52 patients were included in the single and multiple treatment groups, respectively. Short- and long-term outcomes including recurrence-free survival (RFS) and overall survival (OS), were evaluated. All patients were classified as Child-Pugh class A. RFS and OS were not significantly different between the two groups (P=0.176 and P=0.966, respectively). Multivariate analysis revealed that transfusion and intrahepatic metastasis were significantly associated with recurrence (P=0.046 and P=0.005, respectively). Additionally, intrahepatic metastasis was significantly associated with OS (hazard ratio, 1.989; 95% confidence interval, 1.040-3.802; P=0.038). Since there was no significant difference in survival between the single and multiple groups among patients with AJCC 8th stage T2 and T3, surgical resection with curative intent could be considered with acceptable long-term survival for selected patients with multiple HCC of up to three nodules.

PGAM1 Inhibition Promotes HCC Ferroptosis and Synergizes with Anti-PD-1 Immunotherapy.

The combination of immunotherapy and molecular targeted therapy exhibits promising therapeutic efficacy in hepatocellular carcinoma (HCC), but the underlying mechanism is still unclear. Here, phosphoglycerate mutase 1 (PGAM1) is identified as a novel immunometabolic target by using a bioinformatic algorithm based on multiple HCC datasets. PGAM1 is highly expressed in HCC and associated with a poor prognosis and a poor response to immunotherapy. In vitro and in vivo experiments indicate that targeting PGAM1 inhibited HCC cell growth and promoted the infiltration of CD8+ T-cells due to decreased enzymatic activity. Mechanistically, inhibition of PGAM1 promotes HCC cell ferroptosis by downregulating Lipocalin (LCN2) by inducing energy stress and ROS-dependent AKT inhibition, which can also downregulate Programmed death 1-ligand1 (PD-L1). Moreover, an allosteric PGAM1 inhibitor (KH3) exhibits good antitumor effects in patient-derived xenograft (PDX) models and enhanced the efficacy of anti-PD-1 immunotherapy in subcutaneous and orthotopic HCC models. Taken together, the findings demonstrate that PGAM1 inhibition exerts an antitumor effect by promoting ferroptosis and CD8+ T-cell infiltration and can synergize with anti-PD-1 immunotherapy in HCC. Targeting PGAM1 can be a promising new strategy of "killing two birds with one stone" for HCC treatment.

Glucose-induced and ChREBP: MLX-mediated lipogenic program promotes hepatocellular carcinoma development.

The Carbohydrate Response Element (ChoRE) Binding Protein (ChREBP) and its binding partner Max-like protein X (MLX) mediate transcription of lipogenic genes under glucose-rich conditions. Dysregulation of glucose and lipid metabolism frequently occurs in cancers, including Hepatocellular Carcinomas (HCCs). However, it is currently unclear whether the glucose-induced lipogenic program plays a role in the development of HCCs. Here, we show that MLX expression is elevated in HCC specimens and downregulation of MLX expression inhibits proliferation of HCC cells. In mice, liver-specific knockout of Mlx results in dramatic decrease in the expression of lipogenic genes and lipid levels in circulation. Interestingly, in the absence of Mlx, the development of tumors in multiple HCC models, such as diethylnitrosamine (DEN) treatment and hydrodynamic injection of oncogenes (AKT/RAS or CTNNB1/RAS), is robustly blocked. However, a high-fat diet can partially restore tumorigenesis in Mlx-deficient livers, indicating a critical role of lipid synthesis in HCC development. In addition, liver-specific expression of a dominant negative MLX (dnMLX) via adeno-associated virus effectively blocks tumorigenesis in mice. Thus, the glucose-induced lipogenic program is required in the development of HCC, and the ChREBP: MLX transcription factors serve as a potential target for cancer therapies.

Dietary antioxidant quercetin overcomes the acquired resistance of Sorafenib in Sorafenib-resistant hepatocellular carcinoma cells through epidermal growth factor receptor signaling inactivation.

Sorafenib (SOR) is a molecular targeting agent commonly utilized as a primary treatment for advanced and inoperable hepatocellular carcinoma (HCC). Regrettably, the effectiveness of SOR is frequently hindered by the resistance of multiple HCC cases. The current investigation endeavors to examine the potential of the natural product quercetin (QUE) in reversing the acquired resistance of SOR-resistant cells, known as Huh7R, to SOR. Moreover, this study aims to elucidate the underlying molecular mechanism that contributes to this phenomenon. The results demonstrated that QUE significantly impeded proliferation and stimulated apoptosis in Huh7R cells, while also suppressing the growth of transplanted tumors. The impact of QUE enhanced the efficacy of SOR treatment for Huh7R. Additionally, bioinformatic and western blot analyses indicated that the underlying mechanisms may be associated with EGFR tyrosine kinase inhibitor resistance, the PI3K-AKT signaling pathway, and HCC. Furthermore, molecular docking and dynamics simulation assays revealed that QUE exhibited strong affinity and stability towards its hub targets, EGFR and AKT1. It is noteworthy that the activation of EGFR by its ligand, EGF, mitigated the effects of co-treatment with QUE and SOR. These findings suggest that QUE might potentially serve as a therapeutic agent in treating as well as facilitating SOR against Huh7R cells, which has substantial clinical and research implications for the treatment of acquired resistance to SOR in HCC.