Effects In Hepatocellular Carcinoma Research Articles

Abstract CT210: Phase Ib/II study of sorafenib (SOR) and pembrolizumab (PEM) in patients (pts) with advanced hepatocellular carcinoma (HCC)

Abstract Background: SOR is an active agent and has favorable immunomodulatory effects in HCC. Combinations of immune checkpoint inhibitors with SOR may enhance effector T-cell function and lead to improved clinical outcomes for patients with HCC. We tested this hypothesis in an open-label, multicenter study of SOR and PEM in patients with advanced HCC. Methods: Pts with advanced or metastatic HCC, CP Class A, ECOG PS of 0/1 were included. One prior therapy was allowed. Pts were treated with SOR alone for 4 weeks (lead-in) at a stable dose (minimum 200 BID), followed by SOR plus PEM 200mg IV q3 weeks. The phase Ib part included the first 6 pts who completed the SOR lead-in and began SOR+PEM. Treatment continued until disease progression or unacceptable toxicity. Disease assessment was performed q6 weeks using the RECIST 1.1 criteria. The primary endpoint was overall response rate (ORR). The study used an exact one-stage design. With 27 evaluable pts, the study had 80% power to detect an ORR ≥20% with SOR/PEM vs. 5% with SOR alone (a=0.05). Peripheral blood mononuclear cells were collected at baseline and on-treatment for correlative analyses (flow cytometry). Results: Of the 37 total pts enrolled, 27 were evaluable (9 female). Median age was 68 years. Forty-four percent of the pts had viral hepatitis. Four pts were pre-treated with atezolizumab/bevacizumab (n=3) or tivozanib/durvalumab (n=1). The ORR was 33% (95% CI: 18 - 52%) p= 0.08. Best response was PR in 9 patients (33%), 12 patients SD (44%), and 6 (22%) had PD. One patient had a near CR. Median progression-free (PFS) and overall survival (OS) were 4.8 (95% CI: 3.4 - 16.3) and 28.5 (95% CI: 15.2 - 57.7) months respectively. Fifty-two percent of patients had a decrease in AFP >50%. One atezolizumab/bevacizumab pretreated pt with progressive disease as best response had a near-complete response to SOR+PEM. The most common grade ≥3 treatment-related adverse events (AEs) in the safety population (n=37) were hypertension (16%), immune-related AEs (11%), fatigue (8%) and diarrhea (8%). CD8+ T-cells increased by 5.5% (-35.6 - 36.9%; p=0.035) and Tregs decreased by 14.73% (-378.6 - 61%, p=0.049) from baseline to C1D1. The increase in activated Tregs prior to PEM initiation was associated with worse OS (HR: 1.8, p=0.03) while a higher Teff/Treg at C1D1 (prior to PEM initiation) was associated with improved PFS (HR: 0.4, p=0.036). An increase in the percentage of Tregs between baseline and C4D1 was associated with worse PFS (HR: 2; p=0.03). Conclusions: SOR+PEM is a safe and active treatment for pts with advanced HCC. The results of our study confirm the favorable immunomodulatory effects of SOR and support further exploration of this regimen for patients with advanced HCC. Further tissue and blood correlative analyses are ongoing. NCT03211416. Citation Format: Renuka Iyer, Sahithi Sonti, Devalingam Mahalingam, Sarbajit Mukherjee, Sayan Chakraborty, Kristopher Attwood, Anthony George, Orla Maguire, Hans Minderman, Christos Fountzilas. Phase Ib/II study of sorafenib (SOR) and pembrolizumab (PEM) in patients (pts) with advanced hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT210.

Inhibiting Pink1/Parkin-mediated mitophagy enhances the anticancer effects of quercetin in hepatocellular carcinomaf

Quercetin, a naturally occurring flavonoid, has been investigated for its potential anti-cancer effects in various types of cancer, including hepatocellular carcinoma (HCC). However, its suppressing effect on reactive oxygen species (ROS) production might limited its anti-cancer effects. In this study, we aimed to explore the interplay among quercetin, mitochondrial dynamics and mitophagy and whether mitophagy-inhibition synergistically enhances the anti-tumor effects of quercetin. Huh7 and Hep3B cells were utilized for in vitro and in vivo studies. Results showed that quercetin treatment significantly increased the expression of mitochondrial fusion genes (MFN1 and MFN2) and decreased the expression of fission genes (DRP1 and FIS1) in Huh7 and Hep3B cells, leading to a more fused and elongated mitochondrial network. Quercetin upregulated the expression of key mitophagy regulators, PINK1 and PARK2, and enhanced the colocalization of mitochondria with lysosomes, indicating increased mitophagy. Knockdown of PINK1, PARK2, or SIRT1 attenuated quercetin-induced mitophagy and reduction of intracellular ROS levels. Quercetin treatment upregulates SIRT1 expression, which subsequently enhances PINK1 and PARK2 expression in Huh7 and Hep3B cells. In vivo experiments using Hep3B xenograft models revealed that the combination of quercetin with the mitophagy inhibitor hydroxychloroquine or SIRT1 knockdown significantly enhanced the anticancer effects of quercetin, as evidenced by reduced tumor size and weight, increased necrosis and apoptosis, and decreased proliferation in tumor tissues. These findings suggest that quercetin-induced mitochondrial fusion and Pink1/Parkin-dependent mitophagy may negatively influence its anti-cancer effects in HCC. Targeting mitophagy may enhance the therapeutic potential of quercetin in HCC treatment.

Deciphering m6A signatures in hepatocellular carcinoma: Single-cell insights, immune landscape, and the protective role of IGFBP3.

RNA m6 methyladenosine (m6A) modifications impact tumor biology and immune processes, particularly in hepatocellular malignant tumors. Using a consensus clustering algorithm on 371 hepatocellular carcinoma (HCC) samples, we identified three m6A-modified subtypes and correlated them with positive tumor microenvironment (TME) markers for distinct immune phenotypes. Stratifying patients based on m6A scores revealed a low presentation group with better immune penetration, lower tumor mutation load, and increased expression of immune checkpoint markers like CTLA-4 and PD-1, suggesting enhanced responsiveness to immunization therapy. A machine-learning model of 23 m6A genes was constructed. Single-cell analysis revealed a surprising enrichment of IGFBP3 in astrocytes, prompting the exploration of associated signaling pathways. Experimental verification shows that IGFBP3 is significantly enhanced in normal tissues, while immunohistochemical analysis shows that its expression is lower in tumor tissues, indicating its protective effect in HCC and a good prognosis. Importantly, high IGFBP3 expression is associated with better outcomes in patients receiving immunotherapy. Moreover, cytotoxic T lymphocyte (CTL) experiments have confirmed that high expression of IGFBP3 is associated with stronger T cell-killing ability. In summary, the comprehensive evaluation of m6A modification, immune characteristics, and single-cell analysis in this study not only revealed the TME of HCC but also made significant contributions to the progress of personalized HCC immunotherapy targeting IGFBP3. This study provides a solid theoretical foundation for clinical translation and emphasizes its potential impact on developing effective treatment strategies.

Exosome-Related FTCD Facilitates M1 Macrophage Polarization and Impacts the Prognosis of Hepatocellular Carcinoma.

Exosomes are essential for hepatocellular carcinoma (HCC) progression and have garnered significant interest as novel targets for diagnostic, prognostic, and therapeutic approaches. This study aims to identify potential exosome-related biomarkers for the development of useful strategies for HCC diagnosis and therapy. Three datasets obtained from the Gene Expression Omnibus (GEO) were utilized to identify differentially expressed genes (DEGs) in HCC. Through Gene Ontology (GO) analysis and protein-protein interaction (PPI) network, overall survival (OS) analysis, Cox analyses, and diethylnitrosamine (DEN)-induced HCC mouse model detection, exosome-related hub gene was screened out, followed by a prognostic value assessment and immune-correlates analysis based on the Cancer Genome Atlas (TCGA) dataset. The hub gene-containing exosomes derived from Hepa1-6 cells were isolated and characterized using differential ultracentrifugation, transmission electron microscopy scanning, and Western blot. Ultrasound-guided intrahepatic injection, cell co-culture, CCK-8, and flow cytometry were performed to investigate the effects of the hub gene on macrophage infiltration and polarization in HCC. A total of 83 DEGs enriched in the extracellular exosome term, among which, FTCD, HRA, and C8B showed the strongest association with the progression of HCC. FTCD was independently associated with a protective effect in HCC and selected as the hub gene. The presence of FTCD in exosomes was confirmed. FTCD-stimulated macrophages were polarized towards the M1 type and suppressed HCC cells proliferation. FTCD is a potential exosome-related biomarker for HCC diagnosis, prognosis, and treatment. The crosstalk between FTCD-containing exosomes and macrophages in HCC progression deserves further investigation.

NLRP3/IL-1β induced myeloid-derived suppressor cells recruitment and PD-L1 upregulation promotes oxaliplatin resistance of hepatocellular carcinoma.

Oxaliplatin is commonly used as the first-line chemotherapeutic agent for advanced hepatocellular carcinoma (HCC). Unfortunately, the acquired resistance, limits the effectiveness of oxaliplatin and the underlying mechanisms remain unknown. Therefore, we explored the role of NOD-like receptor protein 3 (NLRP3)/IL-1β in mediating oxaliplatin resistance in HCC. We observed that NLRP3/IL-1β expression was much higher in oxaliplatin-resistant HCC cells. To further understand its impact on drug resistance, we knocked down NLRP3 and observed that it sensitized HCC cells to the growth-inhibitory effects of oxaliplatin and induced cell apoptosis. NLRP3/IL-1β overexpressing tumor cells also attracted polymorphonuclear myeloid-derived suppressor cells. Using mouse models, we demonstrated that NLRP3/IL-1β inhibition by short hairpin RNA or MCC950 effectively overcame oxaliplatin resistance. Furthermore, NLRP3/IL-1β inhibition resulted in reduced expression of PD-L1. We also found that PD-L1 antibody combined with NLRP3/IL-1β blockade displayed significant antitumor effect in HCC. Overall, our study provides compelling evidence supporting the essential role of NLRP3/IL-1β in conferring resistance to oxaliplatin and reshaping the immunosuppressive microenvironment in HCC. Targeting NLRP3/IL-1β presents a potential therapeutic target for overcoming oxaliplatin resistance and reshaping microenvironment of HCC.

KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ

BackgroundPrimary hepatocellular carcinoma (HCC) is a malignancy with high morbidity and mortality. KH domain-containing, RNA-binding signal transduction-associated protein 3 (KHDRBS3) is an RNA-binding protein that is aberrantly expressed in multiple tumors; however, its expression and biological function in HCC have not been reported.MethodsKHDRBS3 knockdown and overexpression were performed using the lentiviral vector system to investigate the effects of KHDRBS3 on cell proliferation, apoptosis, chemoresistance, and glycolysis. Murine xenograft tumor models were constructed to study the role of KHDRBS3 on tumor growth in vivo. Furthermore, RNA-Pull Down and RNA immunoprecipitation were utilized to explore the interaction between KHDRBS3 and 14-3-3ζ, a phosphopeptide-binding molecule encoded by YWHAZ.ResultsKHDRBS3 was highly expressed in human HCC tissues and predicted the poor prognosis of patients with HCC. Knockdown of KHDRBS3 exhibited a carcinostatic effect in HCC and impeded proliferation and tumor growth, reduced glycolysis, enhanced cell sensitivity to doxorubicin, and induced apoptosis. On the contrary, forced expression of KHDRBS3 expedited the malignant biological behaviors of HCC cells. The expression of KHDRBS3 was positively correlated with the expression of 14-3-3ζ. RNA immunoprecipitation and RNA pull-down assays demonstrated that KHDRBS3 bound to YWHAZ. We further confirmed that 14-3-3ζ silencing significantly reversed the promotion of proliferation and glycolysis and the inhibition of apoptosis caused by KHDRBS3 overexpression.ConclusionsOur findings suggest that KHDRBS3 promotes glycolysis and malignant progression of HCC through upregulating 14-3-3ζ expression, providing a possible target for HCC therapy.

The double-edged sword role of hydrogen sulfide in hepatocellular carcinoma.

With an increasing worldwide prevalence, hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver in the world. It is also the primary reason for cancer-related death in the world. The pathogenesis of HCC is complex, such as DNA methylation changes, immune regulatory disorders, cell cycle disorders, chromosomal instability, and so on. Although many studies have been conducted on HCC, the molecular mechanisms of HCC are not completely understood. At present, there is no effective treatment for HCC. Hydrogen sulfide (H2S) has long been regarded as a toxic gas with the smell of rotten eggs, but recent studies have shown that it is an important gasotransmitter along with carbon monoxide (CO) and nitric oxide (NO). Increasing evidence indicates that H2S has multiple biological functions, such as anti-inflammation, anti-apoptosis, anti-oxidative stress, and so on. Recently, a lot of evidence has shown that H2S has a "double-edged sword" effect in HCC, but the mechanism is not fully understood. Here, we reviewed the progress on the role and mechanism of H2S in HCC in recent years, hoping to provide a theoretical reference for future related research.

Construction of a stable centromere protein F overexpression cell model of hepatocellular carcinoma using CRISPR activation system

Current studies have shown that centromere protein F (CENPF) was overexpressed in hepatocellular carcinoma (HCC) and might be involved in the pathogenesis of HCC. Specifically, due to the very large molecular weight (358 kDa) of CENPF full length protein, only CENPF knock-down, but not overexpression models, were applied currently to explore the carcinogenicity of CENPF in HCC. Whether CENPF overexpression is a cause or an effect in HCC remains to be illustrated. We aimed to establish a CENPF overexpression cell model using CRISPR/dCas9 synergistic activation mediator (SAM) system with lentiMPHv2 and lentiSAMv2 vectors to explore the role of CENPF overexpression in HCC. Single guide RNAs (sgRNAs) that specifically identify the transcription initiation site of CENPF gene were synthesized and inserted into the lentiSAMv2 plasmid. Huh-7 and HCCLM3 cells were first transduced with lentiMPHv2 and then selected with hygromycin B. The cells were then transduced with lentiSAMv2 carrying specific sgRNA for CENPF gene, followed by blasticidin S selection. The mRNA and protein detection results of Huh-7 and HCCLM3 cells screened by hygromycin B and blasticidin S showed that the endogenous overexpression of CENPF can be induced by sgRNA1 and sgRNA4, especially by sgRNA4. By using the CRISPR/dCas9 technique, stable cell models with overexpressed CENPF were successfully constructed to explore the role of CENPF in tumorigenesis, which provides a reference for the construction of cell models overexpressing large molecular weight protein.

A novel immunogenic cell death-related genes signature for predicting prognosis, immune landscape and immunotherapy effect in hepatocellular carcinoma.

Immunogenic cell death (ICD) has emerged as a promising strategy to activate the adaptive immune response, modulate the tumor microenvironment (TME) and enhance the efficacy of immune therapy. However, the relationship between ICD and TME reprogramming in hepatocellular carcinoma (HCC) remains poorly understood. Transcriptional profiles and clinical spectrum of 486 HCC patients were obtained from TCGA and GEO databases. We utilized consensus clustering analysis to construct two distinct molecular subtypes and established an ICD-based scoring system (named ICD score) via WGCNA and LASSO Cox regression to predict the prognosis of the HCC cohort. Then we employed CIBERSORT and ESTIMATE methods to analyze the immune landscape of ICD score in HCC. Subsequently, the immunophenoscore (IPS) and tumor immune dysfunction and rejection (TIDE) analyses were performed to determine whether the ICD score could influence the immune therapeutic effect. Based on the ICD scoring system, a novel nomogram was generated to provide a numerical probability of HCC patients' overall survival (OS). We identified two independent ICD clusters (cluster A/B), and cluster B possessed a worse prognosis and higher immune cell infiltration. Using ICD scoring system, the HCC patients were divided into high- and low-ICD-score groups. Through integrative analyses, the high-ICD cohort owned advanced TNM stage, high pathologic grade and increased suppressive immune cell enrichment. We developed a nomogram containing the ICD score, demonstrating a high predictive accuracy with a C-index of 0.703. We further discovered that PSMD2 and PSMD14 could serve as ICD-associated prognostic biomarkers and therapeutic targets in HCC. The ICD score exhibits a high degree of reliability for predicting prognosis and may provide valuable guidance for the selection of immunotherapy for HCC patients. This novel scoring system enables the estimation of clinical immunotherapy response for HCC patients, offering new opportunities for personalized immunotherapy.

Combination therapy with dendritic cell loaded-exosomes supplemented with PD-1 inhibition at different time points have superior antitumor effect in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a prevalent cause of cancer-related deaths, is insensitive to traditional treatments. At different time intervals, the combined antitumor effects of DC-TEX and the programmed death protein 1 (PD-1) antibody (Ab) have not been investigated. In this study, HCC models were established and treated at different time intervals with DC-TEX alone or in combination with PD-1 Ab. In addition, we developed an orthotopic HCC model in BALB/c nude mice and restored T cells. Results demonstrated that the PD-1 + CD8 + T-cell population also increased significantly after DC-TEX treatment, in addition to the increased number of CD8 + T cells. The number of CD8 + T cells increased 72h after DC-TEX administration. Similar observations were made for PD-1 + CD8 + T cells. Subsequently, PD-1 Ab was administered in combination with DC-TEX at different time points (0, 24, 72, 96, 120, or 168h). Surprisingly, the combination treatment demonstrated a strong antitumor effect, which was very prominent when PD-1 Ab was administered at 72h. PD-1 Ab significantly reversed the proliferative ability of PD-1 + CD8 + T cells at 72h in vitro. The combined antitumor effects of PD-1 Ab and DC-TEX occurred mainly by stimulating CD8 + T cell proliferation and inhibiting T cell exhaustion. In conclusion, our results indicate that the combination of DC-TEX and PD-1 Ab significantly inhibits tumor growth in a murine HCC model and that the timing of PD-1 Ab administration impacts the antitumor effect.

Co-delivery of curcumin and PTTG1 siRNA by galactose receptor-targeted liposomes for enhanced anti-tumor effects in hepatocellular carcinoma

Co-delivery of curcumin and PTTG1 siRNA by galactose receptor-targeted liposomes for enhanced anti-tumor effects in hepatocellular carcinoma

Characterization of circSEC11A as a novel regulator of Iodine-125 radioactive seed-induced anticancer effects in hepatocellular carcinoma via targeting ZHX2/GADD34 axis

Iodine-125 (I-125) radioactive seed implantation is used for the local treatment of hepatocellular carcinoma (HCC), but the molecular mechanisms regulating its anticancer effects remain incompletely understood. In this study, we report that hsa_circ_0000647 (circSEC11A) is highly expressed after I-125 treatment in HCC cell lines and tissues and is a key regulator of I-125-induced anticancer effects. CircSEC11A acts as a competing endogenous RNA (ceRNA) to sponge miR-3529-3p, promoting the expression of zinc fingers and homeoboxes 2 (ZHX2) and enhancing I-125-induced anticancer effects. Dual-luciferase reporter assay, RNA pull-down, RNA immunoprecipitation, and fluorescence in situ hybridization were thereafter performed to verify the interaction among the molecules. Anticancer effects were detected using CCK-8, flow cytometry, TUNEL, EdU, transwell, and wound healing assays. Furthermore, ZHX2 transcriptionally inhibits GADD34, a negative regulator of endoplasmic reticulum stress (ERS), to enhance I-125- induced anticancer effects in vivo and in vitro. In conclusion, we characterized circSEC11A as a novel regulator of I-125-induced anticancer effects in HCC via miR-3529-3p/ZHX2/GADD34 axis-mediated ERS. Thus, circSEC11A may act as a potential therapeutic target for I-125 implantation in the clinic.

Liver fibrosis-derived exosomal miR-106a-5p facilitates the malignancy by targeting SAMD12 and CADM2 in hepatocellular carcinoma.

The mechanism of hepatocellular carcinoma (HCC) development induced by liver fibrosis is obscure. The objective of this study is to establish miRNAs from exosomes associated with liver fibrosis, and to identify potential biomarkers for the prediction of personalized clinical management effectiveness in HCC. Our research focused on miRNAs from exosomes and mRNA from liver fibrosis, which we found in the gene expression omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) evaluated miRNAs from exosomes associated with liver fibrosis, and Wilcoxon analysis assessed differentially expressed mRNAs (DEGs) across liver fibrosis/normal tissues. Following that, DEGs were assessed through gene set enrichment analysis (GSEA), gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, based on the screened targeted genes, including SAMD12 and CADM2, we further elucidated their correlation in HCC patients from the BEST database. The Kaplan-Meier Plotter platform was applied to evaluate the prognostic values of miRNA in HCC. In vitro and vivo experiments validated our findings. Six miRNAs associated with liver fibrosis were evaluated in our investigation. In-depth research presented exosome-derived miR-106a-5p, SAMD12 and CADM2 could exert valuable predictive implications for HCC treatment and illness assessment. Serum miR-106a-5p derived from liver fibrosis was decreased compared with healthy individuals. SAMD12 and CADM2 were diminished in liver cancer cell lines, and their knockdown of them exacerbated the proliferation capacities of liver cells in vitro. Exosome-derived miRNA of liver fibrosis modulated tumorigenesis by targeting SAMD12 and CADM2 in HCC.

Pseudolaric acid B triggers cell apoptosis by activating AMPK/JNK/DRP1/mitochondrial fission pathway in hepatocellular carcinoma

Pseudolaric acid B (PAB), a natural product isolated from the root bark of Pseudolarix kaempferi, has been reported to exert inhibitory effects in various cancers. However, the underlying mechanisms remain largely unclear. In the present study, we investigated the mechanism through which PAB exert its anticancer effects in hepatocellular carcinoma (HCC). PAB inhibited the viability of and induced apoptosis in Hepa1–6 cells in a dose-dependent manner. It disrupted mitochondrial membrane potential (MMP) and impaired ATP production. Furthermore, PAB induced phosphorylation of DRP1 at Ser616 and mitochondrial fission. Blocking DRP1 phosphorylation by Mdivi-1 inhibited mitochondrial fission and PAB-induced apoptosis. Moreover, c-Jun N-terminal kinase (JNK) was activated by PAB, and blocking JNK activity using SP600125 inhibited PAB-induced mitochondrial fission and cell apoptosis. Furthermore, PAB activated AMP-activated protein kinase (AMPK), and inhibiting AMPK by compound C attenuated PAB-stimulated JNK activation and blocked DRP1-dependent mitochondrial fission and apoptosis. Our in vivo data confirmed that PAB inhibited tumor growth and induced apoptosis in an HCC syngeneic mouse model by inducing the AMPK/JNK/DRP1/mitochondrial fission signaling pathway. Furthermore, a combination of PAB and sorafenib showed a synergistic effect in inhibiting tumor growth in vivo. Taken together, our findings highlight a potential therapeutic strategy for HCC.

Identification of a novel signature based on macrophage-related marker genes to predict prognosis and immunotherapeutic effects in hepatocellular carcinoma.

Tumor-related macrophages (TAMs) have emerged as an essential part of the immune regulatory network in hepatocellular carcinoma (HCC). Constructing a TAM-related signature is significant for evaluating prognosis and immunotherapeutic response of HCC patients. Informative single-cell RNA sequencing (scRNA-seq) dataset was obtained from the Gene Expression Omnibus (GEO) database, and diverse cell subpopulations were identified by clustering dimension reduction. Moreover, we determined molecular subtypes with the best clustering efficacy by calculating the cumulative distribution function (CDF). The ESTIMATE method, CIBERSORT (cell-type identification by estimating relative subsets of RNA transcripts) algorithm and publicly available tumor immune dysfunction and exclusion (TIDE) tools were used to characterize the immune landscape and tumor immune escape status. A TAM-related gene risk model was constructed through Cox regression and verified in multiple datasets and dimensions. We also performed functional enrichment analysis to detect potential signaling pathways related to TAM marker genes. In total, 10 subpopulations and 165 TAM-related marker genes were obtained from the scRNA-seq dataset (GSE149614). After clustering 3 molecular subtypes based on TAM-related marker genes, we found significantly different prognostic survival and immune signatures among the three subtypes. Subsequently, a 9-gene predictive signature (TPP1, FTL, CXCL8, CD68, ATP6V1F, CSTB, YBX1, LGALS3, and APLP2) was identified as an independent prognostic factor for HCC patients. Those patients with high RiskScore had a lower survival rate and benefited less from immunotherapy than those with low RiskScore. Moreover, more samples of the Cluster C subtype were enriched in the high-risk group, with higher tumor immune escape incidence. We constructed a TAM-related signature with excellent efficacy for predicting prognostic survival and immunotherapeutic responses in HCC patients.

Asperulosidic Acid Restrains Hepatocellular Carcinoma Development and Enhances Chemosensitivity Through Inactivating the MEKK1/NF-κB Pathway.

Asperulosidic acid (ASPA) is a plant-extracted iridoid terpenoid with tumor-suppressive and anti-inflammatory properties. At present, the antitumor function of ASPA and its related mechanisms in hepatocellular carcinoma (HCC) cells were explored. Human normal hepatocytes HL-7702 and HCC cells (Huh7 and HCCLM3) were treated with varying concentrations (0 to 200μg/mL) of ASPA. Cell viability, proliferation, apoptosis, migration, and invasion were checked. The expression of proteins was detected by Western blot. Furthermore, the effect of ASPA (100μg/mL) on the sensitivity of HCC cells to chemotherapeutic agents, including doxorubicin and cisplatin, was evaluated. A subcutaneous xenografted tumor model was set up in nude mice, and the antitumor effects of ASPA were evaluated. ASPA hindered HCC cells' proliferation, migration, and invasion, and amplified their apoptosis and sensitivity to chemotherapeutic agents. Additionally, ASPA inactivated the MEKK1/NF-κB pathway. Overexpression of MEKK1 increased HCC proliferation, migration, and invasion and facilitated chemoresistance. ASPA treatment alleviated the carcinogenic effect mediated by MEKK1 overexpression. MEKK1 knockdown slowed down HCC progression. However, ASPA could not exert additional antitumor effects in MEKK1 knockdown cells. In vivo results displayed that ASPA substantially curbed tumor growth and inactivated the MEKK1/NF-κB pathway in mice. All over, ASPA exerts antitumor effects in HCC by suppressing the MEKK1/NF-κB pathway.

PRAME Is a Novel Target of Tumor-Intrinsic Gas6/Axl Activation and Promotes Cancer Cell Invasion in Hepatocellular Carcinoma.

(1) Background: Activation of the receptor tyrosine kinase Axl by Gas6 fosters oncogenic effects in hepatocellular carcinoma (HCC), associating with increased mortality of patients. The impact of Gas6/Axl signaling on the induction of individual target genes in HCC and its consequences is an open issue. (2) Methods: RNA-seq analysis of Gas6-stimulated Axl-proficient or Axl-deficient HCC cells was used to identify Gas6/Axl targets. Gain- and loss-of-function studies as well as proteomics were employed to characterize the role of PRAME (preferentially expressed antigen in melanoma). Expression of Axl/PRAME was assessed in publicly available HCC patient datasets and in 133 HCC cases. (3) Results: Exploitation of well-characterized HCC models expressing Axl or devoid of Axl allowed the identification of target genes including PRAME. Intervention with Axl signaling or MAPK/ERK1/2 resulted in reduced PRAME expression. PRAME levels were associated with a mesenchymal-like phenotype augmenting 2D cell migration and 3D cell invasion. Interactions with pro-oncogenic proteins such as CCAR1 suggested further tumor-promoting functions of PRAME in HCC. Moreover, PRAME showed elevated expression in Axl-stratified HCC patients, which correlates with vascular invasion and lowered patient survival. (4) Conclusions: PRAME is a bona fide target of Gas6/Axl/ERK signaling linked to EMT and cancer cell invasion in HCC.

Bispecific GPC3/PD‑1 CAR‑T cells for the treatment of HCC.

Constantly stimulated by the tumor microenvironment (TME), programmed death1 (PD‑1) is elevated, and it interacts with PD ligand1 (PD‑L1), rendering chimeric antigen receptor (CAR)‑Tcells dysfunctional. Hence, CAR‑Tcells immune to PD‑1‑induced immunosuppression were constructed to improve the function of CAR‑Tcells in hepatocellular carcinoma (HCC). Double‑target CAR‑Tcells, targeting glypican‑3 (GPC3) [a tumour-associated antigen (TAA)] and hindering PD‑1‑PD‑L1 binding, were established. The expression of GPC3, PD‑L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR‑Tcells were determined using lactate dehydrogenase release assay, enzyme‑linked immunosorbent assay, and flow cytometry, respectively. HCC cells were targeted and eliminated by double‑target CAR‑Tcells. These double‑target CAR‑Tcells limit PD‑1‑PD‑L1 binding and sustain cytotoxicity to PD‑L1+ HCC cells. The relatively low IR expression and differentiation level in double‑target CAR‑Tcells in tumour tissues induced tumour‑suppression and extended survival in PD‑L1+ HCC TX models, as opposed to their single‑target counterparts. The results of the present study suggested that the newly constructed double‑target CAR‑T cells exhibit stronger tumour‑suppressing effects in HCC than their single‑target counterparts, which are common, suggesting the potential of strengthening CAR‑T cell activity in HCC treatment.

HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8+T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a fatal malignant tumor, but effective clinical interventions are limited. PLGA/PEI-mediated DNA vaccine encoding the dual targets of high-mobility group box 1 (HMGB1) or GPC3 was developed for HCC treatment. Compared with PLGA/PEI-GPC3 immunization, PLGA/PEI-HMGB1/GPC3 co-immunization significantly inhibited the subcutaneous tumor growth, while increasing the infiltration of CD8+T cells and DCs. Furthermore, the PLGA/PEI-HMGB1/GPC3 vaccine induced a strong CTL effect and promoted functional CD8+T cell proliferation. Intriguingly, the depletion assay proved thatthe therapeutic effect PLGA/PEI-HMGB1/GPC3 vaccine was dependent on antigen-specific CD8+T cell immune responses. In the rechallenge experiment, PLGA/PEI-HMGB1/GPC3 vaccine provided a long-lasting resistance to the growth of the contralateral tumor by inducing the memory CD8+T cell responses. Collectively, PLGA/PEI-HMGB1/GPC3 vaccine could induce a strong and long-lasting CTL effect and inhibit the tumor progression or re-attack. Therefore, the combined co-immunization of PLGA/PEI-HMGB1/GPC3 might be served as an effective anti-tumor strategy against HCC.

Epigenetic inhibition of lncRNA GMDS-AS1 by methyltransferase ESET promoted cell viability and metastasis of hepatocellular carcinoma.

Long noncoding RNA (lncRNAs) GMDS-AS1 has been reported as a tumor regulator in tumor growth and metastasis, but its effect in hepatocellular carcinoma (HCC) remains unclear. ESET, a histone H3K9 methyl-transferase, is involved in epigenomic regulation of tumor progression in multiple cancers. However, the correlation between ESET and lncRNA in HCC is less reported. Quantitative real-time PCR (qRT-PCR) was taken to determine the expression of ESET and GMDS-AS1. Western blot was taken to determine the target protein levels of ESET and GMDS-AS1. Online database and bioinformatics analysis were used to screen abnormally expressed genes. Luciferase assay was performed to confirm the binding of GMDS-AS1 and PSMB1. Ki67 and Edu were used for evaluated the proliferation of tumor cells. ChIP assay was performed to verify the relationship between H3K9me1 and lncRNA GMDS-AS1 promoter. Transwell was taken to determine the migration and invasion ability of tumor cells. CCK-8 was used for determining the viability of tumor cells. Flow cytometry was performed to detect the cell cycle of tumor cells. The expression of GMDS-AS1 was decreased and the expression of ESET was increased in HCC. GMDS-AS1 inhibition contributed to tumor development, and this effect was closely related to epigenetic inhibition of GMDS-AS1 by ESET. PSMB1, a downstream target of GMDS-AS1, promoted the tumor proliferation and was negatively regulated by GMDS-AS1. Our result demonstrates anti-tumorigenic traits of lncRNA GMDS-AS1 in HCC and explains its pattern of regulation mediated by ESET. Our work unmasked an essential role of GMDS-AS1 in HCC progression and detected a novel pathway for ESET to promote HCC.