Human HCC Cell Research Articles

Exosomal miR-6126 as a novel therapeutic target for overcoming resistance of anti-cancer effect in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) stands as the sixth most prevalent cancer globally, presenting a substantial health challenge, particularly due to late-stage diagnoses that limit treatment effectiveness. Sorafenib, a multi-kinase inhibitor, is the primary chemotherapeutic agent for advanced HCC, but it only extends survival by 2–3 months. However, drug resistance remains a major clinical challenge, necessitating the exploration of new molecular mechanisms, including the role of microRNAs (miRNAs) in sorafenib resistance. In this study, we aimed to identify miRNAs within exosomes derived from sorafenib-resistant HCC cells to elucidate the molecular mechanisms underlying resistance.MethodsSorafenib-resistant cells were generated by culturing the human HCC cell line Huh7 in a medium containing 20 µM sorafenib for six months. Exosomes were isolated from the conditioned medium 24 h before cell harvest using exosome-depleted serum medium. miRNA sequencing and western blotting were used to analyze the expression profiles of exosomal miRNAs and proteins, respectively. pH measurement was performed to assess pH changes in response to sorafenib treatment and miRNA modulation.ResultsA total of 180 exosomal miRNAs were found to be dysregulated between sorafenib-treated control Huh7 (Huh7S) and sorafenib-resistant Huh7 (Huh7RS) cells, as well as between untreated control Huh7 and Huh7RS cells. Among these, miR-6126 was significantly downregulated in Huh7RS cells compared to Huh7S cells. Functional studies using 2-dimensional (D) and 3D cell culture systems revealed that miR-6126 overexpression reduced sorafenib resistance in Huh7RS cells, while its inhibition increased resistance in Huh7 cells. miR-6126 downregulated key proteins involved in cancer stem cell maintenance, such as CD44 and HK2. Furthermore, the pH level was elevated in cells overexpressing miR-6126 following sorafenib treatment, whereas inhibiting miR-6126 resulted in a lower pH.ConclusionsExosomal miR-6126 plays a pivotal role in sorafenib resistance and tumorigenesis, highlighting its potential as a novel therapeutic target for overcoming drug resistance in HCC.

-RAMP3 promotes hepatocellular carcinoma tumor cell-mediated CCL2 degradation by supporting membrane distribution of ACKR2

This study aimed to explore the potential bind of Receptor Activity-Modifying Protein 3 (RAMP3) with atypical chemokine receptor 2 (ACKR2), and their cooperative regulation on the degradation of the immunosuppressive chemokine CCL2 in the tumor microenvironment of HCC. Bioinformatic analysis was conducted using available bulk-tissue RNA-seq, single-cell RNA-seq, and protein–protein interaction datasets. Human HCC cell line Huh7 and HepG2 and mouse HCC cell line Hepa1-6 were utilized for experiments. Results showed that RAMP3 binds with ACKR2 in HCC tumor cells and promotes the membrane distribution of ACKR2 through RAB4-positive vesicles. RAMP3 promotes CCL2 scavenging through ACKR2 in HCC cells. Mouse RAMP3 inhibited the proliferation of mouse liver cancer cell line (Hepa1-6)-derived syngeneic tumors through ACKR2, reduced the intratumoral concentration of CCL2 in the tumor, and inhibited the phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) and protein kinase B (AKT). In addition, mouse RAMP3 inhibited CD11b+/Gr-1 + myeloid cell infiltration and neovascularization in the tumors through ACKR2. In TCGA-LIHC, RAMP3low/ACKR2low group had the worst progression-free interval (PFI), while the RAMP3high/ACKR2high group had the best overall survival (OS). In summary, restoring RAMP3 expression in HCC cells may generate synergistic support for the anticancer effect of ACKR2.

Combined neurokinin-1 receptor antagonist and sorafenib is a promising approach for hepatocellular carcinoma therapy

BackgroundHCC (Hepatocellular carcinoma) is the most common primary malignant cancer in the liver. Treatment options to incurable HCC such as sorafenib, an oral multikinase inhibitor, had numerous side effects and questionable effectiveness. Neurokinin-1 receptor (NK1R) have a major role in inflammation and tumour environment including the resistance to cell death, the induction of angiogenesis and the promotion of cell migration and proliferation. Additionally, NK-1R is over-expressed in human tumour cells including HCC. Moreover, Aprepitant, one of the NK-1R antagonists exerts multiple antitumor activities (antiproliferative, apoptotic, antimigration, and antiangiogenesis) in vivo and in vitro.Study aimTo analyze the effectiveness of combining sorafenib with aprepitant in the management of HCC (experimental).Patients and methodsIn this retrospective experimental study, the human HCC cell line, HepG2, cells were exposed to increasing concentrations of sorafenib alone, aprepitant alone and combination of both sorafenib and aprepitant evaluation of cytotoxicity, apoptosis, MMP-9, VEGF, NF-kB p-65, p-AKT and p-ERK were done. Moreover, The extent of the NK-1 receptor expression was assessed by immunocytochemistry on 50 HCC paraffin blocks of Egyptian HCC patients and another 50 paraffin blocks of liver cirrhosis only as a control.ResultsDecreased levels of MMP-9, VEGF, NF-kB p-65, p-AKT and p-ERK was more substantial in the combination therapy compared to sorafenib alone and aprepitant alone. Moreover, the rate of apoptosis and cytotoxicity were significantly higher in the combination treatment group than the monotherapy groups with more anti inflammatory, anti angiogentic and anti metastatic effects. Also, among the 50 HCC paraffin blocks, the majority (60%) showed a strong NK-1 expression; which significantly (p < 0.05) correlated with the progression free survival (PFS) but not the overall survival (OS) of the patients when applying multivariate analysis.ConclusionHCC had strong expression and immunostaining for NK1R.Therefore, combined aprepitant and sorafenib may be a promising approach in HCC treatment compared to each one alone.

Material basis revelation of anti-hepatoma effect of Huachansu (Cinobufacini) through down-regulation of thymidylate synthase

ObjectiveHepatocellular carcinoma (HCC) is a leading cause of mortality worldwide. Huachansu (Cinobufacini) is active extract isolated from the dry skin of Bufo Bufo gargarizans. It has now been widely used in clinical treatment of cancer, this study is to clarify the material basis of down-regulation of thymidylate synthase (TYMS) induced by Huachansu. MethodsOur study utilized UPLC-MS/MS to identify major bioactive components from Huachansu. Cell Counting Kit 8 (CCK-8) assay and clone formation assay were used to examine the cell viability of tumor cells. TYMS and γ-H2AX level were detected by using quantitative real-time RT-PCR and/or western blotting. Small interfering RNA (siRNA) transfection was used to explore whether inhibition of TYMS could enhance the suppressive effect of Huachansu on cell growth of HCC cells. ResultsIn our study, firstly, we identify 21 major bioactive components from Huachansu. CCK-8 assay results showed that Huachansu and its bioactive bufadienolides (Bufalin, Bufotalin, Cinobufotalin, Desacetylcinobufagin, Arenobufagin, Telocinobufagin, and Resibufogenin) significantly inhibited the proliferation of HepG2 and SK-HEP-1 cells in a dose- and time-dependent manner. Further molecular mechanistic investigation demonstrates that Huachansu significantly suppresses thymidylate synthase (TYMS), the enzyme which provides the sole de novo source of thymidylate for DNA synthesis. The inhibition of TYMS could lead to cell-cycle block and DNA damage of HCC cells. Furthermore, we identified that Huachansu markedly increased γ-H2AX expression, which indicated the presence of DNA damage. Moreover, we confirmed that transfection of cells with small interfering RNA specific to TYMS could increase the suppressive effects of Huachansu on the HCC cells proliferation. Quantitative RT-PCR analysis showed that Huachansu treatment had no effect on the transcription level of TYMS. Furthermore, proteasomal inhibitor MG132 could block TYMS inhibition induced by Huachansu, and concomitant administration of protein synthesis inhibitor cycloheximide (CHX) with Huachansu could further suppress the protein level of TYMS, indicating that Huachansu promotes proteasome-dependent degradation of TYMS in liver cancer cells. More importantly, the bioactive bufadienolides of Huachansu such as Bufalin, Bufotalin, Cinobufotalin, Desacetylcinobufagin, Arenobufagin, Telocinobufagin, and Resibufogenin could also significantly restrain the protein level of TYMS, revealing the material basis of inhibition of TYMS exposed to Huachansu. 5-fluorouracil (5-FU) is a TYMS inhibitor, we also evaluate the effects of the combined treatment of Huachansu with 5-FU, the results show that interactions between Huachansu and 5-FU are synergistic or antagonistic. Thus, in clinical, attention should be paid to the dosage of Huachansu in combination with 5-FU. ConclusionHuachansu inhibits the growth and induces DNA damage of human HCC cells through proteasome-dependent degradation of TYMS, bioactive bufadienolides are the material basis of down-regulation of TYMS induced by Huachansu.

Cyclovirobuxine D inhibits hepatocellular carcinoma growth by inducing ferroptosis of hepatocellular carcinoma cells

ObjectiveHepatocellular carcinoma (HCC) is one cancer with high death rates. Nowadays, there are no effective drugs to treat it. Cyclovirobuxine D (CVB-D) is the primary ingredient of the traditional Chinese medicine (TCM) Buxus microphylla. Here, we try to explore the impacts of CVB-D on human HCC cells and explain the potential mechanisms.MethodsHepG2 and Huh-7 cells were used for our experiments. The cell viability and half inhibitory concentration (IC50) were detected by MTT assays. The apoptosis ratio was examined by Annexin V-FITC/7AAD staining and flow cytometry (FCM). The Fe2+ content was examined by ferrous ion content assays. The malondialdehyde (MDA) content was evaluated by lipid peroxidation MDA assays. The reactive oxygen species (ROS) level was examined by the DCFH-DA probe. The expression of apoptotic markers (Bax and Bcl-2) and ferroptosis-related proteins (GPX4 and FSP1) was detected by western blotting. The in vivo curative effect of CVB was explored using xenograft models established in C-NKG mice.ResultsThe cell viability could be inhibited by CVB-D in HepG2 and Huh-7 cells. The IC50 value of CVB-D on HepG2 and Huh-7 cells are 91.19 and 96.29 µM at 48 h, and 65.60 and 72.80 µM at 72 h. FCM showed that the apoptosis rate was increased by CVB-D in HepG2 and Huh-7 cells. Next, ferrous ion content assays showed that the level of Fe2+ was increased by CVB-D in HepG2 and Huh-7 cells. Then, we found the level of MDA and ROS was increased by CVB-D. And the Fe2+ promotion by CVB-D could be reversed by Fer-1. Additionally, western blotting assays showed that the expression of GPX4 and FSP1 was inhibited by CVB-D in HepG2 and Huh-7 cells. Moreover, in vivo, CVB-D displayed excellent anticancer effects in HCC tumor-bearing C-NKG mice.ConclusionCVB-D suppresses the growth in HCC cells through ferroptosis.

Abstract 4283: NAMPT-enriched small extracellular vesicle promotes liver cancer via activation of SLC27A4-mediated glycolysis

Abstract Introduction: Tumor-derived small extracellular vesicles (sEV) act as a major mediator of the tumor microenvironment (TME) and are reported to regulate various metabolic pathways. Methods: Small extracellular vesicles (sEV) were collected from normal human hepatocyte and metastatic HCC cell lines using differential ultracentrifugation. Mass Spectrometry proteomic analysis was employed to identify NAMPT to be enriched in metastatic HCC-sEV. The glycolytic effect of NAMPT-sEV was analyzed by seahorse glycolytic assay. Result: Mass spectrometry protein analysis revealed nicotinamide phosphoribosyl transferase (NAMPT) to be upregulated in metastatic HCC-sEV. NAMPT is a rate-limiting enzyme essential for maintaining cellular level of nicotinamide dinucleotide (NAD+). However, the underlying pathway of sEV-NAMPT-induced metabolic consequence in HCC remains largely unknown. This study elucidated sEV-NAMPT’s carcinogenic role by establishing NAMPT-knockdown (NAMPT-KD) stable clone in metastatic HCC cell lines, which significantly hampered the promoting effect of HCC-sEV in glycolysis. sEV-mediated in vitro and in vivo tumor growth was also inhibited. Through sEV treatment, we found that sEV-NAMPT significantly elevate SLC27A4 in both transcription and protein level of recipient cells. Mechanistically, it is demonstrated that SLC27A4 expression was enhanced by NF-κB transcription factor, which is activated upon binding of sEV-NAMPT to toll-like receptor 4 (TLR4). SLC27A4 mainly function as a long-chain fatty acid transporter or acyl-CoA synthetase. Lipidomic and metabolomic analysis unveiled SLC27A4 to be positively correlated with intracellular triacylglycerol (TG) and dihydroxyacetone phosphate (DHAP) level. Elevation of TG aggravates lipolysis by hepatic lipase and promotes conversion of glycerol-3-P to DHAP, which is an intermediate metabolite that transit between lipid metabolism and glycolysis. This study uncovers a regulatory axis of sEV-NAMPT-mediated SLC27A4 in glycolysis, apart from conventional fatty acid-related metabolisms. Clinically, we explored the therapeutic potential of targeting sEV-NAMPT by inhibitor FK866. Treatment of NAMPT-inhibitor significantly hampered tumor growth in various HCC in vivo models, which suggested an alternative therapeutic strategy for HCC. Conclusion: NAMPT is enriched in metastatic HCC sEV. sEV-NAMPT regulates the promoting effect of HCC-sEV in glycolysis and oncogenesis. SLC27A4 function as the downstream target that promotes HCC progression via glycolysis. NAMPT inhibitor FK866 are proven to reduce in vitro and in vivo tumorigenic ability of metastatic HCC-sEV, which may serve as potential HCC treatment option. Citation Format: Lot Sum Cherlie Yeung, Tu Him ng, Judy Wai Ping Yam. NAMPT-enriched small extracellular vesicle promotes liver cancer via activation of SLC27A4-mediated glycolysis [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 4283.

Abstract 7065: AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer, with emerging evidence highlighting the role of cancer stem cells (CSCs) in its progression. AMBRA1 has been implicated to exert both tumor-promoting and tumor-suppressive roles through regulating cell cycle and mitophagy in various solid tumors. However, the functional roles of AMBRA1 in modulating cancer stemness and tumorigenesis in HCC remain elusive. Initial analysis of The Cancer Genome Atlas data revealed a significant inverse correlation between AMBRA1 expression and stemness indices (TSS) in HCC patients, with low AMBRA1 levels associated with an upregulated oxidative phosphorylation (OXPHOS) gene signature. To elucidate the functional consequences of AMBRA1 modulation in liver tumor development and stemness maintenance, we exploited both hydrodynamic mouse models and human HCC cell lines. Our findings indicate that hepatic AMBRA1 deletion enhances cancer stemness and tumorigenesis both in vivo and in vitro. Moreover, HCC cells with reduced AMBRA1 expression demonstrated heightened mitochondrial activity, characterized by increased oxygen consumption, respiration, mitochondrial membrane potential, and intracellular ATP levels. Apart from OXPHOS, we also observed a concomitant up-regulation and enrichment of MYC targets in TSS-high and AMBRA1-low HCC patients, pointing towards a deregulated c-MYC signaling to promote cancer stemness under AMBRA1 regulation in HCC. Collectively, our results demonstrate that AMBRA1 loss enhances mitochondrial function to support hepatic tumorigenesis in HCC. Citation Format: Yan Liu, Yunong Xie, Yimiao He, Stephanie Ma, Man Tong. AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis 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 7065.

Abstract 630: The role of AKT pathway in β-catenin mutated hepatocellular carcinoma

Abstract The Wnt/β-catenin and PI3K/AKT pathways are pivotal contributors to the development of the malignant phenotype in cancer. The activated Wnt/β-catenin pathway synergizes with various signaling cascades to propel HCC formation, operating through its downstream effectors. It has been well-established that the mammalian target of rapamycin complex 2 (mTORC2) serves as an upstream regulator of AKT, orchestrating the phosphorylation and activation of AKT. Once activated, AKT phosphorylates and inhibits canonical downstream targets like forkhead box O protein (FOXO) and tuberous sclerosis complex 2 (TSC2). Studies indicated that AKT-mediated phosphorylation of TSC2 activates mTORC1, subsequently suppressing autophagy. However, there are limited data exist on the interplay between Wnt/β-catenin and AKT pathways in HCC. In this study, we sought to explore the role of the AKT pathway in β-catenin activated HCC tumors. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (N90-β-catenin) and c-MET in the livers of mice to establish the HCC model (c-Met/β-catenin model). Notably, the knockout of Rictor, a component of the mTORC2 complex, significantly impeded tumor growth, underscoring the pivotal role of AKT in c-Met/β-catenin tumor development. AKT1 and AKT2 are the two main isoforms expressed in the liver. We determined that both AKT1 and AKT2 isoforms are critical for c-Met/β-catenin tumor growth. Intriguingly, simultaneous knockout of Rictor and activation of mTORC1 in the c-Met/β-catenin model resulted in delayed tumor growth, indicating that mTORC1 is not the sole downstream determinant of AKT signaling during c-Met/β-Catenin tumor development. Furthermore, overexpression of the activated FOXO1 (FOXO1AAA) significantly delayed the tumor growth, which highlighted the importance of FOXO1. Given the limited understanding of FOXO1 in HCC, we extended our investigation on FOXO1 to human HCC cell lines. Overexpression of FOXO1AAA notably delayed tumor growth in vitro. RNAseq analysis of FOXO1AAA-overexpressed human HCC cell lines revealed a significant upregulation of "metabolic pathways", suggesting metabolic reprogramming post FOXO1 activation. This study, for the first time, unveils the role of the AKT pathway in β-catenin-mutated HCC growth and highlights the potential therapeutic value of targeting the AKT pathway in patients with β-catenin-mutated HCC. Citation Format: Xue Wang, Xin Chen. The role of AKT pathway in β-catenin mutated 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 630.

USP40 promotes hepatocellular carcinoma cell proliferation, migration and stemness by deubiquitinating and stabilizing Claudin1

BackgroundHepatocellular carcinoma (HCC) is a prevalent malignant tumor that poses a major threat to people’s lives and health. Previous studies have found that multiple deubiquitinating enzymes are involved in the pathogenesis of HCC. The purpose of this work was to elucidate the function and mechanism of the deubiquitinating enzyme USP40 in HCC progression.MethodsThe expression of USP40 in human HCC tissues and HCC cell lines was investigated using RT-qPCR, western blotting and immunohistochemistry (IHC). Both in vitro and in vivo experiments were conducted to determine the crucial role of USP40 in HCC progression. The interaction between USP40 and Claudin1 was identified by immunofluorescence, co-immunoprecipitation and ubiquitination assays.ResultsWe discovered that USP40 is elevated in HCC tissues and predicts poor prognosis in HCC patients. USP40 knockdown inhibits HCC cell proliferation, migration and stemness, whereas USP40 overexpression shows the opposite impact. Furthermore, we confirmed that Claudin1 is a downstream gene of USP40. Mechanistically, USP40 interacts with Claudin1 and inhibits its polyubiquitination to stabilize Claudin1 protein.ConclusionsOur study reveals that USP40 enhances HCC malignant development by deubiquitinating and stabilizing Claudin1, suggesting that targeting USP40 may be a novel approach for HCC therapy.

A traditional prescription comprising Astragali Radix and Schisandra chinensis Fructus induces apoptosis and protective autophagy in hepatocellular carcinoma cells

Ethnopharmacological relevanceHepatocellular carcinoma (HCC) poses a growing challenge to global health efforts. The 5-year survival rate of HCC patients is still dismal. A traditional prescription Qi-Wei-Wan (QWW) comprising Astragali Radix and Schisandra chinensis Fructus has traditionally been used for HCC treatment according to traditional Chinese medicine theory, but the pharmacological basis is not clear. Aim of the studyThis study aims to investigate the anti-HCC effects of an ethanolic extract of QWW (hereafter, QWWE) and the mechanism of action. Materials and methodsAn UPLC-Q-TOF-MS/MS method was developed to control the quality of QWWE. Two human HCC cell lines (HCCLM3 and HepG2) and a HCCLM3 xenograft mouse model were employed to investigate the anti-HCC effects of QWWE. The anti-proliferative effect of QWWE in vitro was determined by MTT, colony formation and EdU staining assays. Apoptosis and protein levels were examined by flow cytometry and Western blotting, respectively. Nuclear presence of signal transducer and activator of transcription 3 (STAT3) was examined by immunostaining. Transient transfection of pEGFP-LC3 and STAT3C plasmids was performed to assess autophagy and determine the involvement of STAT3 signaling in QWWE’s anti-HCC effects, respectively. ResultsWe found that QWWE inhibited the proliferation of and triggered apoptosis in HCC cells. Mechanistically, QWWE inhibited the activation of SRC and STAT3 at Tyr416 and Tyr705, respectively; inhibited the nuclear translocation of STAT3; lowered Bcl-2 protein levels, while increased Bax protein levels in HCC cells. Over-activating STAT3 attenuated the cytotoxic and apoptotic effects of QWWE in HCC cells. Moreover, QWWE induced autophagy in HCC cells by inhibiting mTOR signaling. Blocking autophagy with autophagy inhibitors (3-methyladenine and chloroquine) enhanced the cytotoxicity, apoptotic effect and the inhibitory effect on STAT3 activation of QWWE. Intragastric administration of QWWE at 10 mg/kg and 20 mg/kg potently repressed tumor growth and inhibited STAT3 and mTOR signaling in tumor tissues, but did not significantly affect mouse body weight. ConclusionQWWE exhibited potent anti-HCC effects. Inhibiting the STAT3 signaling pathway is involved in QWWE-mediated apoptosis, while blocking mTOR signaling contributes to QWWE-mediated autophagy induction. Blockade of autophagy enhanced the anti-HCC effects of QWWE, indicating that the combination of an autophagy inhibitor and QWWE might be a promising therapeutic strategy for HCC management. Our findings provide pharmacological justifications for the traditional use of QWW in treating HCC.

Single-cell transcriptome analysis reveals the metabolic changes and the prognostic value of malignant hepatocyte subpopulations and predict new therapeutic agents for hepatocellular carcinoma.

The development of HCC is often associated with extensive metabolic disturbances. Single cell RNA sequencing (scRNA-seq) provides a better understanding of cellular behavior in the context of complex tumor microenvironments by analyzing individual cell populations. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data was employed to investigate the metabolic pathways in HCC. Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) analysis were applied to identify six cell subpopulations, namely, T/NK cells, hepatocytes, macrophages, endothelial cells, fibroblasts, and B cells. The gene set enrichment analysis (GSEA) was performed to explore the existence of pathway heterogeneity across different cell subpopulations. Univariate Cox analysis was used to screen genes differentially related to The Overall Survival in TCGA-LIHC patients based on scRNA-seq and bulk RNA-seq datasets, and LASSO analysis was used to select significant predictors for incorporation into multivariate Cox regression. Connectivity Map (CMap) was applied to analysis drug sensitivity of risk models and targeting of potential compounds in high risk groups. Analysis of TCGA-LIHC survival data revealed the molecular markers associated with HCC prognosis, including MARCKSL1, SPP1, BSG, CCT3, LAGE3, KPNA2, SF3B4, GTPBP4, PON1, CFHR3, and CYP2C9. The RNA expression of 11 prognosis-related differentially expressed genes (DEGs) in normal human hepatocyte cell line MIHA and HCC cell lines HCC-LM3 and HepG2 were compared by qPCR. Higher KPNA2, LAGE3, SF3B4, CCT3 and GTPBP4 protein expression and lower CYP2C9 and PON1 protein expression in HCC tissues from Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) databases. The results of target compound screening of risk model showed that mercaptopurine is a potential anti-HCC drug. The prognostic genes associated with glucose and lipid metabolic changes in a hepatocyte subpopulation and comparison of liver malignancy cells to normal liver cells may provide insight into the metabolic characteristics of HCC and the potential prognostic biomarkers of tumor-related genes and contribute to developing new treatment strategies for individuals.

LncRNA RPPH1 acts as a molecular sponge for miR-122 to regulate Wnt1/β-catenin signaling in hepatocellular carcinoma

This study aimed to explore the role of lncRNA RPPH1 in hepatocellular carcinoma. The expression of RPPH1 and miR-122 was determined by Real-time PCR. Cell proliferation and colony formation assays were employed to monitor cell growth in vitro. Wound healing and Transwell assays were applied to detect cell migration and invasion. A dual-luciferase reporter assay was used to verify the interaction between RPPH1 and miR-122. The in vivo function of RPPH1 was illustrated by xenograft tumor models. The results showed that the expression of RPPH1 was markedly upregulated in human HCC specimens and cell lines compared to normal controls. However, the trend of miR-122 was the opposite. RPPH1 facilitates the proliferation, migration, and invasion of HCC cells and synchronously suppresses cell apoptosis. The dual-luciferase assay confirmed the relationship between RPPH1 and miR-122. Rescue experiments showed that RPPH1 acted as a competing endogenous RNA (ceRNA) by sponging miR-122 in HCC cells. Moreover, RPPH1 positively regulated the expression of Wnt1 and its downstream targets through miR-122. Our study demonstrates for the first time that RPPH1 promotes HCC progression via the miR-122/Wnt1/β-catenin axis, which may represent a valuable therapeutic target for patients with HCC.

Genomic and transcriptomic profiling reveals key molecules in metastatic potentials and organ-tropisms of hepatocellular carcinoma

Metastasis is a landmark event for rapid postsurgical relapse and death of HCC patients. Although distinct genomic and transcriptomic profiling of HCC metastasis had been reported previously, the causal relationships of somatic mutants, mRNA levels and metastatic potentials were difficult to be established in clinic. Therefore, 11 human HCC cell lines and 7 monoclonal derivatives with definite metastatic potentials and tropisms were subjected to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). TP53, MYO5A, ROS1 and ARID2 were the prominent mutants of metastatic drivers in HCC cells. During HCC clonal evaluation, TP53, MYO5A and ROS1 mutations occurred in the early stage, EXT2 and NIN in the late stage. NF1 mutant was unique in lung tropistic cell lines, RNF126 mutant in lymphatic tropistic ones. PER1, LMO2, GAS7, NR4A3 expression levels were positively associated with relapse-free survival (RFS) of HCC patients. The integrative analysis revealed 58 genes exhibited both somatic mutation and dysregulated mRNA levels in high metastatic cells. Altogether, metastatic drivers could accumulate gradually at different stages during HCC progression, some drivers might modulate HCC metastatic potentials and the others regulate metastatic tropisms.

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance. MethodsTaking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry. ResultsThe success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90–100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4+ T cells were significantly reduced. ConclusionsThrough the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.

STING pathway contributes to the prognosis of hepatocellular carcinoma and identification of prognostic gene signatures correlated to tumor microenvironment

BackgroundHepatocellular carcinoma (HCC) is one of the most malignant solid tumors worldwide. Recent evidence shows that the stimulator of interferon genes (STING) pathway is essential for anti-tumor immunity via inducing the production of downstream inflammatory cytokines. However, its impact on the prognosis and tumor microenvironment of HCC was still limited known.MethodsWe obtained gene expression profiles of HCC from GEO, TCGA, and ICGC databases, and immune-related genes (IRGs) from the ImmPort database. Multivariate Cox regression was performed to identify independent prognostic factors. Nomogram was established to predict survival probability for individual patients. Kaplan–Meier curve was used to evaluate the survival difference. Afterward, ESTIMATE, TISCH, and TIMER databases were combined to assess the immune cell infiltration. Furthermore, the qPCR, western blotting, and immunohistochemistry were done to evaluate gene expression, and in vitro cell models were built to determine cell migratory ability.ResultsWe found that gene markers of NLRC3, STING1, TBK1, TRIM21, and XRCC6 within STING pathway were independent prognostic factors in HCC patients. Underlying the finding, a predictive nomogram was constructed in TCGA-training cohort and further validated in TCGA-all and ICGC datasets, showing credible performance. Experimentally, up-regulated TBK1 promotes the ability of HCC cell migration. Next, the survival-related immune-related co-expressed gene signatures (IRCGS) (VAV1, RHOA, and ZC3HAV1) were determined in HCC cohorts and their expression was verified in human HCC cells and clinical samples. Furthermore, survival-related IRCGS was associated with the infiltration of various immune cell subtypes in HCC, the transcriptional expression of prominent immune checkpoints, and immunotherapeutic response.ConclusionCollectively, we constructed a novel prognostic nomogram model for predicting the survival probability of individual HCC patients. Moreover, an immune-related prognostic gene signature was determined. Both might function as potential therapeutic targets for HCC treatment in the future.

Radiosensitization effect by HDAC inhibition improves NKG2D-dependent natural killer cytotoxicity in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Radiotherapy (RT) controls HCC unsatisfactorily and temporarily. Histone deacetylase inhibitor (HDACi) is a heterogeneous group of epigenetic therapeutics with promising anticancer effects and synergism in combination with RT. HDACi modulates natural killer (NK) cell ligand expression on tumor cells, and leads to immune evasion of cancer cells. Expressions of NK group 2D (NKG2D) ligands on cancer cells determine the cytotoxic effect by interacting with NKG2D receptor on NK cells. However, the role of NKG2D signaling in HCC upon combined RT and HDACi remains unclear.Method In vitro co-culture system with NK cells was tested for human and murine HCC cell lines. Pan-HDACi (panobinostat) and specific HDAC4 knockdown (HDAC4-KD) were used for HDAC inhibition. Clonogenic assay and flow cytometry examined HCC cell survival and NKG2D ligand expression, respectively. Syngeneic mouse model was used to validate the radiosensitizing effect in vivo.ResultsCombined RT and HDACi/HDAC4-KD significantly enhanced NK cell-related cytotoxicity and increased NKG2D ligands, MICA/MICB expressions in human and RAE-1/H60 expressions in murine HCC cells. Delayed tumor growth in vivo by the combinational treatment of RT and HDACi/HDAC4-KD was shown with the associated NKG2D ligand expressions. However, NKG2D receptor did not significantly change among tumors.ConclusionRadiosensitizing effect with combined RT and HDAC inhibition increased the expression of NKG2D ligands in HCC cells and enhanced their susceptibility to NK cell-mediated cytotoxicity. These findings imply the potential use of combined RT/HDACi and NK cell-directed immunotherapy.

Innovative signature establishment using lymphangiogenesis-related lncRNA pairs to predict prognosis of hepatocellular carcinoma

AimsHepatocellular carcinoma (HCC) remains a major tumoral burden globally, and its heterogeneity encumbers prognostic prediction. The lymphangiogenesis-related long non-coding RNAs (lrlncRNAs) reported to be implicated in immune response regulation show potential importance in predicting the prognostic and therapeutic outcome. Hence, this study aims to establish a lrlncRNA pairs-based signature not requiring specific expression levels of transcripts, which displays promising clinical practicality and satisfactory predictive capability. Main methodsTranscriptomic and clinical information of the Liver Hepatocellular Carcinoma (LIHC) project retrieved from the TCGA portal were used to find differently expressed lrlncRNA (DElrlncRNA) via analysis performed between lymphangiogenesis-related genes (lr-genes) and lncRNAs(lrlncRNA), and to ultimately construct the signature based on lrlncRNA pairs screened out via Lasso and Cox regression analyses. Akaike information criterion (AIC) values were computed to find the cut-off point optimum for high-risk and low-risk group allocation. The signature then underwent trials in terms of its predictive value for survival, clinicopathological features, immune cells infiltration in tumoral microenvironment, selected checkpoint biomarkers and chemosensitivity. Key findingsA novel lymphangiogenesis-related lncRNA pair signature was established using nine lrlncRNA pairs identified and significantly related to overall survival, clinicopathological features, immune cells infiltration and susceptibility to chemotherapy. Moreover, the signature efficacy was verified in acknowledged clinicopathological subgroups and partially validated by qRT-PCR assay in various human HCC cell lines. SignificanceThe novel lrlncRNA-pairs based signature was shown to effectively and independently estimate HCC prognosis and help screen patients suitable for anti-tumor immunotherapy and chemotherapy.

SLC38A6, regulated by EP300-mediated modifications of H3K27ac, promotes cell proliferation, glutamine metabolism and mitochondrial respiration in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common form of liver cancer. The incidence of HCC is increasing and effective prevention methods are needed. The solute carrier family 38 member 6 (SLC38A6) plays an important role in the metabolism of glutamine, which is a central nutrient for many cancers. However, the regulation and function of SLC38A6 in HCC are unclear. SLC38A6 levels in human HCC tissue arrays and cells were determined. SLC38A6 was silenced or overexpressed to determine its role in regulating cell viability, colony formation, cell cycle progression, glutamine metabolism and mitochondrial respiration. A luminescence assay was used to study the interaction between SLC38A6 and EP300. The interactions between SLC38A6, H3K27ac and EP300 were determined using chromatin immunoprecipitation assays. Quantitative RT-PCR and immunoblots were performed to measure mRNAs and proteins, respectively. SLC38A6 expression was higher in HCC compared with expression in normal tissue. Silencing SLC38A6 inhibited cell viability, colony formation, cell cycle progression, glutamine metabolism and mitochondrial respiration, while SLC38A6 overexpression had the opposite effects. Silencing SLC38A6 also inhibited tumor growth in vivo. Silencing EP300 significantly suppressed the interaction between H3K27ac and the SLC38A6 promoter, leading to decreased SLC38A6. SLC38A6 is regulated by EP300-mediated modifications of H3K27ac and promotes viability, colony formation, cell cycle progression, glutamine metabolism and mitochondrial respiration in HCC cells.

MiR-200a-3p- and miR-181-5p-Mediated HOXB5 Upregulation Promotes HCC Progression by Transcriptional Activation of EGFR.

BackgroundHepatocellular carcinoma (HCC) remains a worldwide burden. However, the mechanisms behind the malignant biological behavior of HCC remain unclear. The homeobox (HOX) family could act as either promoters or suppressors in different kinds of malignancies. Our study discovered the role of HOXB5 in regulating HCC progression.MethodsThe HOXB5 expression was assessed by RT-PCR analysis in human HCC samples and cell lines. HOXB5 transcriptional regulation of the EGFR was verified by the luciferase reporter assay and chromatin immunoprecipitation experiment. The oncogenic role of HOXB5 in HCC progression was analyzed by CCK8, colony-forming, and transwell assays.ResultsUpregulation of HOXB5 was found in human HCC, and was strongly correlated with HCC tumor size, tumor-nodule metastasis, TNM stage, and relatively unfavorable OS and DFS. Ectopic expression of HOXB5 promoted the capacity of cell growth and clonogenicity, while the inhibition of HOXB5 decreased the proliferation and clonogenicity potential in vitro by CCK8 and colony-forming assays. In addition, HOXB5 also promoted cell migration by transwell experiment. Mechanism studies elucidated that HOXB5 triggers HCC progression via direct transcriptional activation of EGFR. The upregulation of HOXB5 is regulated by miR-200a-3p and miR-181-5p. Transfection of miR-200a-3p and miR-181-5p mimics blocked the cell proliferation and migration regulated by HOXB5, while overexpression of the 3′-UTR mutant HOXB5 abolished the suppressive effect of miR-200a-3p and miR-181-5p, but not the wild-type HOXB5.ConclusionHOXB5 is a promising prognostic factor in human HCC. Targeting miR-200a-3p and the miR-181-5p/HOXB5/EGFR signaling pathway may provide new options for the treatment strategies of HCC.

Antitumor effects of rhamnazinon sorafenib-treated human hepatocellular carcinoma cell lines via modulation of VEGF signaling and PI3K/NF-κB p38/caspase-3 axes cross talk

AimsHepatocellular carcinoma (HCC) is the most common liver malignancy,characterized by dysregulation of multiple oncogenic signaling pathways, including the VEGF/PI3K/NF-κB and p38 MAPK axes.Sorafenib is a multikinase inhibitor that targets Raf kinases and receptor tyrosine kinases,which mediate HCC angiogenesis.Rhamnazin is a VEGFR2 signaling inhibitor, which inhibits the phosphorylation of Vascular endothelial growth factor receptor 2(VEGFR2) and its downstream signaling regulators. This study was designed to assess the antitumor effects of rhamnazin on human HCC cell lines treated with sorafenib, and to investigate the molecular mechanisms mediating this effect. Main methodsHepG2 and HUH-7 HCC cell lines were used.Cell viability was assessed by MTT assay. NF-κB, p38MAPK, VEGF, VEGFR2, PI3K, and Ki67 levels were assessed using ELISA. Caspase-3 activity was measured colorimetrically. VEGFR2 expression was detected by RT-PCR. Key findingsMTT assay revealed that the sorafenib–rhamnazin combination showed significant cytotoxicity compared with sorafenib or rhamnazin alone. The sorafenib–rhamnazin combination also showed significant inhibition of the angiogenicVEGF/VEGFR2/PI3K/NF-κBsignaling axis associated with significant upregulation of the apoptotic p38MAPK/caspase-3 axis and inhibition of Ki67, a proliferation marker in HepG2 and HUH-7 cells. SignificanceRhamnazin potentiates the chemotherapeutic effect of sorafenib via modulation ofthe VEGF/PI3K/NF-κBsignaling axis, downregulation of VEGFR2 expression, and upregulation of the p38MAPK/caspase-3 axis in human HCC cell lines.