Human Hepatocellular Carcinoma Research Articles

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

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

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

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

Unravelling the potential of natural chelating agents in the control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

Iron is essential for the formation, maturation and dispersal of bacterial biofilms, playing a crucial role in the physiological and metabolic functions of bacteria as well as in the regulation of virulence. Limited availability of iron can impair the formation of robust biofilms by altering cellular motility, hydrophobicity and protein composition of the bacterial surface. In this study, the antibiofilm activity of two natural iron chelating agents, kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) and maltol (3-hydroxy-2-methyl-4-pyrone), were investigated against Staphylococcus aureus and Pseudomonas aeruginosa. In addition, the ability of these 2-hydroxy-4-pyrone derivatives in preventing and eradicating S. aureus and P. aeruginosa biofilms through the enhancement of the efficacy of two antibiotics (tobramycin and ciprofloxacin) was explored. The iron binding capacity of the kojic acid and maltol was confirmed by their affinity for iron (III) which was found to be about 90 %, comparable to the regular chelating agent ethylenediaminetetraacetic acid (EDTA, 89 %). The antibiofilm efficacy of 2-hydroxy-4-pyrone derivatives, alone and in combination with antibiotics, was evaluated by measuring the total biomass, metabolic activity, and culturability of biofilm cells. Furthermore, their impact on the membrane integrity of S. aureus biofilm cells was investigated using flow cytometry and epifluorescence microscopy with propidium iodide staining. It was also examined the ability of 2-hydroxy-4-pyrone derivatives and 2-hydroxy-4-pyrone derivate-antibiotic dual-combinations in inhibiting the production of virulence factors (total proteases, lipases, gelatinases and siderophores) by S. aureus. Regarding biofilm formation, the results showed that 2-hydroxy-4-pyrone derivatives alone reduced the metabolic activity of S. aureus biofilm cells by over 40 %. When combined with tobramycin, a 2-log (CFU cm-2) reduction in S. aureus biofilm cells was observed. Moreover, the combination of maltol and kojic acid with ciprofloxacin prevented P. aeruginosa biomass production by 60 %, compared to 36 % with ciprofloxacin alone. In pre-established S. aureus and P. aeruginosa biofilms, selected compounds reduced the metabolic activity by over 75 %, and a 3-log (CFU cm-2) reduction in the culturability of biofilm cells was noted when kojic acid and maltol were combined with antibiotics. Moreover, 2-hydroxy-4-pyrone derivatives alone and in combination with tobramycin, damaged the cell membranes of pre-established biofilms and completely inhibited total proteases production. Despite the increasing of reactive oxygen species production caused by the cellular treatment of maltol, both 2-hydroxy-4-pyrone derivatives showed good safe profile when tested in human hepatocarcinoma (HepG2) cells. The pre-treatment of HepG2 cells with both compounds was crucial to prevent the cellular damage caused by iron (III). This study demonstrates for the first time that the selected 2-hydroxy-4-pyrone derivatives significantly enhance the antibiofilm activity of tested antibiotics against S. aureus and P. aeruginosa, highlighting their potential as antibiotic adjuvants in preventing and eradicating biofilm-related infections.

Mitochondrial damage and associated combined toxicity induced by deoxynivalenol and Alternaria toxins co-exposure

Deoxynivalenol and Alternaria toxins are common food contaminants posing significant threats to human and animal health. Although their individual toxicities have been extensively studied, the combined effects of co-exposure remain largely unexplored. Our findings revealed combined toxins displayed concentration-dependent synergistic and antagonistic interactions on human hepatocellular carcinoma cells. Co-exposure significantly reduced mitochondrial activity, increased intracellular ROS levels, and activated the mitochondrial-dependent caspase signaling pathway, ultimately leading to enhanced apoptosis. Metabolomics analysis revealed that combined exposure severely disrupted multiple key metabolic processes, including those related to energy (NAD+, adenosine, AMP, and ADP), amino acids (l-aspartate, L-glutamate), and carbohydrate (Glucose-6-phosphate) metabolisms. These findings highlight that co-occurrence exacerbates the disruption of overall metabolic balance, potentially impairing the normal function of cells by affecting energy production, protein synthesis, and cell proliferation. This study underscores the importance of considering combined mycotoxin exposure and provided possible ideas for inhibiting or mitigating toxicity to ensure food safety.

Therapeutic Effects of Crocin Nanoparticles Alone or in Combination with Doxorubicin against Hepatocellular Carcinoma In vitro.

Crocin (CRO), the primary antioxidant in saffron, is known for its anticancer properties. However, its effectiveness in topical therapy is limited due to low bioavailability, poor absorption, and low physicochemical stability. This study aimed to prepare crocin nanoparticles (CRO-NPs) to enhance their pharmaceutical efficacy and evaluate the synergistic effects of Cro-NPs with doxorubicin (DOX) chemotherapy on two cell lines: human hepatocellular carcinoma cells (HepG2) and non-cancerous cells (WI38). CRO-NPs were prepared using the emulsion diffusion technique and characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Zeta potential, and Fourier transform infrared spectroscopy (FT-IR). Cell proliferation inhibition was assessed using the MTT assay for DOX, CRO, CRO-NPs, and DOX+CRO-NPs. Apoptosis and cell cycle were evaluated by flow cytometry, and changes in the expression of apoptotic gene (P53) and autophagic genes (ATG5 & LC3) were analyzed using real-time polymerase chain reaction. TEM and SEM revealed that CRO-NPs exhibited a relatively spherical shape with an average size of 9.3 nm, and zeta potential analysis indicated better stability of CRO-NPs compared to native CRO. Significantly higher antitumor effects of CRO-NPs were observed against HepG2 cells (IC50 = 1.1 mg/ml and 0.57 mg/ml) compared to native CRO (IC50 = 6.1 mg/ml and 3.2 mg/ml) after 24 and 48 hours, respectively. Annexin-V assay on HepG2 cells indicated increased apoptotic rates across all treatments, with the highest percentage observed in CRO-NPs, accompanied by cell cycle arrest at the G2/M phase. Furthermore, gene expression analysis showed upregulation of P53, ATG5, and LC3 genes in DOX/CRO-NPs co-treatment compared to individual treatments. In contrast, WI38 cells exhibited greater sensitivity to DOX toxicity but showed no adverse response to CRONPs. Although more in vivo studies in animal models are required to corroborate these results, our findings suggest that CRO-NPs can be a potential new anticancer agent for hepatocellular carcinoma. Moreover, they have a synergistic effect with DOX against HepG2 cells and mitigate the toxicity of DOX on normal WI38 cells.

Antioxidant and anti-inflammatory function of walnut green husk aqueous extract (WNGH-AE) on human hepatocellular carcinoma cells (HepG2) treated with t-BHP.

Oxidative damage, oxidative inflammation, and a range of downstream diseases represent significant threats to human health. The application of natural antioxidants and anti-inflammatory agents can help prevent and mitigate these associated diseases. In this study, we aimed to investigate the effectiveness of walnut green husk (WNGH) as an antioxidant and anti-inflammatory agent in an in vitro setting. HepG2 cells were treated with tert-butyl hydroperoxide (t-BHP) to establish a cellular model of oxidative damage and inflammation. We assessed the biocompatibility of walnut green husk aqueous extract (WNGH-AE) on HepG2 cells using MTT and LDH assays (WNGH-AE concentration: 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mg/mL). Additionally, we measured intracellular oxidative stress indicators, such as ROS and 8-OHdG, along with inflammatory factors TNF-α and IL-1β through ELISA to evaluate the antioxidant and anti-inflammatory capacity of WNGH-AE (concentration: 0.025, 0.05, 0.1 mg/mL) in HepG2 cells. We also determined the free radical scavenging capacity of various extracts of WNGH using DPPH and ·OH methods. The total phenols, total polysaccharides, and total flavonoids in WNGH-AE were analyzed using the Folin-Ciocalteu's reagent, the phenol-sulfuric acid method, and the spectrophotometry, respectively. The bioactive components of WNGH-AE were analyzed using LC-MS/MS. Our results demonstrated that WNGH-AE was highly biocompatible with HepG2 cells. The antioxidant effect of WNGH-AE involved the scavenging of intracellular ROS, while its anti-inflammatory effect was linked to the down-regulation of the NF-κB pathway. Compared to other extractants (ethyl acetate, n-butanol, 75% ethanol, and petroleum ether), WNGH-AE exhibited the strongest free radical scavenging ability. Through LC-MS/MS analysis, we identified 403 compounds in WNGH-AE, with gentisic acid being the most abundant and possessing high antioxidant capacity, suggesting it may be a key active component contributing to the antioxidant activity of WNGH-AE. In conclusion, our findings indicate that WNGH-AE is a natural, high-quality antioxidant and anti-inflammatory biomaterial deserving further research and development, with significant potential applications in healthcare. (311 words).

The protection of UCK2 protein stability by GART maintains pyrimidine salvage synthesis for HCC growth under glucose limitation.

Overexpression of uridine-cytidine kinase 2 (UCK2), a key enzyme in the pyrimidine salvage pathway, is implicated in human cancer development, while its regulation under nutrient stress remains to be investigated. Here, we show that under glucose limitation, AMPK phosphorylates glycinamide ribonucleotide formyltransferase (GART) at Ser440, and this modification facilitates its interaction with UCK2. Through its binding to UCK2, GART generates tetrahydrofolate (THF) and thus inhibits the activity of integrin-linked kinase associated phosphatase (ILKAP) for removing AKT1-mediated UCK2-Ser254 phosphorylation under glucose limitation, in which dephosphorylation of UCK2-Ser254 tends to cause Trim21-mediated UCK2 polyubiquitination and degradation. In this way, both UCK2 binding ability and THF producing catalytic activity of GART protect protein stability of UCK2 and pyrimidine salvage synthesis, and sustain tumor cell growth under glucose limitation. In addition, UCK2-Ser254 phosphorylation level displays a positive relationship with GART-Ser440 phosphorylation level and its enhancement is correlated with poor prognosis of human hepatocellular carcinoma (HCC) patients. These findings reveal a non-canonical role of GART in regulating pyrimidine salvage synthesis under nutrient stress, and raise the potential for alternative treatments in targeting pyrimidine salvage-dependent tumor growth.

Impact of Daily High Ergosterol Intake for 14 Weeks in Ovariectomized Rats on Cholesterol and Vitamin D3 Biosynthesis Pathways.

Postmenopausal women are at a higher risk of developing dyslipidemia and osteoporosis due to estrogen deficiency, necessitating regular vitamin D supplementation and the use of cholesterol inhibitors, respectively, to prevent these conditions. Despite current treatments, alternatives are needed to address both conditions simultaneously. Ergosterol, a precursor of vitamin D2, is a fungal sterol converted to brassicasterol by 7-dehydrocholesterol reductase, a cholesterol biosynthesis enzyme that converts 7-dehydrocholesterol (a precursor of vitamin D3) into cholesterol. Our previous research demonstrated that ergosterol decreases cholesterol levels and increases 7-dehydrocholesterol levels in human HepG2 hepatoma cells. Furthermore, we reported that ergosterol increases vitamin D2 levels, inhibits the cholesterol biosynthetic pathway, and potentially promotes vitamin D3 biosynthesis in male rats fed a high-fat and high-sucrose diet. In this study, we investigated the effects of daily high ergosterol intake on cholesterol, vitamin D2, and D3 biosynthetic pathways in ovariectomized (OVX) rats using GC-MS and LC with tandem mass spectrometry. OVX rats treated with ergosterol for 14 weeks exhibited significantly decreased plasma cholesterol levels and markers of cholesterol absorption, such as the plant sterol sitosterol. Furthermore, 7-dehydrocholesterol levels increased in these rats compared to untreated OVX rats. We observed that 1α, 25-dihydroxyvitamin D3 levels slightly increased in OVX rats treated with ergosterol. Additionally, ergosterol improved bone strength and increased OVX-induced bone resorption. These results indicate that daily ergosterol intake may aid in the simultaneous prevention of dyslipidemia and osteoporosis in postmenopausal women.

TGF-β signaling orchestrates cancer-associated fibroblasts in the tumor microenvironment of human hepatocellular carcinoma: unveiling insights and clinical significance

Liver cancer, specifically hepatocellular carcinoma (HCC), stands out as one of the most formidable solid tumors, characterized by a dauntingly low survival rate. At the forefront of the tumor microenvironment (TME) orchestrating the initiation and advancement of HCC are cancer-associated fibroblasts (CAFs). TGF-β, widely recognized as a potent activator of CAFs, not only regulates their activity but also assumes a pivotal role in the metastatic journey of the tumor. In our recent study, drawing from the GEO database, we identified two fibroblast subtypes in HCC through single-cell RNA sequencing (scRNA-seq) and explore the expression and distribution of TGF-β and its receptors in the TME of HCC. Subsequently, we investigated the interactions between tumor cells expressing high levels (TGFB1high) and low levels (TGFB1low) of TGF-β in the HCC TME and the two subtypes of CAFs. We also employed multi-color immunohistochemistry (mIHC) technology to examine the expressions of FAP, α-SMA, CD4, Foxp3, and TGF-β in HCC tissues within a tissue microarray. Additionally, we analyzed clinical associations, prognostic values, and the correlation of these molecules. These insights advance our understanding of the molecular mechanisms driving HCC progression and underscore the intricate interplay between tumor cells and the stromal components of the TME.

A hyaluronic acid modified copper-based metal-organic framework overcomes multidrug resistance via two-way redox dyshomeostasis under hypoxia.

Multidrug resistance (MDR) has become a major challenge in tumor chemotherapy, primarily associated with the overexpression of P-glycoprotein (P-gp). Inhibiting P-gp expression and function through redox dyshomeostasis has shown great potential for reversing MDR. Here, a nanometer system of copper-based metal-organic framework (HA-CuMOF@DOX) modified with hyaluronic acid (HA) was constructed to overcome MDR via two-way regulation of redox homeostasis under hypoxia. HA-CuMOF@DOX is a spherical glutathione (GSH) responsive nanoparticle with a drug loading capacity of 20.69 %, which could deplete GSH through Cu2+ and electrophilic ligands, and generate •OH via a Fenton-like reaction. In vitro experiments suggested that the nanoparticles had good targetability to cancer cells and biocompatibility to normal cells. HA-CuMOF@DOX was successfully internalized by drug-resistant human hepatoma carcinoma cell line (HepG2-ADR cells). It aggravated redox dyshomeostasis via dual regulation, inducing mitochondrial damage, reducing intracellular adenosine triphosphate (ATP) levels, and downregulating P-gp to overcome HepG2-ADR drug resistance. More importantly, in vivo experiments demonstrated an 80.69 % tumor growth inhibition in nude mice bearing HepG2-ADR cells. This work represents a significant advancement in the development of effective treatments for drug-resistant tumors.

Natural Compounds from Alhagi maurorum as Potential HCC and HepG2 Inhibitors: An Integrated Study using Pharmacophore Development, Molecular Docking, MD Simulation, and DFT Approaches.

The rise in the frequency of liver cancer all over the world makes it a prominent area of research in the discovery of new drugs or repurposing of existing drugs. This article describes the pharmacophore-based structure-activity relationship (3DQSAR) on the secondary metabolites of Alhagi maurorum to inhibit human liver cancer cell lines Hepatocellular carcinoma (HCC) and hepatoma G2 (HepG2) which represents the molecular level understanding for isolated phytochemicals of Alhagi maurorum. The definite features, such as hydrophobic regions, average shape, and active compounds' electrostatic patterns, were mapped to screen phytochemicals. The 3D-QSAR model generates pharmacophore-based descriptors and alignment of active compounds. Further, docking studies were performed on the active compounds to check out their binding affinity with the active site of the target proteins. It was further validated by applying molecular simulations, and the results were found to be accurate. The geometrical optimization and energy gap of the hit compound were calculated by the density functional theory (DFT). Then, ADMET was performed on this hit compound for drug-like features and toxicity. Out of 59 compounds, eight ligands were found active after the 3D-QSAR study. After that, molecular docking was performed on the active compounds F72, F52, F54, F29, F37, F38, F25, and F29, which were recognized as potential targets, and the docking results showed that compound F52 (also an FDA-approved drug) was the best hit. F52 was found to be the best hit against liver cancer cell lines HCC and HepG2. This study would be helpful for early drug discovery optimization and lead identification.

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

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

TroTNFα, a teleost tumour necrosis factor of golden pompano (Trachinotus ovatus), enhances pathogen clearance and acts as an immune adjuvant.

Tumour necrosis factor (TNF) is one of the most pivotal factors of the TNF family and plays an essential biological role in immunity. However, the antibacterial function and mechanism of TNFα in teleosts are relatively poorly understood. In this study, a novel TNFα from Trachinotus ovatus (TroTNFα) was characterized. TroTNFα is widely expressed in immune tissues and increased after Vibrio harveyi infection. The recombinant protein TroTNFα facilitated the proliferation and chemotaxis of T. ovatus head kidney lymphocytes, induced apoptosis in human hepatocellular carcinoma cells (HepG2), and enhanced NF-κB promoter activity, whereas mutants with altered conserved receptor binding sites (Phe and Tyr mutated to Ala) lost these functions. Similarly, in vivo research revealed that, compared with the control, TroTNFα overexpression significantly reduced bacterial colonization, whereas the bacterial colonization of the mutants was similar to that of the control. Furthermore, our results showed that TroTNFα increased the vaccine-induced immune responses induced by the DNA vaccine pCTssJ against V. harveyi. Taken together, our results indicate that TroTNFα plays an indispensable role in antibacterial immunity, providing the first evidence that the binding sites (Phe144 and Tyr216) of TroTNFα are crucial in these processes in teleosts and enhances DNA vaccine efficacy as an immune adjuvant.

Transcriptomic-Based Identification of miR-125a Novel Targets in Human Hepatocarcinoma Cells.

Hepatocellular carcinoma (HCC) is among the most aggressive and lethal human tumors. Many functional studies have demonstrated the role of non-coding RNAs (ncRNA), particularly microRNAs (miRNA), in the regulation of hepatocarcinogenesis driving pathways. MiR-125a-5p (miR-125a) has been consistently reported as an oncosuppressive miRNA, as demonstrated in vivo and in vitro. However, its HCC relevant targets and molecular mechanisms are still largely unknown. Here, a genome-wide perspective of the whole miR-125a targetome has been achieved. In particular, two different HCC cell lines were subjected to a miRNA boosting by mimic transfections, and consequently many genes were de-regulated, as observed by a transcriptomic approach. The merging of down-regulated genes with results from bioinformatic predictive tools yielded a number of candidate direct targets that were further experimentally validated by luciferase-based reporter assays. Different novel targets were found, in particular ARID3A, CCNJ, LIPA, NR6A1, and NUP210, oncogenes in various tumors and here also related to HCC through miR-125a regulation. The RNA interactions investigated in this work could pave the way to piece together the RNA regulatory networks governed by the miRNA impacting on hepatocarcinogenesis, and be exploited in the future for identifying novel biomarkers and therapeutic targets in HCC.

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

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

ERK-USP9X-coupled regulation of thymidine kinase 1 promotes both its enzyme activity-dependent and its enzyme activity-independent functions for tumor growth.

Thymidine kinase 1 (TK1), a crucial enzyme in DNA synthesis, is highly expressed in various cancers. However, the mechanisms underlying its elevated expression and the implications for tumor metabolism remain unclear. Here we demonstrate that activation of growth factor receptors enhances TK1 expression. Treatment with epidermal growth factor or insulin-like growth factor 1 induces the binding of ERK1/2 to TK1 and subsequent TK1 S13/231 phosphorylation by ERK1/2. This modification recruits ubiquitin carboxyl-terminal hydrolase 9X to deubiquitylate TK1, preventing its proteasomal degradation. Stabilized TK1 not only enhances its enzyme activity-dependent deoxythymidine monophosphate production for DNA synthesis but also promotes glycolysis independently of its enzymatic activity by upregulating phosphofructokinase/fructose bisphosphatase type 3 expression. This dual role of TK1 drives the proliferation of human hepatocellular carcinoma cells and liver tumor growth in mice. Our findings reveal a crucial mechanism by which growth factors promote tumor development through TK1-mediated DNA synthesis and glycolysis and highlight TK1 as a potential molecular target for cancer treatment.

(-)-Epigallocatechin-3-Gallate and Quercetin Inhibit Quiescin Sulfhydryl Oxidase 1 Secretion from Hepatocellular Carcinoma Cells.

Liver cancer is one of the most prevalent cancers worldwide. The first-line therapeutic drug sorafenib offers only a moderate improvement in patients' conditions. Therefore, an approach to enhancing its therapeutic efficacy is urgently needed. It has been revealed that hepatocellular carcinoma (HCC) cells with heightened intracellular quiescin sulfhydryl oxidase 1 (QSOX1) exhibit increased sensitivity to sorafenib. QSOX1 is a secreted disulfide catalyst, and it is widely recognized that extracellular QSOX1 promotes the growth, invasion, and metastasis of cancer cells through its participation in the establishment of extracellular matrix. Inhibiting QSOX1 secretion can increase intracellular QSOX1 and decrease extracellular QSOX1. Such an approach would sensitize HCC cells to sorafenib but remains to be established. Since (-)-epigallocatechin-3-gallate (EGCG) has been demonstrated to be an effective inhibitor of α-fetal protein secretion from HCC cells, we screened QSOX1 secretion inhibition using polyphenolic compounds. We examined eight dietary polyphenols (EGCG, quercetin, fisetin, myricetin, caffeic acid, chlorogenic acid, resveratrol, and theaflavin) and found that EGCG and quercetin effectively inhibited QSOX1 secretion from human HCC cells (HepG2 or Huh7), resulting in high intracellular QSOX1 and low extracellular QSOX1. The combination of EGCG or quercetin, both of which change the cellular distribution of QSOX1, with sorafenib, which has no influence on the cellular distribution of QSOX1, exhibited multiple synergistic effects against the HCC cells, including the induction of apoptosis and inhibition of invasion and metastasis. In conclusion, our current results suggest that dietary EGCG and quercetin have the potential to be developed as adjuvants to sorafenib in the treatment of HCC by modulating the cellular distribution of QSOX1.

The Cell Polarity Protein MPP5/PALS1 Controls the Subcellular Localization of the Oncogenes YAP and TAZ in Liver Cancer.

The oncogenes yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are potent liver oncogenes. Because gene mutations cannot fully explain their nuclear enrichment, we aim to understand which mechanisms cause YAP/TAZ activation in liver cancer cells. The combination of proteomics and functional screening identified numerous apical cell polarity complex proteins interacting with YAP and TAZ. Co-immunoprecipitation (Co-IP) experiments confirmed that membrane protein palmitoylated 5 (MPP5; synonym: PALS1) physically interacts with YAP and TAZ. After removing different MPP5 protein domains, Co-IP analyses revealed that the PDZ domain plays a crucial role in YAP binding. The interaction between YAP and MPP5 in the cytoplasm of cancer cells was demonstrated by proximity ligation assays (PLAs). In human hepatocellular carcinoma (HCC) tissues, a reduction in apical MPP5 expression was observed, correlating with the nuclear accumulation of YAP and TAZ. Expression data analysis illustrated that MPP5 is inversely associated with YAP/TAZ target gene signatures in human HCCs. Low MPP5 levels define an HCC patient group with a poor clinical outcome. In summary, MPP5 facilitates the nuclear exclusion of YAP and TAZ in liver cancer. This qualifies MPP5 as a potential tumor-suppressor gene and explains how changes in cell polarity can foster tumorigenesis.

ADARp110 promotes hepatocellular carcinoma progression via stabilization of CD24 mRNA

ADAR is highly expressed and correlated with poor prognosis in hepatocellular carcinoma (HCC), yet the role of its constitutive isoform ADARp110 in tumorigenesis remains elusive. We investigated the role of ADARp110 in HCC and underlying mechanisms using clinical samples, a hepatocyte-specific Adarp110 knock-in mouse model, and engineered cell lines. ADARp110 is overexpressed and associated with poor survival in both human and mouse HCC. It creates an immunosuppressive microenvironment by inhibiting total immune cells, particularly cytotoxic GZMB+CD8+ T cells infiltration, while augmenting Treg cells, MDSCs, and exhausted CD8+ T cells ratios. Mechanistically, ADARp110 interacts with SNRPD3 and RNPS1 to stabilize CD24 mRNA by inhibiting STAU1-mediated mRNA decay. CD24 protects HCC cells from two indispensable mechanisms: macrophage phagocytosis and oxidative stress. Genetic knockdown or monoclonal antibody treatment of CD24 inhibits ADARp110-overexpressing tumor growth. Our findings unveil different mechanisms for ADARp110 modulation of tumor immune microenvironment and identify CD24 as a promising therapeutic target for HCCs.

N6-methyladenosine-modification of USP15 regulates chemotherapy resistance by inhibiting LGALS3 ubiquitin-mediated degradation via AKT/mTOR signaling activation pathway in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is among the most malignant tumors and seriously threatens human health worldwide, and its incidence rate is increasing annually. USP15 is a member of the ubiquitination-specific protease (USP) family, which can regulate protein ubiquitination, thereby affecting their stability, and is dysregulated in many cancers, but its expression and regulatory mechanism in HCC are unclear. The aims of this study were to explore the role and mechanism of USP15 in regulating HCC cell stemness, proliferation, and lenvatinib resistance. Immunohistochemistry and high-throughput sequencing analyses of tumor and adjacent normal tissue samples from 52 patients with HCC were conducted. Functional analyses of immortalized human liver and HCC cell lines were conducted, including quantitative real-time PCR; western blot; plasmid, lentivirus, and siRNA transfection; co-immunoprecipitation; mass spectrometry; MeRIP-qPCR; and ubiquitination, cell growth, colony formation, and spheroid formation assays. HCC tumor growth was also assessed using cell transplantation in nude mice. We found that USP15 is upregulated in HCC and affects patient prognosis. Our results demonstrated that USP15 can increase LGALS3 stability in HCC through deubiquitination modification, and affect the stemness, proliferation, and lenvatinib resistance of HCC cells by activating the AKT/mTOR pathway. USP15 expression levels were positively correlated with HCC cell stemness, proliferation, and lenvatinib resistance. In addition, methyltransferase-like protein 3 (Mettl3) N6-methyladenosine (m6A) modified USP15 to upregulate its levels by increasing its mRNA stability. These findings provide a theoretical basis for the potential discovery of new HCC oncogenes, as well as the identification of effective targets and development of novel anti-HCC drugs and clinical applications.