Cytoskeletal gene alterations linked to sorafenib resistance in hepatocellular carcinoma.

BACKGROUND: Although sorafenib has been consistently used as a first-line treatment for advanced hepatocellular carcinoma (HCC), most patients will develop resistance, and the mechanism of resistance to sorafenib needs further study. METHODS: Using KAS-seq technology, we obtained the ssDNA profiles within the whole genome range of SMMC-7721 cells treated with sorafenib for differential analysis. We then intersected the differential genes obtained from the analysis of hepatocellular carcinoma patients in GSE109211 who were ineffective and effective with sorafenib treatment, constructed a PPI network, and obtained hub genes. We then analyzed the relationship between the expression of these genes and the prognosis of hepatocellular carcinoma patients. RESULTS: In this study, we identified 7 hub ERGs (ACTB, CFL1, ACTG1, ACTN1, WDR1, TAGLN2, HSPA8) related to drug resistance, and these genes are associated with the cytoskeleton. CONCLUSIONS: The cytoskeleton is associated with sorafenib resistance in hepatocellular carcinoma. Using KAS-seq to analyze the early changes in tumor cells treated with drugs is feasible for studying the drug resistance of tumors, which provides reference significance for future research.

Phosphorylated FOXQ1, a novel substrate of JNK1, inhibits sorafenib-induced ferroptosis by activating ETHE1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly heterogeneous and malignant cancer with poor overall survival. The application of sorafenib is a major breakthrough in the treatment of HCC. In our study, FOXQ1 was significantly overexpressed in sorafenib-resistant HCC cells and suppressed sorafenib-induced ferroptosis. We found that phosphorylation of FOXQ1 at serine 248 is critical for the suppression of sorafenib-induced ferroptosis. Furthermore, as the upstream phosphorylation kinase of FOXQ1, JNK1, which is activated by sorafenib, can directly phosphorylate the serine 248 site of FOXQ1. Then, the phosphorylated FOXQ1 got a high affinity for the promoter of ETHE1 and activates its transcription. Further flow cytometry results showed that ETHE1 reduced intracellular lipid peroxidation and iron levels. Collectively, our study implicated the JNK1-FOXQ1-ETHE1 axis in HCC ferroptosis induced by sorafenib, providing mechanistic insight into sensitivity to sorafenib therapy of HCC.

Ex Vivo Evaluation of Antiangiogenic Drugs in Oral Cancer: Potential Implications for Targeting Vasculogenic Mimicry.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is characterized by early metastasis, clinical resistance and poor prognosis. Recently, we showed that aggressive OSCC cells co-express endothelial cell markers and can form tube-like structures, known as vasculogenic mimicry (VM), a process associated with poor prognosis in head and neck cancers. Given the limited success of current antiangiogenic therapy in treating OSCC, this study sought to explore the efficiency of these drugs in targeting an ex vivo model of VM. MATERIALS AND METHODS: OSCC cell lines from the tongue and floor of the mouth in addition to human endothelial cells were used. The treatments comprised a set of clinically relevant antiangiogenic drugs: sorafenib, sunitinib, and axitinib, which were administered in different doses. Multiple ex vivo approaches including cell tubulogenesis, proliferation, apoptosis, and migration assays were used. RESULTS: Although these drugs inhibited the formation of endothelial cell capillaries, they showed clear differential effects on OSCC cell-derived VM and cell morphology. Sorafenib inhibited the tubulogenesis of aggressive OSCC cells compared with the limited effect of sunitinib and axitinib. Furthermore, our data consistently demonstrated a preferential efficacy of certain drugs over others. Sorafenib and sunitinib exhibited anti-cancer effects on tumor cell proliferation, apoptosis, and cell migration, compared with the limited effect of axitinib. CONCLUSION: The antiangiogenic drugs, except sorafenib, had limited effect on VM formation in vitro and exhibited varying anti-cancer effects on OSCC cells. These data support the notion that VM formation may in part explain the development of drug resistance in OSCC cells.

Exosome-derived circUPF2 enhances resistance to targeted therapy by redeploying ferroptosis sensitivity in hepatocellular carcinoma.

BACKGROUND: Advanced hepatocellular carcinoma (HCC) can be treated with sorafenib, which is the primary choice for targeted therapy. Nevertheless, the effectiveness of sorafenib is greatly restricted due to resistance. Research has shown that exosomes and circular RNAs play a vital role in the cancer's malignant advancement. However, the significance of exosomal circular RNAs in the development of resistance to sorafenib in HCC remains uncertain. METHODS: Ultracentrifugation was utilized to isolate exosomes (Exo-SR) from the sorafenib-resistant HCC cells' culture medium. Transcriptome sequencing and differential expression gene analysis were used to identify the targets of Exo-SR action in HCC cells. To identify the targets of Exo-SR action in HCC cells, transcriptome sequencing and analysis of differential expression genes were employed. To evaluate the impact of exosomal circUPF2 on resistance to sorafenib in HCC, experiments involving gain-of-function and loss-of-function were conducted. RNA pull-down assays and mass spectrometry analysis were performed to identify the RNA-binding proteins interacting with circUPF2. RNA immunoprecipitation (RIP), RNA pull-down, electrophoretic mobility shift assay (EMSA), immunofluorescence (IF) -fluorescence in situ hybridization (FISH), and rescue assays were used to validate the interactions among circUPF2, IGF2BP2 and SLC7A11. Finally, a tumor xenograft assay was used to examine the biological functions and underlying mechanisms of Exo-SR and circUPF2 in vivo. RESULTS: A novel exosomal circRNA, circUPF2, was identified and revealed to be significantly enriched in Exo-SR. Exosomes with enriched circUPF2 enhanced sorafenib resistance by promoting SLC7A11 expression and suppressing ferroptosis in HCC cells. Mechanistically, circUPF2 acts as a framework to enhance the creation of the circUPF2-IGF2BP2-SLC7A11 ternary complex contributing to the stabilization of SLC7A11 mRNA. Consequently, exosomal circUPF2 promotes SLC7A11 expression and enhances the function of system Xc- in HCC cells, leading to decreased sensitivity to ferroptosis and resistance to sorafenib. CONCLUSIONS: The resistance to sorafenib in HCC is facilitated by the exosomal circUPF2, which promotes the formation of the circUPF2-IGF2BP2-SLC7A11 ternary complex and increases the stability of SLC7A11 mRNA. Focusing on exosomal circUPF2 could potentially be an innovative approach for HCC treatment.

Biomimetic Cancer-Targeting Nanoplatform Boosting AIEgens-Based Photodynamic Therapy and Ferroptosis by Disrupting Redox-Homeostasis.

Photodynamic therapy (PDT) using aggregation-induced emission photosensitizer (AIE-PS) holds tremendous potential but is limited by its inherent disadvantages and the high concentrations of reduced glutathione (GSH) in tumor cells that can neutralize ROS to weaken PDT. Herein, we designed a nanodelivery system (CM-HSADSP@[PS-Sor]) in which albumin was utilized as a carrier for hydrophobic drug AIE-PS and Sorafenib, cross-linkers with disulfide bonds were introduced to form a nanogel core, and then cancer cell membranes were wrapped on its surface to confer homologous tumor targeting ability. A two-way strategy was employed to disturb redox-homeostasis through blocking GSH synthesis by Sorafenib and consuming excess GSH via abundant disulfide bonds, thereby promoting the depletion of GSH, which in turn increased the ROS levels in cancer cells to amplify the efficacy of ferroptosis and PDT, achieving an efficient in vivo antibreast cancer effect. This study brings a new strategy for ROS-based cancer therapy and expands the application of an albumin-based drug delivery system.

Targeting NG2 relieves the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitors.

BRAFV600E represents a constitutively active onco-kinase and stands as the most prevalent genetic alteration in thyroid cancer. However, the clinical efficacy of small-molecule inhibitors targeting BRAFV600E is often limited by acquired resistance. Here, we find that nerve/glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4), is up-regulated in thyroid cancers, and its expression is increased with tumor progression in a BRAFV600E-driven thyroid cancer mouse model. Functional studies show that NG2 knockout almost does not affect tumor growth, but significantly improves the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4720. Mechanistically, the blockade of ERK-dependent feedback by BRAF inhibitor can activate receptor tyrosine kinase (RTK) signaling, causing the resistance to this inhibitor. NG2 knockout attenuates the PLX4720-mediated feedback activation of several RTKs, improving the sensitivity of BRAF-mutant thyroid cancer cells to this inhibitor. Based on this finding, we propose and demonstrate an alternative strategy for targeting NG2 to effectively treat BRAF-mutant thyroid cancers by combining multiple kinase inhibitor (MKI) Sorafenib or Lenvatinib with PLX4720. Thus, this study uncovers a new mechanism in which NG2 contributes to the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitor, and provides a promising therapeutic option for BRAF-mutant thyroid cancers.

Targeted degradation of NDUFS1 by agrimol B promotes mitochondrial ROS accumulation and cytotoxic autophagy arrest in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a global public health problem with increased morbidity and mortality. Agrimol B, a natural polyphenol, has been proved to be a potential anticancer drug. Our recent report showed a favorable anticancer effect of agrimol B in HCC, however, the mechanism of action remains unclear. Here, we found agrimol B inhibits the growth and proliferation of HCC cells in vitro as well as in an HCC patient-derived xenograft (PDX) model. Notably, agrimol B drives autophagy initiation and blocks autophagosome-lysosome fusion, resulting in autophagosome accumulation and autophagy arrest in HCC cells. Mechanistically, agrimol B downregulates the protein level of NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) through caspase 3-mediated degradation, leading to mitochondrial reactive oxygen species (mROS) accumulation and autophagy arrest. NDUFS1 overexpression partially restores mROS overproduction, autophagosome accumulation, and growth inhibition induced by agrimol B, suggesting a cytotoxic role of agrimol B-induced autophagy arrest in HCC cells. Notably, agrimol B significantly enhances the sensitivity of HCC cells to sorafenib in vitro and in vivo. In conclusion, our study uncovers the anticancer mechanism of agrimol B in HCC involving the regulation of oxidative stress and autophagy, and suggests agrimol B as a potential therapeutic drug for HCC treatment.

CARMIL1 regulates liver cancer cell proliferation by activating the ERK/mTOR pathway through the TRIM27/p53 axis.

Capping protein regulatory factor and myosin 1 linker 1 is termed CARMIL1. CARMIL1 is involved in several physiological processes; it forms an actin filament network and plasma membrane-bound cellular projection tissues and positively regulates the cellular components and tissues. CARMIL1 exhibits important biological functions in cancer; nonetheless, these functions have not been completely explored. We aimed to investigate the novel functions of CARMIL1 in liver cancer, particularly in cell proliferation. The cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Component A experiments, and subcutaneous tumor formation model suggest that CARMIL1 is central to the proliferation of liver cancer cells both in vivo and in vitro. We extracted CARMIL1 samples from The Cancer Genome Atlas Program and analyzed its enrichment. CARMIL1 regulated the pathway activity by affecting the expression of star molecular proteins of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Moreover, it influenced the proliferation ability of liver cancer cells. Western blotting suggested that CARMIL1 downregulation could affect ERK and mTOR phosphorylation. Results of the co-immunoprecipitation demonstrated that CARMIL1 binds to tripartite motif (TRIM)27, which in turn binds to p53. Subsequently, CARMIL1 can regulate p53 stability and promote its degradation through TRIM27. Additionally, CARMIL1 inhibition enhanced the sensitivity of liver cancer cells to sorafenib. Tumor growth was significantly inhibited in the group treated with sorafenib and CARMIL1, compared with the group treated with CARMIL1 alone. Sorafenib is a first-line targeted chemotherapeutic drug for hepatocellular carcinoma treatment. It increases the long-term survival of hepatocellular carcinoma by 44%. In this study, downregulated CARMIL1 combined with sorafenib significantly reduced the tumor volume and weight of the mouse subcutaneous tumor model, indicating the potential possibility of combining CARMIL1 with sorafenib in hepatocellular carcinoma treatment. In summary, CARMIL1 promotes liver cancer cell proliferation by regulating the TRIM27/p53 axis and activating the ERK/mTOR pathway.

DDX39B protects against sorafenib-induced ferroptosis by facilitating the splicing and cytoplasmic export of GPX4 pre-mRNA in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the main histological subtype of primary liver cancer and remains one of the most common solid malignancies globally. Ferroptosis was recently defined as an iron-catalyzed form of regulated necrosis. Because cancer cells exhibit higher iron requirements than noncancer cells, treatment with ferroptosis-inducing compounds may be a feasible strategy for cancer therapy. However, cancer cells develop acquired resistance to evade ferroptosis, and the mechanisms responsible for ferroptosis resistance are not fully clarified. In the current study, we reported that DDX39B was downregulated during sorafenib-induced ferroptosis in a dose- and time-dependent manner. Exogenous introduction of DDX39B ensured the survival of HCC cells upon exposure to sorafenib, while the opposite phenomenon was observed in DDX39B-silenced HCC cells. Mechanistically, we demonstrated that DDX39B increased GPX4 levels by promoting the splicing and cytoplasmic translocation of GPX4 pre-mRNA, which was sufficient to detoxify sorafenib-triggered excess lipid ROS production, lipid peroxidation accumulation, ferrous iron levels, and mitochondrial damage. Inhibition of DDX39B ATPase activity by CCT018159 repressed the splicing and cytoplasmic export of GPX4 pre-mRNA and synergistically assisted sorafenib-induced ferroptotic cell death in HCC cells. Taken together, our data uncover a novel role for DDX39B in ferroptosis resistance by modulating the maturation of GPX4 mRNA via a posttranscriptional approach and suggest that DDX39B inhibition may be a promising therapeutic strategy to enhance the sensitivity and vulnerability of HCC cells to sorafenib.

Synthesis and biological evaluation of novel pyrazole scaffold.

synthesis of a pyrazole containing compound was achieved by reacting phenyl hydrazine with (E)-2-((4-bromophenyl) diazinyl)-1-phenylbutane-1,3-dione to produce 4-((4-bromophenyl) diazinyl)-5-methyl-1,3-diphenyl-pyrazole and characterization using mass spectrometer, 1H NMR and 13C NMR. The pharmacological evaluation of the synthesized compound, denoted as (KA5), against Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 29213 and Clostridiums sporogeneses ATCC 19404, indicate that there is no promising antibacterial activity. However, KA5 shows a competitive anticancer activity (IC50: 8.5µM) upon its evaluation against hepatocellular carcinoma cell line (HepG 2) compared to sorafenib (IC50: 4.51µM). Moreover, human skin fibroblast (HSF) was used to investigate the effect of KA5 on normal cell lines, (IC50: 5.53µM). The presented biological evaluations resulted in better understanding of structure-activity relationship for 1, 3, 4-trisubstituted pyrazoles and revealed a great opportunity for more investigations for novel pyrazole-containing anticancer agents.

Stress Granule Core Protein-Derived Peptides Inhibit Assembly of Stress Granules and Improve Sorafenib Sensitivity in Cancer Cells.

Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.

METTL14 decreases FTH1 mRNA stability via m6A methylation to promote sorafenib-induced ferroptosis of cervical cancer.

Cervical cancer (CC) is a prevalent malignancy among women worldwide. This study was designed to investigate the role of METTL14 in sorafenib-induced ferroptosis in CC. METTL14 expression and m6A methylation were determined in CC tissues, followed by analyzes correlating these factors with clinical features. Subsequently, METTL14 was knocked down in CC cell lines, and the effects on cell proliferation, mitochondrial morphology and ferroptosis were assessed using CCK-8, microscopy, and markers associated with ferroptosis, respectively. The regulatory relationship between METTL14 and FTH1 was verified using qRT-PCR and luciferase reporter assays. The functional significance of this interaction was further investigated both in vitro and in vivo by co-transfecting cells with overexpression vectors or shRNAs targeting METTL14 and FTH1 after sorafenib treatment. METTL14 expression and m6A methylation were significantly reduced in CC tissues, and lower METTL14 expression levels were associated with a poorer CC patients' prognosis. Notably, METTL14 expression increased during sorafenib-induced ferroptosis, and METTL14 knockdown attenuated the ferroptotic response induced by sorafenib in CC cells. FTH1 was identified as a direct target of METTL14, with METTL14 overexpression leading to increased m6A methylation of FTH1 mRNA, resulting in reduced stability and expression of FTH1 in CC. Furthermore, FTH1 overexpression or treatment with LY294002 partially counteracted the promotion of sorafenib-induced ferroptosis by METTL14. In vivo xenograft experiments demonstrated that inhibiting METTL14 reduced the anticancer effects of sorafenib, whereas suppression of FTH1 significantly enhanced sorafenib-induced ferroptosis and increased its anticancer efficacy. METTL14 reduces FTH1 mRNA stability through m6A methylation, thereby enhancing sorafenib-induced ferroptosis, which contributes to suppressing CC progression via the PI3K/Akt signaling pathway.

PLAG1 interacts with GPX4 to conquer vulnerability to sorafenib induced ferroptosis through a PVT1/miR-195-5p axis-dependent manner in hepatocellular carcinoma.

BACKGROUND: Sorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), yet its effectiveness is often constrained. Emerging studies reveal that sorafenib triggers ferroptosis, an iron-dependent regulated cell death (RCD) mechanism characterized by lipid peroxidation. Our findings isolate the principal target responsible for ferroptosis in HCC cells and outline an approach to potentially augment sorafenib's therapeutic impact on HCC. METHODS: We investigated the gene expression alterations following sgRNA-mediated knockdown induced by erastin and sorafenib in HCC cells using CRISPR screening-based bioinformatics analysis. Gene set enrichment analysis (GSEA) and the "GDCRNATools" package facilitated the correlation studies. We employed tissue microarrays and cDNA microarrays for validation. Ubiquitination assay, Chromatin immunoprecipitation (ChIP) assay, RNA immunoprecipitation (RIP) assay, and dual-luciferase reporter assay were utilized to delineate the specific mechanisms underlying ferroptosis in HCC cells. RESULTS: Our study has revealed that pleiomorphic adenoma gene 1 (PLAG1), a gene implicated in pleomorphic adenoma, confers resistance to ferroptosis in HCC cells treated with sorafenib. Sorafenib leads to the opposite trend of protein and mRNA levels of PLAG1, which is not caused by affecting the stability or ubiquitination of PLAG1 protein, but by the regulation of PLAG1 at the transcriptional level by its upstream competitive endogenous long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1). Data from 139 HCC patients showed a significant positive correlation between PLAG1 and GPX4 levels in tumor samples, and PLAG1 is instrumental in redox homeostasis by driving the expression of glutathione peroxidase 4 (GPX4), the enzyme that reduces lipid peroxides (LPOs), which further leads to ferroptosis inhibition. CONCLUSIONS: Ferroptosis is a promising target for cancer therapy, especially for patients resistant to standard chemotherapy or immunotherapy. Our findings indicate that PLAG1 holds therapeutic promise and may enhance the efficacy of sorafenib in treating HCC.

DEAD-Box Helicase 17 circRNA (circDDX17) Reduces Sorafenib Resistance and Tumorigenesis in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of liver malignancy. Despite significant progress in HCC treatment, resistance to chemotherapy and tumor metastasis are the main reasons for the unsatisfactory prognosis of HCC. Circular RNAs (circRNAs) have been extensively documented to play a role in the development of various types of cancer. AIMS: Here, we investigated the role of DEAD-box helicase 17 circRNA (circDDX17) in HCC and its underlying molecular mechanisms. METHODS: Our research employed various techniques including reverse transcription-quantitative polymerase chain reaction (RT-qPCR), cell counting kit-8 (CCK-8), flow cytometry, dual luciferase reporter assay, RNA immunoprecipitation (RIP), and western blot analysis. Additionally, we conducted a tumor xenograft assay to investigate the in vivo function of circDDX17. RESULTS: Firstly, the expression of circDDX17 was downregulated in HCC tissues and cells. Through functional experiments, it was observed that the overexpression of circDDX17 enhanced the sensitivity of sorafenib, promoted apoptosis, and inhibited the process of epithelial-mesenchymal transition (EMT) in vitro. Additionally, in vivo studies revealed that circDDX17 reduced tumor growth and increased sorafenib sensitivity. Mechanically, circDDX17 competitively combined miR-21-5p to suppress PTEN expression and activate the PI3K/AKT pathway. Furthermore, our rescue assays demonstrated that circDDX17 act as a tumor suppressor by blocking sorafenib resistance and tumorigenesis, while the inhibitory effect caused by circDDX17 upregulation was neutralized when miR-21-5p was overexpressed, PTEN was silenced, or the PI3K/AKT pathway was activated. CONCLUSION: Our findings firstly confirmed that circDDX17 suppressed sorafenib resistance and HCC progression by regulating miR-21-5p/PTEN/PI3K/AKT pathway, which may provide novel biomarkers for the diagnosis, treatment and prognosis of HCC.

Oxygen Self-Supplied Perfluorocarbon-Modified Micelles for Enhanced Cancer Photodynamic Therapy and Ferroptosis.

Photodynamic therapy (PDT) and ferroptosis show significant potential in tumor treatment. However, their therapeutic efficacy is often hindered by the oxygen-deficient tumor microenvironment and the challenges associated with efficient intracellular drug delivery into tumor cells. Toward this end, this work synthesized perfluorocarbon (PFC)-modified Pluronic F127 (PFC-F127), and then exploits it as a carrier for codelivery of photosensitizer Chlorin e6 (Ce6) and the ferroptosis promoter sorafenib (Sor), yielding an oxygen self-supplying nanoplatform denoted as Ce6-Sor@PFC-F127. The PFCs on the surface of the micelle play a crucial role in efficiently solubilizing and delivering oxygen as well as increasing the hydrophobicity of the micelle surface, giving rise to enhanced endocytosis by cancer cells. The incorporation of an oxygen-carrying moiety into the micelles enhances the therapeutic impact of PDT and ferroptosis, leading to amplified endocytosis and cytotoxicity of tumor cells. Hypotonic saline technology was developed to enhance the cargo encapsulation efficiency. Notably, in a murine tumor model, Ce6-Sor@PFC-F127 effectively inhibited tumor growth through the combined use of oxygen-enhanced PDT and ferroptosis. Taken together, this work underscores the promising potential of Ce6-Sor@PFC-F127 as a multifunctional therapeutic nanoplatform for the codelivery of multiple cargos such as oxygen, photosensitizers, and ferroptosis inducers.

Integrated analyses for identification of a three-gene signature associated with Chaihu Shugan San formula for hepatocellular carcinoma treatment.

Chaihu Shugan San (CSS) is a well-known traditional herbal formula that has the potential to ameliorate hepatocellular carcinoma (HCC); however, its mechanism of action remains unknown. Here, we identified the key targets of CSS against HCC and developed a prognostic model to predict the survival of patients with HCC. The effect of CSS plus sorafenib on HCC cell proliferation was evaluated using the MTT assay. LASSO-Cox regression was used to establish a three-gene signature model targeting CSS. Correlations between immune cells, immune checkpoints and risk score were determined to evaluate the immune-related effects of CSS. The interactions between the components and targets were validated using molecular docking and Surface Plasmon Resonance (SPR) assays. CSS and sorafenib synergistically inhibited HCC cell proliferation. Ten core compounds and 224 targets were identified using a drug compound-target network. The prognostic model of the three CSS targets (AKT1, MAPK3 and CASP3) showed predictive ability. Risk scores positively correlated with cancer-promoting immune cells and high expression of immune checkpoint proteins. Molecular docking and SPR analyses confirmed the strong binding affinities of the active components and the target genes. Western blot analysis confirmed the synergistic effect of CSS and sorafenib in inhibiting the expression of these three targets. In conclusion, CSS may regulate the activity of immune-related factors in the tumour microenvironment, reverse immune escape, enhance immune responses through AKT1, MAPK3, and CASP3, and synergistically alleviate HCC. The co-administration of sorafenib with CSS has a strong clinical outlook against HCC.

Identification of differentially expressed mRNA/lncRNA modules in acutely regorafenib-treated sorafenib-resistant Huh7 hepatocellular carcinoma cells.

The multikinase inhibitor sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but many patients become sorafenib-resistant (SR). This study investigated the efficacy of another kinase inhibitor, regorafenib (Rego), as a second-line treatment. We produced SR HCC cells, wherein the PI3K-Akt, TNF, cAMP, and TGF-beta signaling pathways were affected. Acute Rego treatment of these cells reversed the expression of genes involved in TGF-beta signaling but further increased the expression of genes involved in PI3K-Akt signaling. Additionally, Rego reversed the expression of genes involved in nucleosome assembly and epigenetic gene expression. Weighted gene co-expression network analysis (WGCNA) revealed four differentially expressed long non-coding RNA (DElncRNA) modules that were associated with the effectiveness of Rego on SR cells. Eleven putative DElncRNAs with distinct expression patterns were identified. We associated each module with DEmRNAs of the same pattern, thus obtaining DElncRNA/DEmRNA co-expression modules. We discuss the potential significance of each module. These findings provide insights and resources for further investigation into the potential mechanisms underlying the response of SR HCC cells to Rego.

IL-22 signaling promotes sorafenib resistance in hepatocellular carcinoma via STAT3/CD155 signaling axis.

Introduction: Sorafenib is currently the first-line treatment for patients with advanced hepatocellular carcinoma (HCC). Nevertheless, sorafenib resistance remains a huge challenge in the clinic. Therefore, it is urgent to elucidate the mechanisms underlying sorafenib resistance for developing novel treatment strategies for advanced HCC. In this study, we aimed to investigate the role and mechanisms of interleukin-22 (IL-22) in sorafenib resistance in HCC. Methods: The in vitro experiments using HCC cell lines and in vivo studies with a nude mouse model were used. Calcium staining, chromatin immunoprecipitation, lactate dehydrogenase release and luciferase reporter assays were employed to explore the expression and roles of IL-22, STAT3 and CD155 in sorafenib resistance. Results: Our clinical results demonstrated a significant correlation between elevated IL-22 expression and poor prognosis in HCC. Analysis of transcriptomic data from the phase-3 STORM-trial (BIOSTORM) suggested that STAT3 signaling activation and natural killer (NK) cell infiltration may associate sorafenib responses. STAT3 signaling could be activated by IL-22 administration in HCC cells, and then enhanced sorafenib resistance in HCC cells by promoting cell proliferation and reducing apoptosis in vitro and in vivo. Further, we found IL-22/STAT3 axis can transcriptionally upregulate CD155 expression in HCC cells, which could significantly reduce NK cell-mediated HCC cell lysis in a co-culture system. Conclusions: Collectively, IL-22 could contribute to sorafenib resistance in HCC by activating STAT3/CD155 signaling axis to decrease the sensitivities of tumor cells to sorafenib-mediated direct cytotoxicity and NK cell-mediated lysis. These findings deepen the understanding of how sorafenib resistance develops in HCC in terms of IL-22/STAT3 signaling pathway, and provide potential targets to overcome sorafenib resistance in patients with advanced HCC.

Oxyberberine sensitizes liver cancer cells to sorafenib via inhibiting NOTCH1-USP7-c-Myc pathway.

BACKGROUND: Sorafenib is the first-line therapy for patients with advanced-stage HCC, but its clinical cure rate is unsatisfactory due to adverse reactions and drug resistance. Novel alternative strategies to overcome sorafenib resistance are urgently needed. Oxyberberine (OBB), a major metabolite of berberine in vivo, exhibits potential antitumor potency in various human malignancies, including liver cancer. However, it remains unknown whether and how OBB sensitizes liver cancer cells to sorafenib. METHODS: Cell viability, trypan blue staining and flow cytometry assays were employed to determine the synergistic effect of OBB and sorafenib on killing HCC cells. PCR, western blot, co-immunoprecipitation and RNA interference assays were used to decipher the mechanism by which OBB sensitizes sorafenib. HCC xenograft models and clinical HCC samples were utilized to consolidate our findings. RESULTS: We found for the first time that OBB sensitized liver cancer cells to sorafenib, enhancing its inhibitory effect on cell growth and induction of apoptosis in vitro. Interestingly, we observed that OBB enhanced the sensitivity of HCC cells to sorafenib by reducing ubiquitin-specific peptidase 7 (USP7) expression, a well-known tumor-promoting gene. Mechanistically, OBB inhibited notch homolog 1-mediated USP7 transcription, leading to the downregulation of V-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), which synergized with sorafenib to suppress liver cancer. Furthermore, animal results showed that cotreatment with OBB and sorafenib significantly inhibited the tumor growth of liver cancer xenografts in mice. CONCLUSIONS: These results indicate that OBB enhances the sensitivity of liver cancer cells to sorafenib through inhibiting notch homolog 1-USP7-c-Myc signaling pathway, which potentially provides a novel therapeutic strategy for liver cancer to improve the effectiveness of sorafenib.

NFKBIZ regulates NFκB signaling pathway to mediate tumorigenesis and metastasis of hepatocellular carcinoma by direct interaction with TRIM16.

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence and mortality rates. NFKBIZ, a member of the nuclear factor kappa B inhibitory family, is closely related to tumor progression. However, the precise role of NFKBIZ in HCC remains unclear. To explore this, we conducted a series of experiments from clinic to cells. Western blot and qPCR revealed a significant downregulation of NFKBIZ in human HCC tissues. Clinical character analysis showed that the patients with lower NFKBIZ expression had poorer prognosis and higher clinical stage. By using CCK-8, wound healing, transwell invasion and migration assay, we discovered that NFKBIZ expression was reversely associated with the proliferation, invasion, and migration ability of HCC cells in vitro. Additionally, the results obtained from xenograft assay and lung metastasis models showed that NFKBIZ overexpression inhibited the growth and metastasis of HCC cells in vivo. Western blot and immunofluorescence assay further revealed that NFKBIZ mediated HCC cell growth and migration by regulating NFκB signaling transduction. Finally, flow cytometry, protein degradation assay and Co-immunoprecipitation indicated that TRIM16 can enhance NFKBIZ ubiquitination by direct interactions at its K48 site, which may thereby alleviate HCC cell apoptosis to induce the insensitivity to sorafenib. In conclusion, our study demonstrated that NFKBIZ regulated HCC tumorigenesis and metastasis by mediating NFκB signal transduction and TRIM16/NFKBIZ/NFκB axis may be the underlying mechanism of sorafenib insensitivity in HCC.