Sorafenib-treated Hepatocellular Carcinoma Research Articles

Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC

Background and aimsSorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC.Materials and methodsA clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array.ResultsCRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPARα is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPARα inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC.ConclusionOur study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.

The Clinical Significance of Myosteatosis in Survival Outcomes in Patients with Hepatocellular Carcinoma Treated with Sorafenib.

The role of body composition parameters in sorafenib-treated hepatocellular carcinoma (HCC) patients is still not fully elucidated. Here, we aimed to evaluate the impact of computed tomography (CT)-based body composition parameters on the survival of such patients. In this multicenter study, we analyzed the data of 245 sorafenib-treated HCC patients from January 2008 to December 2019. Sarcopenia, visceral obesity, and myosteatosis were defined by using cross-sectional CT images at the third lumbar vertebra level. The effects of these parameters on overall survival (OS) and progression-free survival (PFS) were evaluated. The median age was 67.0 years (interquartile range: 61.0-78.0 year), and 211 patients (86.1%) were male. The median OS and PFS were 7.9 months and 4.8 months, respectively. Vascular invasion (hazard ratio (HR), 1.727; 95% confidence interval (CI), 1.258-2.371; p = 0.001), extrahepatic metastasis (HR, 1.401; 95% CI, 1.028-1.908; p = 0.033), alpha-fetoprotein level > 200 ng/mL (HR, 1.559; 95% CI, 1.105-2.201; p = 0.012), and myosteatosis (HR, 1.814; 95% CI, 1.112-2.960; p = 0.017) were associated with OS. Patient mortality was significantly higher in the group with two or more risk factors than in the group with fewer risk factors. In conclusion, myosteatosis may be a novel prognostic CT-based radiological biomarker in sorafenib-treated HCC patients.

Immunogenic landscape and risk score prediction based on unfolded protein response (UPR)-related molecular subtypes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of cancer and causes a significant number of cancer-related deaths worldwide. The molecular mechanisms underlying the development of HCC are complex, and the heterogeneity of HCC has led to a lack of effective prognostic indicators and drug targets for clinical treatment of HCC. Previous studies have indicated that the unfolded protein response (UPR), a fundamental pathway for maintaining endoplasmic reticulum homeostasis, is involved in the formation of malignant characteristics such as tumor cell invasiveness and treatment resistance. The aims of our study are to identify new prognostic indicators and provide drug treatment targets for HCC in clinical treatment based on UPR-related genes (URGs). Gene expression profiles and clinical information were downloaded from the TCGA, ICGC and GEO databases. Consensus cluster analysis was performed to classify the molecular subtypes of URGs in HCC patients. Univariate Cox regression and machine learning LASSO algorithm were used to establish a risk prognosis model. Kaplan-Meier and ROC analyses were used to evaluate the clinical prognosis of URGs. TIMER and XCell algorithms were applied to analyze the relationships between URGs and immune cell infiltration. Real time-PCR was performed to analyze the effect of sorafenib on the expression levels of four URGs. Most URGs were upregulated in HCC samples. According to the expression pattern of URGs, HCC patients were divided into two independent clusters. Cluster 1 had a higher expression level, worse prognosis, and higher expression of immunosuppressive factors than cluster 2. Patients in cluster 1 were more prone to immune escape during immunotherapy, and were more sensitive to chemotherapeutic drugs. Four key UPR genes (ATF4, GOSR2, PDIA6 and SRPRB) were established in the prognostic model and HCC patients with high risk score had a worse clinical prognosis. Additionally, patients with high expression of four URGs are more sensitive to sorafenib. Moreover, ATF4 was upregulated, while GOSR2, PDIA6 and SRPRB were downregulated in sorafenib-treated HCC cells. The UPR-related prognostic signature containing four URGs exhibits high potential application value and performs well in the evaluation of effects of chemotherapy/immunotherapy and clinical prognosis.

Astaxanthin Supplementation Assists Sorafenib in Slowing Skeletal Muscle Atrophy in H22 Tumor-Bearing Mice via Reversing Abnormal Glucose Metabolism.

Cachexia, which is often marked by skeletal muscular atrophy, is one of the leading causes of death in cancer patients. Astaxanthin, a carotenoid obtained from marine organisms that can aid in the prevention and treatment of a variety of disorders. In this study, to assess whether astaxanthin ameliorates weight loss and skeletal muscle atrophy in sorafenib-treated hepatocellular carcinoma mice is aimed. H22 mice are treated with 30mgkg-1 day-1 of sorafenib and 60mgkg-1 day-1 of astaxanthin by gavage lasted for 18 days. Sorafenib does not delay skeletal muscle atrophy and weight loss, although it does not reduce tumor burden. Astaxanthin dramatically delays weight loss and skeletal muscle atrophy in sorafenib-treating mice, without affecting the food intake. Astaxanthin inhibits the tumor glycolysis, slows down gluconeogenesis, and improves insulin resistance in tumor-bearing mice. Astaxanthin increases glucose competition in skeletal muscle by targeting the PI3K/Akt/GLUT4 signaling pathway, and enhances glucose utilization efficiency in skeletal muscle, thereby slowing skeletal muscle atrophy. The findings show the significant potential of astaxanthin as nutritional supplements for cancer patients, as well as the notion that nutritional interventions should be implemented at the initiation of cancer treatment, as instead of waiting until cachexia sets in.

MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

BackgroundMetabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker.MethodsBioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats.ResultsMiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells.ConclusionsMiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.

Inhibition of stress proteins TRIB3 and STC2 potentiates sorafenib sensitivity in hepatocellular carcinoma

Sorafenib resistance is one of the main obstacles to the treatment of advanced hepatocellular carcinoma (HCC). Stress proteins TRIB3 and STC2 confer cell resistance to a variety of stresses, including hypoxia, nutritional deprivation, and other perturbations, which induce endoplasmic reticulum stress. However, the role of TRIB3 and STC2 in sorafenib sensitivity to HCC remains unclear. In this study, our results indicated that the common differentially expressed genes (DEGs) in sorafenib-treated HCC cells obtained from the NCBI-GEO database (Huh7 and Hep3B cells; GSE96796) included TRIB3, STC2, HOXD1, C2orf82, ADM2, RRM2, and UNC93A. The most significantly upregulated DEGs were TRIB3 and STC2, which were both stress protein genes. Bioinformatic analysis in NCBI public databases indicated that TRIB3 and STC2 were highly expressed in HCC tissues and closely associated with poor prognoses in HCC patients. Further investigation showed that inhibition of TRIB3 or STC2 with siRNA could enhance the anti-cancer effect of sorafenib in HCC cell lines. In conclusion, our study showed that stress proteins TRIB3 and STC2 are closely associated with sorafenib resistance in HCC. The combination of TRIB3 or STC2 inhibition and sorafenib may be a promising therapeutic strategy for HCC.

Blocking Autophagy by the Two-Pore Channels Antagonist Tetrandrine Improves Sorafenib-Induced Death of Hepatocellular Carcinoma Cells

Sorafenib, an oral multi-kinase inhibitor, used to treat hepatocellular carcinoma (HCC). However, drug resistance is still common in several HCC patients. This complex mechanism is not yet fully elucidated, driving the search for new therapeutic targets to potentiate the antitumoral effect of sorafenib. Recent findings have linked the expression of Two-Pore Channels (TPCs) receptors with the development and progression of cancer. TPCs receptors are stimulated by NAADP, a Ca2+ messenger, and inhibited by their antagonists Ned-19 and tetrandrine. Here, we investigate the participation of TPCs inhibition in cell death and autophagy in sorafenib-treated HCC cells. Here, we show that the association of sorafenib with tetrandrine increased sorafenib-induced cell death accompanied by increased lysotracker fluorescence intensity. In contrast, these effects were not observed after treating these cells with Ned-19. The pharmacological TPC antagonists by Ned-19 and tetrandrine or siRNA-mediated TPC1/2 inhibition decreased sorafenib-induced Ca2+ release, reinforcing the participation of TPCs in sorafenib HCC responses. Furthermore, the association tetrandrine and sorafenib blocked autophagy through ERK1/2 pathway inhibition, which represents a putative target for potentiating HCC cell death. Therefore, our study proposes the use of tetrandrine analogs with the aim of improving sorafenib therapy. Also, our data also allow us to suggest that TPCs may be a new target in anticancer therapies.

Iron deficiency in hepatocellular carcinoma cells induced sorafenib resistance by upregulating HIF-1α to inhibit apoptosis

Sorafenib is the first-line therapeutic agent for hepatocellular carcinoma (HCC), but the drug resistance has become a major impediment. Previously we found that the abnormal iron metabolism in HCC led to iron deficiency, whether it induces sorafenib resistance during the treatment of HCC is not yet disclosed. In this study, we observed the effects of iron deficiency on sorafenib resistance and explored the underlying mechanisms. The results revealed that the killing effects of sorafenib on HCC cells were weakened by iron deficiency but effectively restored by iron re-supplementation. The ferroptosis indicators, including the contents of lipid hydroperoxide (LPO) and malondialdehyde (MDA), the level of intracellular reactive oxygen species (ROS), and the expression of glutathione peroxidase 4 (GPX4), were not significantly changed by iron deficiency in sorafenib-treated HCC cells. However, the sorafenib-induced apoptosis of HCC cells was inhibited by iron deficiency. Notably, the expression of anti-apoptotic protein B-cell lymphoma-2 (BCL-2) was elevated, and the expressions of other apoptotic proteins, BCL2-associated X (Bax), caspase-3, and caspase-9, were inhibited by iron deficiency. Mechanistically, iron deficiency upregulated hypoxia-inducible factor 1 alpha (HIF-1α) to increase BCL-2. Inhibition of HIF-1α suppressed the iron deficiency-induced BCL-2 and sorafenib resistance. In summary, iron deficiency in HCC cells generated sorafenib resistance by increasing HIF-1α and BCL-2, which therefore inhibited the sorafenib-induced apoptosis of HCC cells. These results identified iron deficiency as a new factor of sorafenib resistance in HCC cells, which would be an effective target to alleviate sorafenib resistance.

Midkine inhibition enhances anti-PD-1 immunotherapy in sorafenib-treated hepatocellular carcinoma via preventing immunosuppressive MDSCs infiltration

Sorafenib, a multiple-target tyrosine kinase inhibitor, is the standard of care for patients with advanced hepatocellular carcinoma (HCC), but provides limited benefits. Emerging evidences suggest that prolonged sorafenib treatment induces an immunosuppressive HCC microenvironment, but the underling mechanism is undetermined. In the present study, the potential function of midkine, a heparin-binding growth factor/cytokine, was evaluated in sorafenib-treated HCC tumors. Infiltrating immune cells of orthotopic HCC tumors were measured by flow cytometry. Differentially expressed genes in sorafenib-treated HCC tumors were evaluated by transcriptome RNA sequencing. The potential function of midkine were evaluated by western blot, T cell suppression assay, immunohistochemistry (IHC) staining and tumor xenograft model. We found that sorafenib treatment increased intratumoral hypoxia and altered HCC microenvironment towards an immune-resistant state in orthotopic HCC tumors. Sorafenib treatment promoted midkine expression and secretion by HCC cells. Moreover, forced midkine expression stimulated immunosuppressive myeloid-derived suppressor cells (MDSCs) accumulation in HCC microenvironment, while knockdown of midkine exhibited opposite effects. Furthermore, midkine overexpression promoted CD11b+CD33+HLA-DR− MDSCs expansion from human PBMCs, while midkine depletion suppressed this effect. PD-1 blockade showed no obvious inhibition on tumor growth of sorafenib-treated HCC tumors, but the inhibitory effect was greatly enhanced by midkine knockdown. Besides, midkine overexpression promoted multiple pathways activation and IL-10 production by MDSCs. Our data elucidated a novel role of midkine in the immunosuppressive microenvironment of sorafenib-treated HCC tumors. Mikdine might be a potential target for the combination of anti-PD-1 immunotherapy in HCC patients.

Protocadherin 20 promotes ferroptosis by suppressing the expression of Sirtuin 1 and promoting the acetylation of nuclear factor erythroid 2-related factor 2 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is characterized by a poor prognosis. Our previous work suggested that Protocadherin 20 (PCDH20) promoted ferroptosis in HCC. Nevertheless, the underlying mechanism remains elusive. Recently, we found that both the mRNA and protein levels of PCDH20 were upregulated in erastin- or sorafenib-treated HCC cells. Meanwhile, data showed that Sirtuin 1 (SIRT1) was markedly downregulated in PCDH20-SNU-449 cells. Additionally, overexpression of PCDH20 or erastin-treated cells dramatically decreased cell viability and colony-forming capacity of HCC cells, whereas blocking PCDH20 reversed these effects. Moreover, PCDH20 overexpression or treatment with erastin significantly downregulated the expression of SIRT1, Solute carrier family 7 member 11 (SLC7A11), as well as the ferroptosis-related protein glutathione peroxidase 4 (GPX4) and glutathione (GSH), while elevated malondialdehyde (MDA), 2'-7'-dichlorofluorescein (DCF) and intercellular iron levels. Conversely, knockdown of PCDH20 upregulated SIRT1 and SLC7A11. Immunoprecipitation assay demonstrated that PCDH20 or erastin increased the amount of acetylated nuclear factor erythroid 2-related factor-2 (NRF2). This reducing effect of NRF2 deacetylation by PCDH20 was counteracted by restoring the expression of SIRT1. In addition, PCDH20 lowered the levels of GPX4, GSH, and cell viability, as well as resulted in an elevation in intercellular iron level, MDA, and DCF. These effects were reversed by SIRT1 expression. Besides, PCDH20 could promote ferroptosis by inhibiting SIRT1 from deacetylating NRF2, which led to the downregulation of SLC7A11, GPX4, and GSH both in vivo and in vitro. Our results signals that PCDH20 promotes ferroptosis by suppressing the expression of SIRT1 and thus, promoting the acetylation of NRF2in HCC.

A novel polypeptide encoded by the circular RNA ZKSCAN1 suppresses HCC via degradation of mTOR

Backgroundhsa_circ_0001727 (circZKSCAN1) has been reported to be a tumor-associated circRNA by sponging microRNAs. Intriguingly, we found that circZKSCAN1 encoded a secretory peptide (circZKSaa) in the liver. The present study aims to elucidate the potential role and molecular mechanism of circZKSaa in the regulation of hepatocellular carcinoma (HCC) progression.MethodsThe circRNA profiling datasets (RNA-seq data GSE143233 and GSE140202) were reanalyzed and circZKSCAN1 was selected for further study. Mass spectrometry, polysome fractionation assay, dual-luciferase reporter, and a series of experiments showed that circZKSCAN1 encodes circZKSaa. Cell proliferation, apoptosis, and tumorigenesis in nude mice were examined to investigate the functions of circZKSaa. Mechanistically, the relationship between the circZKSaa and mTOR in HCC was verified by immunoprecipitation analyses, mass spectrometry, and immunofluorescence staining analyses.ResultsReceiver operating characteristic (ROC) analysis demonstrated that the secretory peptide circZKSaa encoded by circZKSCAN1 might be the potential biomarker for HCC tissues. Through a series of experiments, we found that circZKSaa inhibited HCC progression and sensitize HCC cells to sorafenib. Mechanistically, we found that the sponge function of circZKSCAN1 to microRNA is weak in HCC, while overexpression of circZKSaa promoted the interaction of FBXW7 with the mammalian target of rapamycin (mTOR) to promote the ubiquitination of mTOR, thereby inhibiting the PI3K/AKT/mTOR pathway. Furthermore, we found that the high expression of cicZKSCAN1 in sorafenib-treated HCC cells was regulated by QKI-5.ConclusionsThese results reveal that a novel circZKSCAN1-encoded peptide acts as a tumor suppressor on PI3K/AKT/mTOR pathway, and sensitizes HCC cells to sorafenib via ubiquitination of mTOR. These findings demonstrated that circZKSaa has the potential to serve as a therapeutic target and biomarker for HCC treatment.

Pembrolizumab as Second-Line Therapy for Advanced Hepatocellular Carcinoma: Longer Term Follow-Up from the Phase 3 KEYNOTE-240 Trial

Introduction: KEYNOTE-240 showed a favorable benefit/risk profile for pembrolizumab versus placebo in patients with sorafenib-treated advanced hepatocellular carcinoma (HCC); however, prespecified statistical significance criteria for overall survival (OS) and progression-free survival (PFS) superiority were not met at the final analysis. Outcomes based on an additional 18 months of follow-up are reported. Methods: Adults with sorafenib-treated advanced HCC were randomized 2:1 to pembrolizumab 200 mg intravenously every 3 weeks or placebo. Dual primary endpoints were OS and PFS assessed per RECIST v1.1 by blinded independent central review (BICR). Secondary endpoints included objective response rate (ORR), assessed per RECIST v1.1 by BICR, and safety. Results: 413 patients were randomized (pembrolizumab, n = 278; placebo, n = 135). As of July 13, 2020, median (range) time from randomization to data cutoff was 39.6 (31.7–48.8) months for pembrolizumab and 39.8 (31.7–47.8) months for placebo. Estimated OS rates (95% CI) were 17.7% (13.4–22.5%) for pembrolizumab and 11.7% (6.8–17.9%) for placebo at 36 months. The estimated PFS rate (95% CI) for pembrolizumab was 8.9% (5.3–13.6%) and 0% for placebo at 36 months. ORR (95% CI) was 18.3% (14.0–23.4%) for pembrolizumab and 4.4% (1.6–9.4%) for placebo. Immune-mediated hepatitis events did not increase with follow-up. No viral hepatitis flare events were reported. Conclusion: With extended follow-up, pembrolizumab continued to maintain improvement in OS and PFS and was associated with a consistent adverse event profile compared with placebo in patients with sorafenib-treated advanced HCC. Although KEYNOTE-240 did not meet prespecified statistical significance criteria at the final analysis, these results together with the antitumor activity of second-line pembrolizumab observed in KEYNOTE-224 and the statistically significant and clinically meaningful OS and PFS benefits of second-line pembrolizumab in patients from Asia observed in KEYNOTE-394 reinforce the clinical activity of pembrolizumab in previously treated patients with advanced HCC.

HMGCS2 Mediation of Ketone Levels Affects Sorafenib Treatment Efficacy in Liver Cancer Cells.

Primary liver cancer is the fifth leading death of cancers in men, and hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver cancer cases. Sorafenib is a first-line drug for advanced-stage HCC patients. Sorafenib is a multi-target kinase inhibitor that blocks tumor cell proliferation and angiogenesis. Despite sorafenib treatment extending survival, some patients experience side effects, and sorafenib resistance does occur. 3-Hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme for ketogenesis, which synthesizes the ketone bodies, β-hydroxybutyrate (β-HB) and acetoacetate (AcAc). β-HB is the most abundant ketone body which is present in a 4:1 ratio compared to AcAc. Recently, ketone body treatment was found to have therapeutic effects against many cancers by causing metabolic alternations and cancer cell apoptosis. Our previous publication showed that HMGCS2 downregulation-mediated ketone body reduction promoted HCC clinicopathological progression through regulating c-Myc/cyclin D1 and caspase-dependent signaling. However, whether HMGCS2-regulated ketone body production alters the sensitivity of human HCC to sorafenib treatment remains unclear. In this study, we showed that HMGCS2 downregulation enhanced the proliferative ability and attenuated the cytotoxic effects of sorafenib by activating expressions of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-P38, and p-AKT. In contrast, HMGCS2 overexpression decreased cell proliferation and enhanced the cytotoxic effects of sorafenib in HCC cells by inhibiting ERK activation. Furthermore, we showed that knockdown HMGCS2 exhibited the potential migratory ability, as well as decreasing zonula occludens protein (ZO)-1 and increasing c-Myc expression in both sorafenib-treated Huh7 and HepG2 cells. Although HMGCS2 overexpression did not alter the migratory effect, expressions of ZO-1, c-Myc, and N-cadherin decreased in sorafenib-treated HMGCS2-overexpressing HCC cells. Finally, we investigated whether ketone treatment influences sorafenib sensitivity. We showed that β-HB pretreatment decreased cell proliferation and enhanced antiproliferative effect of sorafenib in both Huh7 and HepG2 cells. In conclusion, this study defined the impacts of HMGCS2 expression and ketone body treatment on influencing the sorafenib sensitivity of liver cancer cells.

DDX24 regulates the chemosensitivity of hepatocellular carcinoma to sorafenib via mediating the expression of SNORA18

ABSTRACT Sorafenib (SFN) is a multi-kinase inhibitor drug for the treatment of advanced hepatocellular carcinoma (HCC), but its limited efficacy is a major obstacle to the clinical outcomes of patients with HCC. We aimed to explore a novel molecular mechanism underlying the chemosensitivity of HCC to SFN, and to identify a promising therapeutic target for HCC treatment. In this study, bioinformatic analysis revealed that DDX24 was associated with poor survival in HCC cases, and significantly related to the pathways modulating tumor development. DDX24 regulated HCC cell proliferation and migration potentials. Moreover, reduction of DDX24 promoted the sorafenib-mediated inhibition of HCC cell growth and migration, the elevation of sorafenib-induced HCC cell apoptosis. DDX24 overexpression suppressed the inhibitory effect of SFN on cell proliferation and migration and reduced the apoptosis induced by SFN. Further, DDX24, combined with SFN treatment, presented a synergistic enhancement of the sensitivity of SFN to the growth and migration of HCC cells via AKT/ERK and the epithelial-mesenchymal transition (EMT) pathways, and that it modulated apoptosis via the caspase/PARP pathway. Mechanistically, SNORA18 served as a target gene for DDX24, regulating the chemosensitivity of sorafenib-treated HCC cells. Furthermore, SNORA18 knockdown or overexpression could partially reverse the inhibition or elevation of cell viability, colony formation and migration induced by DDX24 in sorafenib-treated HCC cells, respectively. Collectively, our results suggest that DDX24 regulates the chemosensitivity of HCC to SFN by mediating the expression of SNORA18, which may act as an effective therapeutic target for improving SFN efficiency in HCC treatment.

CircFMN2 Boosts Sorafenib Resistance in Hepatocellular Carcinoma Cells via Upregulating CNBP by Restraining Ubiquitination.

Purpose Noncoding RNAs exert critical biological effects in hepatocellular carcinoma (HCC). The role of circFMN2, a newly discovered functional RNA in prostate cancer and colorectal cancer, was investigated for the first time in sorafenib-resistance HCC cells. Methods The level of circFMN2 was assessed via quantitative real-time PCR (qRT-PCR). Cell proliferation was detected via CCK-8 and colony formation assay. Cell apoptosis was measured via the TUNEL assay and flow cytometry analysis. A Western blot assay was conducted to detect the CCHC-type zinc finger nucleic acid binding protein (CNBP) level and ubiquitination. RNA pull-down assay and RNA immunoprecipitation were carried out to explore the interaction between circFMN2 and CNBP. Results CircFMN2 was highly expressed in multidrug-resistant (MDR) cells. CircFMN2 overexpression exerted pro-proliferation effects in sorafenib-treated HCC cells, while depletion of circFMN2 displayed negative effect on sorafenib-treated MDR cells. Moreover, CNBP was verified as the binding protein of circFMN2. CNBP was upregulated in MDR cells, which was achieved by inhibition of ubiquitination by circFMN2. Besides, CNBP overexpression was found to boost sorafenib resistance in HCC cells. Conclusions CircFMN2 is aberrantly expressed in sorafenib-resistant HCC cells and contributes to sorafenib resistance in HCC cells via upregulation of CNBP by restraining ubiquitination.

Child-Pugh Score and ABCG2-rs2231142 Genotype Independently Predict Survival in Advanced Hepatoma Patients Treated with Sorafenib.

Patients with advanced hepatocellular carcinoma (HCC) are treated by immunotherapy and/or targeted agents, such as sorafenib. Several single nucleotide polymorphisms (SNPs) and clinical scores have been proposed as prognostic markers in HCC patients treated with sorafenib. This study aimed to validate the prognostic values of these markers in a tertiary referral medical center. Two independent cohorts (cohort-1 [n = 97] and cohort-2 [n = 60]) of advanced HCC patients treated with sorafenib monotherapy were enrolled. Univariate followed by multivariate Cox proportional hazard analysis identified Child–Pugh (CP) score (p < 0.001) and renal insufficiency during treatment (p < 0.001) as independent predictors in cohort-1 patients. The same analytic method revealed ascites (p = 0.000), CP score (p = 0.001), infection during treatment (p < 0.001), and ATP-binding cassette subfamily G member 2 (ABCG2)-rs2231142 genotype (p = 0.003) as independent predictors in cohort-2 patients. ABCG2-rs2231142 genotype “CC” was associated with unfavorable overall survival in sorafenib-treated HCC patients. In conclusion, the CP score and ABCG2-rs2231142 genotype served as independent survival predictors for advanced HCC patients receiving sorafenib treatment.

Macropinocytosis is an alternative pathway of cysteine acquisition and mitigates sorafenib-induced ferroptosis in hepatocellular carcinoma

BackgroundMacropinocytosis, an important nutrient-scavenging pathway in certain cancer cells, allows cells to compensate for intracellular amino acid deficiency under nutrient-poor conditions. Ferroptosis caused by cysteine depletion plays a pivotal role in sorafenib responses during hepatocellular carcinoma (HCC) therapy. However, it is not known whether macropinocytosis functions as an alternative pathway to acquire cysteine in sorafenib-treated HCC, and whether it subsequently mitigates sorafenib-induced ferroptosis. This study aimed to investigate whether sorafenib drives macropinocytosis induction, and how macropinocytosis confers ferroptosis resistance on HCC cells.MethodsMacropinocytosis, both in HCC cells and HCC tissues, was evaluated by measuring TMR-dextran uptake or lysosomal degradation of DQ-BSA, and ferroptosis was evaluated via C11-BODIPY fluorescence and 4-HNE staining. Sorafenib-induced ferroptosis and macropinocytosis were validated in tumor tissues taken from HCC patients who underwent ultrasound-guided needle biopsy.ResultsSorafenib increased macropinocytosis in human HCC specimens and xenografted HCC tissues. Sorafenib-induced mitochondrial dysfunction was responsible for activation of PI3K-RAC1-PAK1 signaling, and amplified macropinocytosis in HCC. Importantly, macropinocytosis prevented sorafenib-induced ferroptosis by replenishing intracellular cysteine that was depleted by sorafenib treatment; this rendered HCC cells resistant to sorafenib. Finally, inhibition of macropinocytosis by amiloride markedly enhanced the anti-tumor effect of sorafenib, and sensitized resistant tumors to sorafenib.ConclusionIn summary, sorafenib induced macropinocytosis, which conferred drug resistance by mitigating sorafenib-induced ferroptosis. Thus, targeting macropinocytosis is a promising therapeutic strategy to facilitate ferroptosis-based therapy for HCC.Graphic

The hsa_circRNA_102049 mediates the sorafenib sensitivity of hepatocellular carcinoma cells by regulating Reelin gene expression

ABSTRACT A growing body of research has illuminated that non-coding RNAs (ncRNAs) plays an important role in the development of drug resistance in hepatocellular carcinoma (HCC) cells. The expression profiles of differential expressed genes (DEGs) and ncRNAs related to the sorafenib resistance in HCC cells were analyzed according to the Gene Expression Omnibus (GEO) dataSets and The Cancer Genome Atlas (TCGA) datasets. Bioinformatics technology was used to construct the interaction network of DEGs and ncRNAs. Cell transfection, dual-luciferase reporter assay, Western blot, cell counting kit-8 (CCK-8), flow cytometry and quantitative real-time polymerase chain reaction(qRT-PCR) were used to study the mechanism of sorafenib resistance in HepG2 cells and Huh-7 cells. The expression of reelin (RELN) and secretagogin (SCGN) were the only down-regulated in sorafenib-resistant HCC cells. The results showed that RELN gene demethylation reversed the cytotoxic of sorafenib on HepG2 cells and Huh-7 cells. Hsa_circRNA_102049 over-expression promoted the sensitivity of HepG2 cells and Huh-7 cells to sorafenib, hsa_circRNA_102049 up-regulated the expression of RELN gene by sponging hsa-miR-214-3p. The resistance to sorafenib in RELN knockout HepG2 cells and Huh-7 cells could be reverted by has-circRNA_102049. These findings support targeting of hsa_circRNA_102049 and RELN in sorafenib-treated HCC cells as a novel intervention, which is expected to overcome sorafenib resistance of HCC cells.

Epigenetic regulation of ferroptosis via ETS1/miR-23a-3p/ACSL4 axis mediates sorafenib resistance in human hepatocellular carcinoma

BackgroundDrug resistance to sorafenib greatly limited the benefits of treatment in patients with hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in the development of drug resistance. The key miRNA regulators related to the clinical outcome of sorafenib treatment and their molecular mechanisms remain to be identified.MethodsThe clinical significance of miRNA-related epigenetic changes in sorafenib-resistant HCC was evaluated by analyzing publicly available databases and in-house human HCC tissues. The biological functions of miR-23a-3p were investigated both in vitro and in vivo. Proteomics and bioinformatics analyses were conducted to identify the mechanisms that regulating miR-23a-3p. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to validate the binding relationship of miR-23a-3p and its targets.ResultsWe found that miR-23a-3p was the most prominent miRNA in HCC, which was overexpressed in sorafenib non-responders and indicated poor survival and HCC relapse. Sorafenib-resistant cells exhibited increased miR-23a-3p transcription in an ETS Proto-Oncogene 1 (ETS1)-dependent manner. CRISPR-Cas9 knockout of miR-23a-3p improved sorafenib response in HCC cells as well as orthotopic HCC tumours. Proteomics analysis suggested that sorafenib-induced ferroptosis was the key pathway suppressed by miR-23a-3p with reduced cellular iron accumulation and lipid peroxidation. MiR-23a-3p directly targeted the 3′-untranslated regions (UTR) of ACSL4, the key positive regulator of ferroptosis. The miR-23a-3p inhibitor rescued ACSL4 expression and induced ferrotoptic cell death in sorafenib-treated HCC cells. The co-delivery of ACSL4 siRNA and miR-23a-3p inhibitor abolished sorafenib response.ConclusionOur study demonstrates that ETS1/miR-23a-3p/ACSL4 axis contributes to sorafenib resistance in HCC through regulating ferroptosis. Our findings suggest that miR-23a-3p could be a potential target to improve sorafenib responsiveness in HCC patients.

Coupling HDAC4 with transcriptional factor MEF2D abrogates SPRY4-mediated suppression of ERK activation and elicits hepatocellular carcinoma drug resistance.

Hepatocellular carcinoma (HCC) lacks effective treatment, and the patients rapidly develop the acquired resistance to sorafenib with less defined mechanisms. Here, we demonstrate that transcriptional factor myocyte enhancer factor 2D (MEF2D) overexpression is detected in sorafenib-resistant HCC specimens and HCC cell lines and predicts poor prognosis of sorafenib-treated HCC patients. Mechanistically, MEF2D in complex with histone deacetylase HDAC4 directly binds to the SPRY4 promoter regions and suppresses the transcriptional expression of SPRY4, which is a negative regulator of MAPK/ERK signaling pathway. Inhibition of HDAC4 with its clinically used inhibitor induces SPRY4 expression and inhibition of ERK activity, resulting in sensitization of HCC cells to sorafenib-induced apoptosis and greatly improved inhibition of liver tumor growth in mice with sorafenib treatment. These findings highlight the critical role of coupling HDAC4 with MEF2D in activation of ERK by suppressing SPRY4 and underscore the great potential to improve HCC treatment by combined administration of sorafenib with HDAC4 inhibitors.