Sorafenib-resistant Hepatocellular Carcinoma Cells Research Articles

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.

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.

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

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. 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. 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. 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.

SPARC stabilizes ApoE to induce cholesterol-dependent invasion and sorafenib resistance in hepatocellular carcinoma.

Dysregulation of cholesterol homeostasis is implicated in the development and progression of hepatocellular carcinoma (HCC) that is characterized by intrahepatic and early extrahepatic metastasis. A better understanding of the underlying mechanisms regulating cholesterol metabolism in HCC could help identify strategies to circumvent the aggressive phenotype. Here, we found that high expression of intracellular SPARC was significantly associated with elevated cholesterol levels and an enhanced invasive phenotype in HCC. SPARC potentiated cholesterol accumulation in HCC cells during tumor progression by stabilizing the ApoE protein. Mechanistically, SPARC competitively bound to ApoE, impairing its interaction with the E3 ligase tripartite motif containing 21 (TRIM21) and preventing its ubiquitylation and subsequent degradation. ApoE accumulation led to cholesterol enrichment in HCC cells, stimulating PI3K-AKT signaling and inducing epithelial-mesenchymal transition (EMT). Importantly, sorafenib-resistant HCC cells were characterized by increased expression of intracellular SPARC, elevated cholesterol levels, and enhanced invasive capacity. Inhibiting SPARC expression or reducing cholesterol levels enhanced the sensitivity of HCC cells to sorafenib treatment. Together, these findings unveil interplay between SPARC and cholesterol homeostasis. Targeting SPARC-triggered cholesterol-dependent oncogenic signaling is a potential therapeutic strategy for advanced HCC.

STIM1 promotes acquired resistance to sorafenib by attenuating ferroptosis in hepatocellular carcinoma

Dysregulated calcium (Ca2+) signaling pathways are associated with tumor cell death and drug resistance. In non-excitable cells, such as hepatocellular carcinoma (HCC) cells, the primary pathway for Ca2+ influx is through stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE). Previous studies have demonstrated the involvement of STIM1-mediated SOCE in processes such as genesis, metastasis, and stem cell self-renewal of HCC. However, it remains unclear whether STIM1-mediated SOCE plays a role in developing acquired resistance to sorafenib in HCC patients. In this study, we established acquired sorafenib-resistant (SR) HCC cell lines by intermittently exposing them to increasing concentrations of sorafenib. Our results showed higher levels of STIM1 and stronger SOCE in SR cells compared with parental cells. Deleting STIM1 significantly enhanced sensitivity to sorafenib in SR cells, while overexpressing STIM1 promoted SR by activating SOCE. Mechanistically, STIM1 increased the transcription of SLC7A11 through the SOCE-CaN-NFAT pathway. Subsequently, up-regulated SLC7A11 increased glutathione synthesis, resulting in ferroptosis insensitivity and SR. Furthermore, combining the SOCE inhibitor SKF96365 with sorafenib significantly improved the sensitivity of SR cells to sorafenib both in vitro and in vivo. These findings suggest a potential strategy to overcome acquired resistance to sorafenib in HCC cells.

LINC00540 promotes sorafenib resistance and functions as a ceRNA for miR-4677-3p to regulate AKR1C2 in hepatocellular carcinoma

Sorafenib resistance is one of the main causes of poor prognosis in patients with advanced hepatocellular carcinoma (HCC). Long noncoding RNAs (lncRNAs) function as suppressors or oncogenic factors during tumor progression and drug resistance. Here, to identify therapeutic targets for HCC, the biological mechanisms of abnormally expressed lncRNAs were examined in sorafenib-resistant HCC cells. Specifically, we established sorafenib-resistant HCC cell lines (Huh7-S and SMMC7721-S), which displayed an epithelial-mesenchymal transition (EMT) phenotype. Transcriptome sequencing (RNA-Seq) was performed to established differential lncRNA expression profiles for sorafenib-resistant cells. Through this analysis, we identified LINC00540 as significantly up-regulated in sorafenib-resistant cells and a candidate lncRNA for further mechanistic investigation. Functionally, LINC00540 knockdown promoted sorafenib sensitivity and suppressed migration, invasion, EMT and the activation of PI3K/AKT signaling pathway in sorafenib-resistant HCC cells, whereas overexpression of LINC00540 resulted in the opposite effects in parental cells. LINC00540 functions as a competing endogenous RNA (ceRNA) by competitively binding to miR-4677-3p , thereby promoting AKR1C2 expression. This is the first study that demonstrates a role for LINC00540 in enhancing sorafenib resistance, migration and invasion of HCC cells through the LINC00540/miR-4677-3p/AKR1C2 axis, suggesting that LINC00540 may represent a potential therapeutic target and prognosis biomarker for HCC.

Glycyrrhizic acid attenuates sorafenib resistance by inducing ferroptosis via targeting mTOR signaling in hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) is the most malignant cancer worldwide. Sorafenib (SRF) is a common therapeutic drug used for patients with advanced HCC. Nevertheless, drug resistance frequently occurs in patients treated with sorafenib. Glycyrrhizic acid (GRA) is a natural compound that is identified to exhibit anti-cancer effects. In this work, we aimed to investigate the effects of GRA on SRF-resistant HCC cells and the potential regulatory mechanisms. Methods We established SRF-resistant HCC cell lines and administrated GRA treatment. We performed CCK-8 and colony formation experiments to detect cell proliferation. The accumulation of lipid reactive oxygen species (ROS) and iron levels were measured to evaluate ferroptosis. The protein levels of ferroptosis suppressor glutathione peroxidase 4 (GPX4) and SLC7A11, and the activation of AKT and mTOR were measured with western blotting assay. Results GRA treatment notably suppressed the viability and proliferation of SRF-resistant HCC cells. SRF-resistant HCC cells exhibited repressed ferroptosis level activated AKT/mTOR cascade, and GRA treatment reversed these effects. Inhibition of ferroptosis and activation of mTOR reversed the anti-proliferation effects of GRA on SRF-resistant HCC cells. Conclusion Treatment with GRA could effectively reverse the SRF resistance of HCC cells via inducing ferroptosis and inactivating the AKT/mTOR cascade.

Exosomal miR-93 derived from hepatocellular carcinoma cell promotes the sorafenib resistance of hepatocellular carcinoma through PTEN/PI3K/Akt pathway.

Exosomal microRNAs (miRNAs) derived from cancer cell is an important regulatory molecule that mediates the formation of tumor drug resistance, but function and mechanisms of exosomal miRNA in sorafenib resistance of hepatocellular carcinoma (HCC) have not been studied. We detected the level and prognosis of miR-93 in HCC by using TCGA HCC database. For confirming the extracted exosome, transmission electron microscopy was used. Cy3-labeled miR-93 and quantitative reverse transcription-polymerase chain reaction were used to prove that exosomal miR-93 derived from HCC cell can be transferred to sensitive HCC cells. CCK8, EdU, and flow cytometer assay were used to confirm the function of exosomal miR-93 in sorafenib resistance of HCC. Bioinformatics software and luciferase reporter assay was used to confirm the direct targeting relationship between PTEN and miR-93. Western blot was used to validate downstream pathways. We found that miR-93 is overexpressed and a prognostic risk factor for the HCC patients. miR-93 was overexpressed in sorafenib resistant HCC cells compared with sensitive cells, and miR-93 contributed to sorafenib resistance of HCC cells through targeting PTEN. miR-93 was enriched in exosomes that secreted from sorafenib resistant cells, and these exosomal miR-93 promote the spread of sorafenib resistant through targeting PTEN to reactivate PI3K/AKT pathway. Therefore, miR-93 can act as a potential therapeutic target for advanced patients with acquired sorafenib resistance.

CircSCMH1 Accelerates Sorafenib Resistance in Hepatocellular Carcinoma by Regulating HN1 Expression via miR-485-5p.

Sorafenib (SOR) is the first-line chemotherapeutic therapy for hepatocellular carcinoma (HCC) treatment, but SOR resistance is a key factor affecting the therapeutic effect. Emerging studies have suggested that circular RNAs (circRNAs) play an important role in the development of drug resistance in HCC cells. This paper aimed to elucidate the potential role and molecular mechanism of circRNA Scm polycomb group protein homolog 1 (circSCMH1) in SOR-resistant HCC cells. CircSCMH1, microRNA-485-5p (miR-485-5p), and hematological and neurological expressed 1 (HN1) contents were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK8) assay was adopted to detect the SOR sensitivity of cells. Cell proliferation, migration, invasion, and apoptosis were assessed using colony formation, 5-Ethynyl-2'-deoxyuridine (EdU), transwell, and flow cytometry assays. Glucose metabolism was analyzed using commercial kits. HN1, B cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) protein levels were assessed using western blot. Binding between miR-485-5p and circSCMH1 or HN1 was verified using a dual-luciferase reporter. Xenograft tumor model was used to explore the function of circSCMH1 in vivo. CircSCMH1 expression and HN1 abundances were increased, but the miR-485-5p level was reduced in SOR-resistant HCC tissues and cells. Deficiency of circSCMH1 enhanced SOR sensitivity by suppressing cell proliferation, migration, invasion, and glucose metabolism and inducing cell apoptosis in SOR-resistant HCC cell lines (Huh7/SOR and Hep3B/SOR). Mechanistically, circSCMH1 sponged miR-485-5p to positively regulate HN1 expression. Importantly, circSCMH1 depletion inhibited tumor growth and increased SOR sensitivity in vivo. CircSCMH1 promoted SOR resistance in HCC cells at least partly through upregulating HN1 expression by sponging miR-485-5p. These findings elucidated a new regulatory pathway of chemo-resistance in SOR-resistant HCC cells and provided a possible circRNA-targeted therapy for HCC.

Targeting C21orf58 is a Novel Treatment Strategy of Hepatocellular Carcinoma by Disrupting the Formation of JAK2/C21orf58/STAT3 Complex.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Functionally uncharacterized genes are an attractive repository to explore candidate oncogenes. It is demonstrated that C21orf58 displays an oncogenic role in promoting cell growth, tumorigenesis and sorafenib resistance of HCC cells by abnormal activation of STAT3 signaling. Mechanistically, a novel manner to regulate STAT3 signaling that adaptor C21orf58 forms a ternary complex is reveal with N-terminal domain of STAT3 and SH2 domain of JAK2, by which C21orf58 overactivates wild-type STAT3 by facilitating its phosphorylation mediated by JAK2, and hyper-activates of constitutively mutated STAT3 due to preferred binding with C21orf58 and JAK2. Moreover, it is validated that inhibition of C21orf58 with drug alminoprofen, selected by virtual screening, could effectively repress the viability and tumorigenesis of HCC cells. Therefore, it is identified that C21orf58 functions as an oncogenic adaptor, reveal a novel regulatory mechanism of JAK2/STAT3 signaling, explain the cause of abnormal activity of activated mutants of STAT3, and explore the attractive therapeutic potential by targeting C21orf58 in HCC.

Targeting AXL induces tumor-intrinsic immunogenic response in tyrosine kinase inhibitor-resistant liver cancer

Hepatocellular carcinoma (HCC) is an aggressive malignancy without effective therapeutic approaches. Here, we evaluate the tumor-intrinsic mechanisms that attenuate the efficacy of immune checkpoint inhibitor (ICI) that is observed in patients with advanced HCC who progress on first-line tyrosine kinase inhibitor (TKI) therapy. Upregulation of AXL observed in sorafenib- and lenvatinib-resistant HCCs is correlated with poor response towards TKI and ICI treatments. AXL upregulation protects sorafenib-resistant HCC cells from oxidative stress, mitochondrial damage, and accompanying immunogenic cell death through suppressed tumor necrosis factor-α (TNF-α) and STING-type I interferon pathways. Pharmacological inhibition of AXL abrogates the protective effect and re-sensitizes TKI-resistant HCC tumors to anti-PD-1 treatment. We suggest that targeting AXL in combination with anti-PD-1 may provide an alternative treatment scheme for HCC patients who progress on TKI treatment.

Exosomal circ_0032704 confers sorafenib resistance to hepatocellular carcinoma and contributes to cancer malignant progression by modulating the miR-514a-3p/PD-L1 pathway.

This study aims to explore the role of circ_0032704 in sorafenib-resistant hepatocellular carcinoma (HCC). The expression of circ_0032704, miR-514a-3p, and programmed death-ligand 1 (PD-L1) mRNA was detected by quantitative real-time PCR (qPCR). The expression of multidrug resistant-related proteins, migration/invasion-related proteins, exosome-related proteins, and PD-L1 protein was detected by western blot. Cell viability was detected by CCK-8 assay. Cell proliferation, migration, and invasion were assessed by EdU assay, wound healing assay, and transwell assay. The binding between miR-514a-3p and circ_0032704 or PD-L1 was verified by RIP assay, pull-down assay, and dual-luciferase reporter assay. Cell- or serum-derived exosomes were isolated and identified by TEM and NTA. Xenograft models were established to determine the effect of circ_0032704 on drug resistance invivo. Circ_0032704 was overexpressed in sorafenib-resistant HCC tissues and cells. Circ_0032704 knockdown reduced sorafenib resistance in HCC cells and inhibited cell proliferation, migration, and invasion of sorafenib-resistant HCC cells, while these effects were reversed by PD-L1 overexpression. We found that circ_0032704 positively regulated PD-L1 expression via targeting miR-514a-3p. Exosomes with circ_0032704 inhibition reduced sorafenib resistance in HCC cells and inhibited cell proliferation, migration, and invasion of sorafenib-resistant HCC cells. Exosomes with circ_0032704 inhibition also inhibited tumor growth invivo. The expression of circ_0032704 in exosomes was stable and possessed diagnostic value. Circ_0032704 enhanced sorafenib resistance in HCC and promoted the malignant development of sorafenib-resistant HCC. Circ_0032704 could be transported by exosomes, and exosomal circ_0032704 had diagnostic value.

Impact of glucose metabolism on PD-L1 expression in sorafenib-resistant hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related mortality worldwide. Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that binds to programmed cell death-1 (PD-1), which is expressed in activated T cells and other immune cells and has been employed in cancer therapy, including HCC. Recently, PD-L1 overexpression has been documented in treatment-resistant cancer cells. Sorafenib is a multikinase inhibitor and the only FDA-approved treatment for advanced HCC. However, several patients exhibit resistance to sorafenib during treatment. This study aimed to assess the effect of glucose deprivation on PD-L1 expression in HCC cells. We used PD-L1-overexpressing HepG2 cells and IFN-γ-treated SK-Hep1 cells to explore the impact of glycolysis on PD-L1 expression. To validate the correlation between PD-L1 expression and glycolysis, we analyzed data from The Cancer Genome Atlas (TCGA) and used immunostaining for HCC tissue analysis. Furthermore, to modulate PD-L1 expression, we treated HepG2, SK-Hep1, and sorafenib-resistant SK-Hep1R cells with rapamycin. Here, we found that glucose deprivation reduced PD-L1 expression in HCC cells. Additionally, TCGA data and immunostaining analyses confirmed a positive correlation between the expression of hexokinase II (HK2), which plays a key role in glucose metabolism, and PD-L1. Notably, rapamycin treatment  decreased the expression of PD-L1 and HK2 in both high PD-L1-expressing HCC cells and sorafenib-resistant cells. Our results suggest that the modulation of PD-L1 expression by glucose deprivation may represent a strategy to overcome PD-L1 upregulation in patients with sorafenib-resistant HCC.

FAT10 mediates the sorafenib-resistance of hepatocellular carcinoma cells by stabilizing E3 ligase NEDD4 to enhance PTEN/AKT pathway-induced autophagy.

Although sorafenib is the first-line therapeutic agent for advanced hepatocellular carcinoma (HCC), the development of drug resistance in HCC cells limits its clinical efficacy. However, the key factors involved in mediating the sorafenib resistance of HCC cells and the underlying mechanisms have not been elucidated. In this study, we generated sorafenib-resistant HCC cell lines, and our data demonstrate that HLA-F locus-adjacent transcript 10 (FAT10), a ubiquitin-like protein, is markedly upregulated in sorafenib-resistant HCC cells and that reducing the expression of FAT10 in sorafenib-resistant HCC cells increases sensitivity to sorafenib. Mechanistically, FAT10 stabilizes the expression of the PTEN-specific E3 ubiquitin ligase NEDD4 that causes downregulation of PTEN, thereby inducing AKT-mediated autophagy and promoting the resistance of HCC cells to sorafenib. Moreover, we screened the small molecule Compound 7695-0983, which increases the sensitivity of sorafenib-resistant HCC cells to sorafenib by inhibiting the expression of FAT10 to inhibit NEDD4-PTEN/AKT axis-mediated autophagy. Collectively, our preclinical findings identify FAT10 as a key factor in the sorafenib resistance of HCC cells and elucidate its underlying mechanism. This study provides new mechanistic insight for the exploitation of novel sorafenib-based tyrosine kinase inhibitor (TKI)-targeted drugs for treating advanced HCC.

Tumor microenvironment responsive nano-platform for overcoming sorafenib resistance of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a malignant tumor, which seriously jeopardizes human health. The 5-year relative survival rate of HCC is only about 18%. Sorafenib, a small molecule multi-targeted tyrosine kinase inhibitor (MTKI), has been classified as the first-line treatment scheme for HCC and has significantly extended the median survival time for patients with advanced HCC. Nevertheless, the emergence of sorafenib resistance has substantially hampered its further clinical application. Herein, the nano-platform based on phototherapy and small molecular targeted therapy (SMTT) was devised to overcome the sorafenib resistance and reduce the adverse effects. Hollow mesoporous manganese dioxide (H–MnO2) was prepared by hard template method, and the prepared H–MnO2 was used to load sorafenib and Chlorin e6 (Ce6). Subsequently, the nanoparticle (NPs) were modified with dopamine to optimize biocompatibility. The final prepared NPs (MCS NPs) exhibit regular spherical shape with a hydrated particle size of approximately 97.02 nm. MCS NPs can not only possess tumor microenvironment (TME) stimuli-responsive drug release performance but also can enhance the efficacy of photodynamic therapy and reverse sorafenib resistance by alleviating tumor hypoxia. Under the action of phototherapy (Ce6) combined with molecular targeted therapy (sorafenib), MCS NPs manifest a satisfactory antitumor effect for sorafenib-sensitive or sorafenib-resistant HCC cells, and retain the antiangiogenic properties of sorafenib. In the nude mouse subcutaneous tumor model constructed with sorafenib-resistant cells, MCS NPs demonstrated superior tumor imaging ability and excellent biocompatibility. The tumor inhibition rate of the MCS NPs group without laser irradiation was 53.4 %, while the MCS NPs group with laser irradiation was as high as 100 %. The novel smart TME-responsive nano-platform shows great potential for overcoming sorafenib resistance and realizes multimodality imaging and therapy of HCC.

Cancer-associated fibroblasts induce sorafenib resistance of hepatocellular carcinoma cells through CXCL12/FOLR1

BackgroundDue to the high drug resistance of hepatocellular carcinoma (HCC), sorafenib has limited efficacy in the treatment of advanced HCC. Cancer-associated fibroblasts (CAFs) play an important regulatory role in the induction of chemoresistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to sorafenib in HCC.MethodsImmunohistochemistry and immunofluorescence showed that the activation of CAFs was enhanced in HCC tissues. CAFs and paracancerous normal fibroblasts (NFs) were isolated from the cancer and paracancerous tissues of HCC, respectively. Cell cloning assays, ELISAs, and flow cytometry were used to detect whether CAFs induced sorafenib resistance in HCC cells via CXCL12. Western blotting and qPCR showed that CXCL12 induces sorafenib resistance in HCC cells by upregulating FOLR1. We investigated whether FOLR1 was the target molecule of CAFs regulating sorafenib resistance in HCC cells by querying gene expression data for human HCC specimens from the GEO database.ResultsHigh levels of activated CAFs were present in HCC tissues but not in paracancerous tissues. CAFs decreased the sensitivity of HCC cells to sorafenib. We found that CAFs secrete CXCL12, which upregulates FOLR1 in HCC cells to induce sorafenib resistance.ConclusionsCAFs induce sorafenib resistance in HCC cells through CXCL12/FOLR1.

Sorafenib Resistance Contributed by IL7 and MAL2 in Hepatocellular Carcinoma Can Be Overcome by Autophagy-Inducing Stapled Peptides.

Drug resistance poses a great challenge in systemic therapy for hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms associated with resistance to anti-cancer drugs, such as Sorafenib, remain unclear. In this study, we use transposon insertional mutagenesis to generate Sorafenib-resistant HCC cell lines in order to identify potential drug resistant causative genes. Interleukin 7 (IL7) and mal, T cell differentiation protein 2 (MAL2) were identified as candidate genes that promote survival by activating JAK/STAT and PI3K/AKT signaling pathways. Sorafenib-resistant cells exhibited higher clonogenic survival and lower drug sensitivity due to IL7 and MAL2 upregulation. Higher anti-apoptotic effect, clonogenic survival and increased PI3K/AKT/STAT3 activities were observed in IL7 and MAL2 co-overexpressing cells compared with controls or cells overexpressing IL7 or MAL2 individually. Given the critical role of MAL2 in endocytosis, we propose that MAL2 might facilitate the endocytic trafficking of IL7 and its cognate receptors to the plasma membrane, which leads to upregulated JAK/STAT and PI3K/AKT signaling pathways and Sorafenib resistance. Additionally, our previous studies showed that an autophagy-inducing stapled peptide promoted the endolysosomal degradation of c-MET oncogene and overcame adaptive Sorafenib resistance in c-MET+ HCC cells. In this study, we demonstrate that these stapled peptides readily induced autophagy and inhibited the proliferation of both wild-type and Sorafenib-resistant HCC cells co-overexpressing both IL7 and MAL2. Furthermore, these peptides showed synergistic cytotoxicity with Sorafenib in drug-resistant HCC cells co-overexpressing both IL7 and MAL2. Our studies suggest that targeting autophagy may be a novel strategy to overcome IL7/MAL2-mediated Sorafenib resistance in HCC.

Physcion increases the sensitivity of hepatocellular carcinoma to sorafenib through miRNA-370/PIM1 axis-regulated glycolysis.

Resistance to sorafenib has become a challenge in clinical treatment of hepatocellular carcinoma (HCC). Physcion is a common bioactive anthraquinone that has potential as an anticancer agent. To study the effect of physcion on sensitizing HCC cells to sorafenib. Sorafenib-resistant HCC cells were established and treated with sorafenib and/or physcion. The cell viability, proliferation and apoptosis were measured by cell counting kit-8, colony formation, flow cytometry, and in vivo xenograft model. Glucose uptake, lactate acid production, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) were measured to analyze glycolysis. Expression of glycolysis-related regulators was assessed by western blotting. The addition of physcion significantly enhanced the antitumor effects of sorafenib on sorafenib-resistant HCC cells, manifested by enhanced apoptosis and suppressed cell growth. The glucose uptake, lactate acid production, and ECAR were elevated, and OCR was suppressed by physcion treatment. The level of PIM1 was elevated and miR-370 was suppressed in sorafenib-resistant HCC cells compared with the parental cells, which was suppressed by physcion treatment. Inhibition of miR-370 notably reversed the effects of physcion on sorafenib-resistant HCC cells. Our data indicated that physcion enhanced the sensitivity of HCC cells to sorafenib by enhancing miR-370 to suppress PIM1-promoted glycolysis.

β-HB treatment reverses sorafenib resistance by shifting glycolysis–lactate metabolism in HCC

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor. Although sorafenib and regorafenib have been approved for first-line and second-line treatment, respectively, of patients with advanced HCC, long-term treatment often results in acquired resistance. Given that glycolysis-mediated lactate production can contribute to drug resistance and impair HCC treatment efficacy, we investigated the effects of ketone body treatment on the metabolic shift in sorafenib-resistant HCC cells. We discovered differential expression of 3-hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) and the ketone body D-β-hydroxybutyrate (β-HB) in four sorafenib-resistant HCC cell lines. In sorafenib-resistant HCC cells, lower HMGCS2 and β-HB levels were correlated with more glycolytic alterations and higher lactate production. β-HB treatment enhanced pyruvate dehydrogenase (PDH) expression and decreased lactate dehydrogenase (LDHA) expression and lactate production in sorafenib-resistant HCC cells. Additionally, β-HB combined with sorafenib or regorafenib promoted the antiproliferative and antimigratory abilities of sorafenib-resistant HCC cells by inhibiting the B-raf/mitogen-activated protein kinase pathway and mesenchymal N-cadherin-vimentin axis. Although the in vivo β-HB administration did not affect tumor growth, the expression of proliferative and glycolytic proteins was inhibited in subcutaneous sorafenib-resistant tumors. In conclusion, exogenous β-HB treatment can reduce lactate production and reverse sorafenib resistance by inducing a glycolytic shift; it can also synergize with regorafenib for treating sorafenib-resistant HCC.

Modelling the Sorafenib-resistant Liver Cancer Microenvironment by Using 3-D Spheroids.

Liver cancer is the third leading cause of cancer-related mortality, and hepatocellular carcinoma (HCC) is the most common form of liver cancer, and it usually occurs in the setting of chronic liver disease and cirrhosis. For patients with advanced HCC, systemic treatment is the first choice - however, resistance occurs frequently. Sorafenib was the first tyrosine kinase inhibitor approved for advanced HCC, and resistance to the therapy is a serious concern. When sorafenib therapy fails in a patient, it can be challenging to decide whether they can undergo a second-line therapy, and to determine which therapy they will be able to tolerate. Thus, physiologically relevant in vitro preclinical models are crucial for screening potential therapies, and 3-D tumour spheroids permit studies of tumour pathobiology. In this study, a drug-resistant 3-D tumour spheroid model was developed, based on sorafenib-resistant hepatocellular carcinoma cells, LX2 stellate cells and THP-1 monocytes. Model tumour spheroids that were formed with the sorafenib-resistant cells demonstrated lower diffusion of doxorubicin and exhibited increased resistance to regorafenib. Moreover, in the sorafenib-resistant spheroids, there was increased presence of CD68-positive cells and a reduction in inflammatory marker secretion. The sorafenib-resistant cell line-derived spheroids also showed a higher expression of FGF-19, PDGF-AA and GDF-15, which are known to be involved in malignancies. This multi-cell type spheroid model represents a potentially useful system to test drug candidates in a microenvironment that mimics the drug-resistant tumour microenvironment in HCC.