Sorafenib-resistant Hepatocellular Carcinoma Cell Lines Research Articles

Suppression of CTC1 inhibits hepatocellular carcinoma cell growth and enhances RHPS4 cytotoxicity.

Although DNA repair mechanisms function to maintain genomic integrity, in cancer cells these mechanisms may negatively affect treatment efficiency. The strategy of targeting cancer cells via inhibiting DNA damage repair has been successfully used in breast and ovarian cancer using PARP inhibitors. Unfortunately, such strategies have not yet yielded results in liver cancer. Hepatocellular carcinoma (HCC), the most common type of liver cancer, is a treatment-resistant malignancy. Despite the development of guided therapies, treatment regimens for advanced HCC patients still fall short of the current need and significant problems such as cancer relapse with resistance still exist. In this paper, we targeted telomeric replication protein CTC1, which is responsible for telomere maintenance. CTC expression was analyzed using tumor and matched-tissue RNA-sequencing data from TCGA and GTEx. In HCC cell lines, q-RT-PCR and Western blotting were used to detect CTC1 expression. The knock-down of CTC1 was achieved using lentiviral plasmids. The effects of CTC1 silencing on HCC cells were analyzed by flow cytometry, MTT, spheroid and colony formation assays. CTC1 is significantly downregulated in HCC tumor samples. However, CTC1 protein levels were higher in sorafenib-resistant cell lines compared to the parental groups. CTC1 inhibition reduced cell proliferation in sorafenib-resistant HCC cell lines and diminished their spheroid and colony forming capacities. Moreover, these cells were more sensitive to single and combined drug treatment with G4 stabilizer RHPS4 and sorafenib. Our results suggest that targeting CTC1 might be a viable option for combinational therapies designed for sorafenib resistant HCC patients.

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.

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.

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.

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.

Loss of LncRNA DUXAP8 synergistically enhanced sorafenib induced ferroptosis in hepatocellular carcinoma via SLC7A11 de-palmitoylation.

Ferroptosis is an important iron-dependent form of cell death in hepatocellular carcinoma (HCC). Sorafenib, a potent ferroptosis inducer, is used to treat advanced HCC but its efficacy is limited by the development of drug resistance. The effects of DUXAP8 expression on HCC progression were evaluated by TCGA database, Kaplan-Meier analysis, and in situ hybridization analysis. Sorafenib resistant HCC cell lines were modeled in vitro to study the regulation of DUXAP8 on ferroptosis in HCC induced by sorafenib. We used RNA pull-down, immunofluorescence assays, acyl-biotinyl exchange assay and mass spectrometry analysis to assess the molecular mechanism of ferroptosis regulation by DUXAP8. Syngeneic subcutaneous and orthotopic CDX models were used to assess whether DUXAP8 inhibition improves HCC in vivo. LncRNA DUXAP8, which is highly expressed in liver cancer and associated with poor prognosis, contributes to sorafenib resistance through suppression of ferroptosis. In vitro tests revealed that DUXAP8 reduced the sensitivity of HCC to sorafenib-induced ferroptosis by acting on SLC7A11, a subunit of the amino acid antiporter system xc-. DUXAP8 facilitates SLC7A11 palmitoylation and impedes its lysosomal degradation, thereby enhancing SLC7A11 action and suppressing ferroptosis. RNA pull-down and immunofluorescence assays confirmed that DUXAP8 decreased membrane translocation and promoted sorting of de-palmitoylated SLC7A11 to lysosomes by binding of DUXAP8 to SLC7A11. In addition, mass spectrometric analysis found that the Cys414 residue of SLC7A11 might be the predominant mutant site responsible for molecular masking of SLC7A11 lysosomal sorting. Further, the antitumor effect of DUXAP8 knockdown was verified in orthotopic and subcutaneous CDX models. Our findings suggest that a novel translational strategy combining sorafenib with DUXAP8 silencing to overcome drug resistance may improve treatment efficacy in patients with advanced HCC.

Abstract 4145: Frizzled-4 expression enhances hepatocellular carcinoma progression, sorafenib resistance and suppresses anti-tumor CD8 T cell immunity

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancers. Sorafenib remains the globally accepted systemic first-line treatment for advanced HCC although acquired drug-resistance is becoming increasingly common. Hopefully, immune-checkpoint inhibitor therapy has recently revolutionized the treatment for advanced HCC that have poor response to sorafenib, but the durable response rate remains low in most patients for unknown reasons. This fuels a wave of research into the molecular mechanisms of tumor-intrinsic resistance to both chemotherapy and immune checkpoint blockade (ICB), which may provide potential systemic treatment options for advanced HCC patients. Methods and Results: RNA sequencing of sorafenib-resistant HCC cell lines was performed to identify Frizzled-4 (FZD4) as a critical gene for drug resistance that was further validated in animal models. Clinical implication of FZD4 overexpression was studied in HCC patients. Using genetic and pharmacological approaches, we found that FZD4 was sufficient to increase clonogenicity, migration and invasion, and prevent sorafenib-induced apoptosis. In addition, FZD4 could reduce secretion of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), as well as intracellular levels of cytotoxic granzyme A and perforin in CD8+ T cells, indicating significant suppression of CD8+ T cell-mediated antitumor immunity in HCC tumors. In vivo studies also revealed that FZD4 expression promoted tumorigenesis and accelerated HCC cell metastatic lung colonization. Combination therapy of FZD4 inhibitor FzM1 with sorafenib in HCC of MYC/sgp53-induced model showed a tendency to significantly impede tumor growth and improve mice survival. Moreover, gene set enrichment analysis (GSEA) identified the cholesterol homeostasis and fatty acid metabolism signaling signatures as the top activated pathways in FZD4-high HCC cells. This evidence suggests a crucial role of FZD4-involved lipid metabolism linked to the development of therapeutic resistance. Conclusion: Our study demonstrates that FZD4 expression plays a pivotal role in HCC progression and holds the potential for expanding the scope of targeted/ICB therapies to tumors that are currently unresponsive, especially in advanced HCC. The resistance mechanisms will be addressed in further work. Citation Format: Jie Luo, Lanqi Gong, Yuma Yang, Qin Liu, Jiao Huang, Xiaona Fang, Xin-Yuan Guan. Frizzled-4 expression enhances hepatocellular carcinoma progression, sorafenib resistance and suppresses anti-tumor CD8 T cell immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4145.

FNDC5 Causes Resistance to Sorafenib by Activating the PI3K/Akt/Nrf2 Pathway in Hepatocellular Carcinoma Cells.

In this study, we aimed to reveal the resistance mechanism of hepatocellular carcinoma (HCC) cells to sorafenib by exploring the effect of FNDC5 on sorafenib-induced ferroptosis in HCC cells. We compared the expression level of FNDC5 between sorafenib-resistant and sorafenib-sensitive HCC cell lines and the level of ferroptosis between the groups after treatment with sorafenib. We knocked down FNDC5 in drug-resistant cell lines and overexpressed it in sorafenib-sensitive HCC cell lines to further demonstrate the role of FNDC5 in sorafenib-induced ferroptosis. Using PI3K inhibitors, we revealed the specific mechanism by which FNDC5 functions. In addition, we verified our findings obtained in in vitro experiments using a subcutaneous tumorigenic nude mouse model. The findings revealed that FNDC5 inhibits sorafenib-induced ferroptosis in HCC cells. In addition, FNDC5 activated the PI3K/Akt pathway, which in turn promoted the nuclear translocation of Nrf2 and increased the intracellular antioxidant response, thereby conferring resistance to ferroptosis. Our study provides novel insights for improving the efficacy of sorafenib.

Low miR-10b-3p associated with sorafenib resistance in hepatocellular carcinoma.

Sorafenib is one of the standard first-line therapies for advanced hepatocellular carcinoma (HCC). Unfortunately, there are currently no appropriate biomarkers to predict the clinical efficacy of sorafenib in HCC patients. MicroRNAs (miRNAs) have been studied for their biological functions and clinical applications in human cancers. In this study, we found that miR-10b-3p expression was suppressed in sorafenib-resistant HCC cell lines through miRNA microarray analysis. Sorafenib-induced apoptosis in HCC cells was significantly enhanced by miR-10b-3p overexpression and partially abrogated by miR-10b-3p depletion. Among 45 patients who received sorafenib for advanced HCC, those with high miR-10b-3p levels, compared to those with low levels, exhibited significantly longer overall survival (OS) (median, 13.9 vs. 3.5 months, p = 0.021), suggesting that high serum miR-10b-3p level in patients treated with sorafenib for advanced HCC serves as a biomarker for predicting sorafenib efficacy. Furthermore, we confirmed that cyclin E1, a known promoter of sorafenib resistance reported by our previous study, is the downstream target for miR-10b-3p in HCC cells. This study not only identified the molecular target for miR-10b-3p, but also provided evidence that circulating miR-10b-3p may be used as a biomarker for predicting sorafenib sensitivity in patients with HCC.

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.

MiR-125b-5p Is Involved in Sorafenib Resistance through Ataxin-1-Mediated Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

Simple SummaryThe mechanism of resistance to multikinase inhibitors in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the responsible miRNAs and target genes involved in the mechanism of resistance. Four miRNAs were significantly upregulated. Among them, we found that miR-125-5p induced sorafenib resistance in HCC cells and in a mouse model. We also revealed that miR-125-5p suppressed ataxin-1 as a target gene and consequently induced Snail-mediated epithelial-mesenchymal transition (EMT) and cancer stemness. Moreover, we demonstrated that ataxin-1 expression has an impact on the prognosis of patients with HCCs. In the future, by comparing the expression status of miR-125b-5p/ataxin-1 and the effect of sorafenib in the clinical setting, it is expected that miR-125b-5p will be established as an effective drug selection marker for treatment selection in patients with HCC.The mechanism of resistance to sorafenib in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the miRNAs responsible for resistance. Drug sensitivity, migration/invasion capabilities, and epithelial-mesenchymal transition (EMT) properties were analyzed by biochemical methods. The clinical relevance of the target genes to survival in HCC patients were assessed using a public database. Four miRNAs were significantly upregulated in PLC/PRF5-R1/-R2 compared with PLC/PRF5. Among them, miR-125b-5p mimic-transfected PLC/PRF5 cells (PLC/PRF5-miR125b) and showed a significantly higher IC50 for sorafenib compared with controls, while the other miRNA mimics did not. PLC/PRF5-miR125b showed lower E-cadherin and higher Snail and vimentin expression—findings similar to those for PLC/PRF5-R2—which suggests the induction of EMT in those cells. PLC/PRF5-miR125b exhibited significantly higher migration and invasion capabilities and induced sorafenib resistance in an in vivo mouse model. Bioinformatic analysis revealed ataxin-1 as a target gene of miR-125b-5p. PLC/PRF5 cells transfected with ataxin-1 siRNA showed a significantly higher IC50, higher migration/invasion capability, higher cancer stem cell population, and an EMT phenotype. Median overall survival in the low-ataxin-1 patient group was significantly shorter than in the high-ataxin-1 group. In conclusion, miR-125b-5p suppressed ataxin-1 and consequently induced Snail-mediated EMT and stemness, leading to a poor prognosis in HCC patients.

MicroRNA-138-1-3p sensitizes sorafenib to hepatocellular carcinoma by targeting PAK5 mediated \u03b2-catenin/ABCB1 signaling pathway

BackgroundSorafenib is a kinase inhibitor that is used as a first-line therapy in advanced hepatocellular carcinoma (HCC) patients. However, the existence of sorafenib resistance has limited its therapeutic effect. Through RNA sequencing, we demonstrated that miR-138-1-3p was downregulated in sorafenib resistant HCC cell lines. This study aimed to investigate the role of miR-138-1-3p in sorafenib resistance of HCC.MethodsIn this study, quantitative real-time PCR (qPCR) and Western Blot were utilized to detect the levels of PAK5 in sorafenib-resistant HCC cells and parental cells. The biological functions of miR-138-1-3p and PAK5 in sorafenib-resistant cells and their parental cells were explored by cell viability assays and flow cytometric analyses. The mechanisms for the involvement of PAK5 were examined via co-immunoprecipitation (co-IP), immunofluorescence, dual luciferase reporter assay and chromatin immunoprecipitation (ChIP). The effects of miR-138-1-3p and PAK5 on HCC sorafenib resistant characteristics were investigated by a xenotransplantation model.ResultsWe detected significant down-regulation of miR-138-1-3p and up-regulation of PAK5 in sorafenib-resistance HCC cell lines. Mechanistic studies revealed that miR-138-1-3p reduced the protein expression of PAK5 by directly targeting the 3′-UTR of PAK5 mRNA. In addition, we verified that PAK5 enhanced the phosphorylation and nuclear translocation of β-catenin that increased the transcriptional activity of a multidrug resistance protein ABCB1.ConclusionsPAK5 contributed to the sorafenib resistant characteristics of HCC via β-catenin/ABCB1 signaling pathway. Our findings identified the correlation between miR-138-1-3p and PAK5 and the molecular mechanisms of PAK5-mediated sorafenib resistance in HCC, which provided a potential therapeutic target in advanced HCC patients.

Abstract 1419: Small molecule inhibitor of NRF2 selectively overcomes sorafenib resistance in KEAP1-deficient hepatocellular carcinoma

Abstract Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. A recent randomized phase III trial of combined immunotherapy (atezolizumab) with antiangiogenesis (bevacizumab) versus the standard sorafenib monotherapy reported an unprecedented response rate of 27%, leading to FDA approval of this combination as the new standard treatment in May 2020. Yet, despite this undisputable progress, the majority of the HCC cases are likely to recur or develop resistance to both first line therapy. In this study, we aimed to identify novel, druggable candidate genes for patients with sorafenib resistant HCC. Method: CRISPR/Cas9 genome (GeCKO v2) library screening was used to identify loss of function mutations conferring sorafenib resistance upon HCC cells. CCK-8 and clonogenic assays were used to measure cell viability and cytotoxicity. Flow cytometry was performed to analyze cellular reactive oxidative species (ROS) and apoptosis level. Multiple animal models were used to study the synergistic efficacy of the combination of nuclear factor erythroid 2- related factor 2 (NRF2) inhibition and sorafenib. Results: In our study, we explored the mechanisms underlying sensitivity of HCC cells to sorafenib using genome-wide CRISPR-Cas9 screening. Kelch-like ECH- associated protein-1(KAEP1) was discovered as a previously unknown factor for determining the sensitivity of HCC cells to sorafenib. Using established sorafenib resistant HCC cell lines and the parental cell, we validated that KEAP1 mediates NRF2 degradation through the ubiquitin-proteasome pathway, thereby conferring sensitivity to sorafenib. Conversely, the loss of function of KEAP1 reduced degradation of NRF2 and conferred increased sorafenib resistance. Analysis of 55 patients with HCC, who had received hepatic resection followed by postoperative standard sorafenib treatment, revealed that high NRF2 expression shortened both overall survival (P<0.05) and disease free survival (P<0.01). CCK-8 and clonogenic assays were used in combination with sorafenib revealed that NRF2 inhibitor substantially enhances cytotoxicity and leads to robust proliferation inhibition of sorafenib resistant HCC cells, as compared with single agents. In both cell derived xenograft (CDX) and patient-derived xenograft (PDX) HCC models with preserved NRF2 function, NRF2 inhibitor, in combination with sorafenib, showed significant antitumor activity (P<0.05). Furthermore, the combination of NRF2 inhibitor and sorafenib shows striking synergy by increasing ROS and inducing apoptosis in HCC cells. Conclusion: Our results uncovered the potential of NRF2 as a drug target for patients with sorafenib resistant HCC. The impressive efficacy of NRF2 inhibitor and sorafenib suggests a potential combination therapy to supplement the existing standard of treatment for sorafenib resistant HCC. Citation Format: Jiang Chen, Shi Jiang, Bixia Li, Tong Ji, Staiculescu Daniel, Jiayan He, Junjie Xu, Xiujun Cai. Small molecule inhibitor of NRF2 selectively overcomes sorafenib resistance in KEAP1-deficient hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1419.

Upregulation of PD-L1 expression promotes epithelial-to-mesenchymal transition in sorafenib-resistant hepatocellular carcinoma cells.

BackgroundThe epithelial-to-mesenchymal transition (EMT) status is associated with programmed death-1 ligand 1 (PD-L1) expression in various cancers. However, the role and molecular mechanism of PD-L1 in the EMT of sorafenib-resistant hepatocellular carcinoma (HCC) cells remain elusive. In this study, we aimed to investigate the regulation of PD-L1 on the EMT in sorafenib-resistant HCC cells.MethodsInitially, the sorafenib-resistant HCC cell lines HepG2 SR and Huh7 SR were established. Western-blot assays were used to detect the expression of PD-L1, E-cadherin, and N-cadherin. The intervention and overexpression of PD-L1 were used to explore the role of PD-L1 in the regulation of EMT in HepG2 SR and Huh7 SR cells. Cell migration and invasion were assessed by transwell assays. PD-L1 or Sterol regulatory element-binding protein 1 (SREBP-1) overexpression and knock-down were performed in order to study the mechanism of PD-L1 in sorafenib-resistant HCC cells.ResultsPD-L1 expression was upregulated, whereas E-cadherin levels were downregulated and N-cadherin expression was increased in HepG2 SR and Huh7 SR cells. The cell viabilities of HepG2 and Huh7 cells were lower than those of HepG2 SR and Huh7 SR cells. PD-L1 overexpression reduced E-cadherin expression and increased N-cadherin levels, whereas PD-L1 knock-down increased E-cadherin expression and decreased N-cadherin expression. PD-L1 expression promoted EMT and the migratory and invasive abilities of HepG2 SR and Huh7 SR cells. PD-L1 promoted the EMT of sorafenib-resistant HCC cells via the PI3K/Akt pathway by activating SREBP-1 expression in HepG2 SR and Huh7 SR cells.ConclusionsThe findings reveal that PD-L1 expression promotes EMT of sorafenib-resistant HCC cells.

LncRNA HEIH Confers Cell Sorafenib Resistance in Hepatocellular Carcinoma by Regulating miR-98-5p/PI3K/AKT Pathway.

BackgroundThe hepatocellular carcinoma up-regulated EZH2-associated long non-coding RNA (HEIH) has been identified to act as an oncogene to promote cell tumorigenesis in hepatocellular carcinoma (HCC); however, the roles of HEIH in sorafenib resistance in HCC cells remain elusive.Materials and MethodsThe expression of HEIH and microRNA (miR)-98-5p was detected using quantitative real-time polymerase chain reaction. Cell viability, apoptosis, migration and invasion were analyzed using cell counting kit-8 assay, flow cytometry and transwell assay. Western blot was used to measure the levels of apoptosis-related protein and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related protein. The interaction between HEIH and miR-98-5p was confirmed by dual-luciferase reporter and RNA immunoprecipitation assay. In vivo experiments were performed using murine xenograft models.ResultsHEIH was up-regulated in sorafenib-resistant HCC tissues and cell lines, and HEIH silence weakened sorafenib resistance by suppressing cell viability, invasion and migration, decreasing the IC50 values to sorafenib, and increasing apoptosis in sorafenib-resistant HCC cells in vitro and reinforced the anti-tumor effects of sorafenib in vivo. HEIH was a sponge of miR-98-5p, and miR-98-5p inhibition reversed the sorafenib sensitivity induced by HEIH deletion in sorafenib-resistant HCC cells. MiR-98-5p inhibition could activate PI3K/AKT pathway, and enhanced sorafenib resistance by regulating the activation of PI3K/AKT pathway in sorafenib-resistant HCC cells. Besides, HEIH also activated PI3K/AKT pathway through regulating miR-98-5p in sorafenib-resistant HCC cells.ConclusionHEIH conferred an advantage to sorafenib resistance in HCC by the activation of PI3K/AKT pathway through miR-98-5p, indicating a potential therapeutic strategy for HCC chemotherapy.

Identification and validation of potential key long noncoding RNAs in sorafenib-resistant hepatocellular carcinoma cells.

As the first-line treatment, sorafenib has been used for advanced hepatocellular carcinoma (HCC), but the chemoresistance commonly restricts to the clinical efficiency. In this study, we intend to investigate the genome-wide expression pattern of long noncoding RNAs (lncRNAs) in sorafenib-resistant HCC. Herein, we identified thousands of differentially expressed lncRNAs in sorafenib-resistant HCC cells by high-throughput sequencing compared to the parental. Besides, based on GO (Gene Ontology) term enrichment analysis, these differentially expressed lncRNAs are mainly related to binding and catalytic activity and biological regulation of metabolic processes in both the sorafenib-resistant Huh7 cells (Huh7-S) and sorafenib-resistant HepG2 cells (HepG2-S) compared to the parental cells. Moreover, when analyzed by KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, the differentially expressed genes were significantly related to the tight junction. Among them, the expression of TCONS_00284048 and TCONS_00006019 was consistently up-regulated in sorafenib-resistant HCC cell lines, whereas when either was knocked down, the sensitivity of Huh7-S and HepG2-S cells to sorafenib was increased. Taken together, our data demonstrate that the lncRNA expression profile is significantly altered in sorafenib-resistant HCC cells as well as differentially expressed lncRNAs may play crucial functions on HCC sorafenib resistance and HCC progression.

LncRNA NEAT1 modulates sorafenib resistance in hepatocellular carcinoma through regulating the miR-149-5p/AKT1 axis.

Background/Aims:The purpose of this study is to explore the expression characteristics of lncRNA NEAT1 in hepatocellular carcinoma (HCC) and the molecular mechanism of its regulation on sorafenib resistance.Materials and Methods:This experimental study was performed from June 2013 to June 2019. The level of NEAT1 was determined using RT-PCR in HCC and matched adjacent tissues from 79 HCC patients in Linyi central hospital. The patients were divided into two groups to compare their prognosis based on the median NEAT1 expressions as a cutoff value. HCC cell line HepG2 negative control (HepG2-NC), sorafenib-resistant HepG2 cells (HepG2-SR) were transfected with or without NEAT1 siRNA, followed by subsequent molecular analysis, to determine the function of NEAT1 on sorafenib resistance in HCC cells. The cell transcripts were determined by RNA-sequencing analysis. The binding site of the NEAT1 and microRNA-149-5p (miR-149-5p) was verified by luciferase assay.Results:We found that NEAT1 was significantly increased in HCC tissues. Furthermore, NEAT1 expressions were significantly associated with HCC prognosis and chemoresistance patterns against sorafenib. Subsequently, the sorafenib-resistant HCC cell lines, together with the controls, were used to determine the regulatory effect of NEAT1 on HCC cells' progression and sorafenib resistance. NEAT1 targets the miR-149-5p, and therefore, decrease the activity of sorafenib against HCC cells. NEAT1 functions were demonstrated to be triggered by the regulation of miR-149-5p/AKT1 axis.Conclusions:NEAT1/miR-149-5p/AKT1 pathway-based therapy might be a potential clinical application for HCC patients.

LXR activation potentiates sorafenib sensitivity in HCC by activating microRNA-378a transcription.

Sorafenib resistance is a major obstacle to the treatment of advanced hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) are multifunctional regulators of gene expression with profound impact for human disease. Therefore, better understanding of the biological mechanisms of abnormally expressed miRNAs is critical to discovering novel, promising therapeutic targets for HCC treatment. This study aimed to investigate the role of miR-378a-3p in the sorafenib resistance of HCC and elucidate the underlying molecular mechanisms.Methods: A novel hub miR-378a-3p was identified based on miRNA microarray and bioinformatics analysis. The abnormal expression of miR-378-3p was validated in different HCC patient cohorts and sorafenib-resistant (SR) HCC cell lines. The functional role of miR-378a-3p and its downstream and upstream regulatory machinery were investigated by gain-of-function and loss-of-function assays in vitro and in vivo. Interactions among miR-378a-3p, LXRα, and IGF1R were examined by a series of molecular biology experiments. Then, the clinical relevance of miR-378a-3p and its targets were evaluated in HCC samples. HCC patient-derived xenograft (PDX) model was used to assess the therapeutic value of LXRα and its downstream miR-378a-3p.Results: miR-378a-3p expression was frequently reduced in established sorafenib-resistant HCC cell lines. The decreased miR-378a-3p levels correlated with poor overall survival of HCC patients following sorafenib treatment. miR-378a-3p overexpression induced apoptosis in SR HCC cells, whereas miR-378a-3p silencing exerted the opposite effects. IGF1R was identified as a novel target of miR-378a-3p. Furthermore, the primary miR-378 level was not consistent with its precursor miRNA level in SR HCC cells, which was attributed to the downregulation of exportin5 (XPO5) and subsequently reduced nuclear export of precursor miR-378 and restrained maturation of miR-378-3p. In this context, we combined an agonist GW3965 of liver X receptor alpha (LXRα), which functioned as a transcription activator of miRNA-378a, and its activation re-sensitized sorafenib-resistant cells to sorafenib treatment in vitro and in vivo.Conclusions: Our finding suggested decreased expression of XPO5 prevents maturation of miR-378a-3p, which leaded to the overexpression of IGF-1R and counteracted the effects of sorafenib-induced apoptosis. LXRα was able to activate miRNA-378a-3p transcription in HCC cells and could be a potential combinable treatment strategy with sorafenib to suppress HCC progression.

Predictors of ribociclib-mediated antitumour effects in native and sorafenib-resistant human hepatocellular carcinoma cells.

The cyclin-dependent kinases (CDKs) CDK4 and CDK6 are important regulators of the cell cycle and represent promising targets in cancer treatment. We aimed to investigate the relevance of CDK4/6 in the development of hepatocellular carcinoma (HCC) and the potential of ribociclib, a novel orally available CDK4/6 inhibitor, as a treatment for HCC. The effect of ribociclib was assessed in native and sorafenib-resistant HCC cell lines using viability assays, colony formation assays and FACS-based analyses. The expression of potential biomarkers of ribociclib response was assessed in cell lines and primary human hepatocytes using Western blotting. In addition, the prognostic relevance of the cyclin D-CDK4/6-retinoblastoma protein (Rb) pathway was assessed by analysing mRNA expression data from The Cancer Genome Atlas (TCGA). We found that ribociclib downregulated Rb and caused a profound loss of cell viability by inducing G1 cell cycle arrest in HCC cell lines exhibiting Rb-high/p16-low protein expression profiles, but not in Rb-low/p16-high cells, regardless their sensitivity to sorafenib. siRNA-based Rb silencing decreased cell proliferation, but did not diminish the sensitivity of HCC cells to ribociclib. Furthermore, we found that ribociclib synergized with sorafenib to cause cell death. mRNA analysis of primary human HCC specimens showed that CDK4 expression was correlated with patient survival and that the expression of Rb and the p16-encoding CDKN2A gene were inversely correlated. From our data we conclude that impairment of the cyclin D-CDK4/6-Rb pathway is a frequent feature of HCC and that it is associated with a unfavourable prognosis. We also found that ribociclib exhibits a preferential antineoplastic activity in Rb-high HCC cells. Our results warrant further investigation of Rb and p16 expression as markers of HCC sensitivity to ribociclib.