Homeobox B13 activates the hypoxia-inducible factor 1 pathway through histone lactylation thereby reprogramming lipid metabolism and promoting sorafenib resistance in hepatocellular carcinoma.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the 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.

Simple SummaryHepatocellular carcinoma (HCC) is one of the major causes of cancer-associated death worldwide. Development of sorafenib resistance presents a major failure of HCC therapy. We applied a combination therapy approach of hydroxychloroquine (HCQ)–sorafenib, both in vitro and in vivo, with data demonstrating the synergistic effect of HCQ in modulating the expression of toll-like receptor (TLR)-9 and regulating the cancer cells stemness, mesenchymal state, autophagy, and sorafenib resistance through inducing the antioxidant superoxide dismutase (SOD)-1 and apoptosis-allied gene expression and reducing the oxidative DNA damage stress in HCC sorafenib-resistant cells via hsa-miR-30a-5p epigenetic regulation axis. Thus, the combination of HCQ–sorafenib provides a suitable approach in improving therapeutic outcomes of sorafenib-resistant HCC patients.Sorafenib is used for treating advanced hepatocellular carcinoma (HCC), but some patients acquire sorafenib resistance. We investigated the mechanisms underlying acquired sorafenib resistance in HCC cells and targeted them to re-sensitize them to sorafenib. In silico analysis indicated that toll-like receptor (TLR)-9 was significantly overexpressed, and that miRNA (hsa-miR-30a-5p) was downregulated in sorafenib-resistant HCC cells, which modulated HCC cell proliferation, oxidative stress, and apoptosis. TLR9 overexpression increased HCC cell proliferation, whereas TLR9 inhibition from hydroxychloroquine (HCQ) decreased HCC cell proliferation, tumor growth, oxidative stress marker (SOD1), and the formation of autophagosome bodies (reduced ATG5 and Beclin-1 expression). Moreover, HCQ treatment reduced epithelial–mesenchymal transition, leading to decreased clonogenicity, migratory ability, and invasiveness. HCQ targeted and reduced the self-renewal capacity phenotype by inhibiting tumorsphere generation. Both in vitro and in vivo results demonstrated the synergistic effect of the HCQ–sorafenib combination on sorafenib-resistant HCC (Huh7-SR) cells, increasing their sensitivity to treatment by modulating TLR9, autophagy (ATG5 and Beclin-1), oxidative stress (SOD1), and apoptosis (c-caspase3) expression and thus overcoming the drug resistance. This study’s findings indicate that TLR9 overexpression occurs in sorafenib-resistant HCC cells and that its downregulation aids HCC suppression. Moreover, HCQ treatment significantly increases sorafenib’s effect on sorafenib-resistant HCC cells.

BackgroundSorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma (HCC). However, resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib. As the activation of AKT/mTOR played a pivotal role in sorafenib resistance, we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells. MethodsThe sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines. The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method. Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting. ResultsSorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells, which underscores the significance of AKT activation. Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction. Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis, leading to the dephosphorylation of Ser136 in BAD protein, and increased the expression of total BAD, which contributed to the apoptosis in sorafenib-resistant HCC cells. ConclusionsIn this study, Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenib-resistant HCC cells, via the suppression of mTORC2-AKT-BAD pathway. Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.

Sorafenib is a small molecule that blocks tumor proliferation by targeting the activity of multi-kinases for the treatment of advanced hepatocellular carcinoma (HCC). Increasing sorafenib resistance following long-term treatment is frequently encountered. Mechanisms underlying sorafenib resistance remain not completely clear. To further understand the mechanism of sorafenib resistance in HCC, we established sorafenib-resistant cell lines by slowly increasing sorafenib concentration in cell culture medium. Upregulation of USP22 and ABCC1 were found in Sorafenib-resistant cells. Sorafenib-resistant cells treated with USP22 siRNA showed significant reduction in endogenous mRNA and protein levels of ABCC1. During sorafenib treatment, upregulation of USP22 increases ABCC1 expression and subsequently contributes to sorafenib resistance in HCC cells. Immunohistochemical analysis revealed a positive correlation between USP22 and ABCC1 expression in tissue samples from sorafenib-resistant patients (Pearson’s correlation = 0.59, p = 0.03). Our findings indicate that upregulation of USP22 and ABCC1 expression during treatment contribute to sorafenib resistance in HCC cells and that USP22 has strong potential as a therapeutic target for overcoming sorafenib resistance in HCC patients.

TRERNA1 upregulation mediated by HBx promotes sorafenib resistance and cell proliferation in HCC via targeting NRAS by sponging miR-22-3p

Sorafenib, an orally active potent tyrosine kinase inhibitor (TKI), represented a primary treatment in patients with advanced hepatocellular carcinoma (HCC). Unfortunately, sorafenib resistance was regarded as a huge obstacle for HCC treatment. RNA-sequencing including circRNA Sequencing (circRNA-Seq) for circular RNAs (circRNAs), miRNA Sequencing (miRNA-Seq) for microRNAs (miRNAs), as well as mRNA Sequencing (mRNA-Seq) for mRNAs in sorafenib-resistant HCC cells vs sorafenib-sensitive HCC cells, were performed. Then, interaction correlation analysis between differentially expressed circRNAs and miRNAs and their target genes in Huh7/SOR and SMMC7721/SOR cells was exhibited. The "circRNA-miRNA-mRNA" network was constructed through the Cytoscape software application, Circular RNA Interactome and Targetscan prediction, RNA binding protein immunoprecipitation (RIP), RNA pull-down, and Dual luciferase reporter assay. Furthermore, mRNA-Seq, Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for the downstream genes involved in the "circRNA-miRNA-mRNA" network was implemented. Iron detection assay, Lipid peroxidation quantification assay, ROS measurement assay, CCK-8 assay, and tumor challenge in vivo were used to determine the mechanisms promoting sorafenib resistance in HCC, where the "circRNA-miRNA-mRNA" network is clearly involved in. circ_0001944 and circ_0078607 with upregulation and 2 downregulated expressed circRNAs (circ_0002874 and circ_0069981), as well as 11 upregulated miRNAs including miR-193a-5p, miR-197-3p, miR-27a-5p, miR-551b-5p, miR-335-3p, miR-767-3p, miR-767-5p, miR-92a-1-5p, miR-92a-3p, miR-3940-3p, and miR-664b-3p and 3 downregulated expressed miRNAs (miR-1292-5p, let-7c-5p, and miR-99a-5p) in sorafenib-resistant HCC cells were determined. Among these non-coding RNAs (ncRNAs), circ_0001944 and miR-1292-5p should not be drop out of sight; circ_0001944has been proved to target miR-1292-5p to inhibit its expression in HCC. Subsequentfindings also raise that miR-1292-5p directly targeted the 3'-noncoding region (3'-UTR) of Fibulin 2 (FBLN2) mRNA. Furthermore, circ_0001944 targets the miR-1292-5p/FBLN2 axis to inhibit cell ferroptosis in which the indicated regulators associated with iron overload and lipid peroxidation were "rearranged". Most importantly, circ_0001944 advanced sorafenib resistance in HCC through mitigating ferroptosis, where the miR-1292-5p/FBLN2 axis cannot be left unrecognized. Circ_0001944 is a putative target for reversing sorafenib resistance in HCC. Our findings are expected to provide new targets and new directions for sorafenib sensitization in the treatment of HCC.

The anti-angiogenic drugs represented by sorafenib over the years have always been the first-line treatment of hepatocellular carcinoma (HCC), but the drug resistance has always been a "bottleneck" in curative effect. Recently, aberrant expression of circular RNA (circRNA) is considered to play a crucial role in many types of cancers. However, the genome-wide expression pattern of circRNAs in sorafenib-resistant HCC cells remains unknown. Herein, we identified 1717 differentially expressed circRNAs with 559 up-regulated and 1158 down-regulated (fold change > 2, P < 0.05) in sorafenib-resistant (HUH7-S) HCC cells along with 582 differentially expressed circRNAs with 272 up-regulated and 310 down-regulated (fold change > 2, P < 0.05) in sorafenib-resistant (HepG2-S) HCC cells, compared to parental sorafenib-sensitive (HUH7, HepG2) HCC cells by high-throughput sequencing. In addition, GO (Gene Ontology) term enrichment analysis results revealed an enrichment for binding and catalytic activity and for biological regulation of metabolic processes in both the Huh7-S and HepG2-S cell lines compared to parental cell lines. Moreover, KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway analysis of the differentially expressed genes were significantly related to pathways in cancer. Among them, hsa_circ_0006294 and hsa_circ_0035944 expression were consistently down-regulated in resistant HCC cells. Taken together, our data demonstrate, using a global transcriptomic network, that the circRNA expression profile is significantly altered in sorafenib-resistant HCC cells and that the differentially expressed circRNAs may play important functions in HCC sorafenib resistance and HCC progression.

BackgroundHepatocellular carcinoma (HCC) is one of the major malignancies and the second most common cause of cancer-related death worldwide. Sorafenib, an approved first-line systematic treatment agent for HCC, is capable to effectively improve the survival of patients with advanced HCC. The long-noncoding RNA (lncRNA) differentiation antagonizing non-protein coding RNA (DANCR) has been reported to exert oncogenic functions in several kinds of human cancers. However, the role of lncRNA DANCR in sorafenib resistance in HCC remains unknown.MethodsThe expression levels of DANCR in HCC tissues were detected by qRT-PCR. DANCR overexpression and knockdown models were established and utilized to investigate the functional role of DANCR on sorafenib resistance in HCC cells. The MS2-binding sequences-MS2-binding protein–based RNA immunoprecipitation assay, RNA pull-down and luciferase reporter assay was used to detect the association between DANCR and PSMD10 mRNA. The activation of DANCR transcription mediated by STAT3 was assessed by luciferase reporter and chromatin immunoprecipitation assays.ResultsWe found that DANCR was significantly overexpressed in HCC tissues and associated with prognosis of HCC patients. Overexpression and knockdown experiments demonstrated that DANCR promoted sorafenib resistance in HCC cells in vitro and in vivo. Mechanistically, the role of DANCR relied largely on the association with PSMD10. DANCR stabilized PSMD10 mRNA through blocking the repressing effect of several microRNAs on PSMD10. Besides, DANCR activated IL-6/STAT3 signaling via PSMD10. Furthermore, we revealed that DANCR transcription was enhanced by the activation of IL-6/STAT3 signaling, indicating a positive feedback loop of DANCR and IL-6/STAT3 signaling.ConclusionCollectively, our study is the first to elucidate the mechanism of DANCR-mediated sorafenib resistance via PSMD10-IL-6/STAT3 signaling axis, which provides a promising target for developing new therapeutic strategy for sorafenib tolerance of HCC.

Hepatocellular carcinoma (HCC) is a lethal human malignancy and a leading cause of cancer‐related death worldwide. Patients with HCC are often diagnosed at an advanced stage, and the prognosis is usually poor. The multikinase inhibitor sorafenib is the first‐line treatment for patients with advanced HCC. However, cases of primary or acquired resistance to sorafenib have gradually increased, leading to a predicament in HCC therapy. Thus, it is critical to investigate the mechanism underlying sorafenib resistance. Transactivation response element RNA‐binding protein 2 (TARBP2) is a multifaceted miRNA biogenesis factor that regulates cancer stem cell (CSC) properties. The tumorigenicity and drug resistance of cancer cells are often enhanced due to the acquisition of CSC features. However, the role of TARBP2 in sorafenib resistance in HCC remains unknown. Our results demonstrate that TARBP2 is significantly downregulated in sorafenib‐resistant HCC cells. The TARBP2 protein was destabilized through autophagic–lysosomal proteolysis, thereby stabilizing the expression of the CSC marker protein Nanog, which facilitates sorafenib resistance in HCC cells. In summary, here we reveal a novel miRNA‐independent role of TARBP2 in regulating sorafenib resistance in HCC cells.

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.

Introduction: Chromatin assembly factor 1B (CHAF1B), a pivotal regulator of chromatin assembly following DNA replication, has been implicated in oncogenic processes. However, its role in sorafenib resistance and potential anti-tumor mechanisms in hepatocellular carcinoma (HCC) remain unclear. This study has sought to elucidate CHAF1B's therapeutic potential and its potential synergistic role with sorafenib in overcoming chemoresistance.Methods: In this study, bioinformatics, immunohistochemistry, western blot, CCK8, colony formation, transwell migration and invasion, and flow cytometry were performed to analyze the correlation between CHAF1B and sorafenib resistance in HCC. Furthermore, RNA sequencing (RNA-seq), combined with signaling pathway-specific inhibitors, was used to elucidate the specific role of CHAF1B in sorafenib resistance of HCC and its related mechanism.Results: CHAF1B was significantly upregulated in HCC tissues and sorafenib-resistant HCC cells, with elevated expression correlated with reduced survival probability in HCC patients. Moreover, high CHAF1B levels predicted poorer clinical outcomes in sorafenib-treated patients. Functional assays revealed that CHAF1B promotes HCC cell proliferation, migration, and invasion, while also enhancing resistance to sorafenib. In contrast, knockdown of CHAF1B significantly increased sorafenib-induced inhibition of proliferation and cell death. Mechanistically, CHAF1B facilitated malignant phenotypes via activation of the PI3K/Akt/HIF-1α pathway. Furthermore, blockade of PI3K/Akt/HIF-1α signaling partially attenuated the CHAF1B-mediated sorafenib resistance.Conclusion: CHAF1B is a key regulator of sorafenib resistance, and targeting CHAF1B in conjunction with sorafenib may represent a promising therapeutic approach for HCC by modulating the PI3K/Akt/HIF-1α signaling axis.

Hepatocellular carcinoma (HCC) is a malignancy that occurs worldwide and is generally associated with poor prognosis. The development of resistance to targeted therapies such as sorafenib is a major challenge in clinical cancer treatment. In the present study, Ten-eleven translocation protein 1 (TET1) was found to be highly expressed in sorafenib-resistant HCC cells and knockdown of TET1 can substantially improve the therapeutic effect of sorafenib on HCC, indicating the potential important roles of TET1 in sorafenib resistance in HCC. Mechanistic studies determined that TET1 and Yes-associated protein 1 (YAP1) synergistically regulate the promoter methylation and gene expression of DNA repair-related genes in sorafenib-resistant HCC cells. RNA sequencing indicated the activation of DNA damage repair signaling was extensively suppressed by the TET1 inhibitor Bobcat339. We also identified TET1 as a direct transcriptional target of YAP1 by promoter analysis and chromatin-immunoprecipitation assays in sorafenib-resistant HCC cells. Furthermore, we showed that Bobcat339 can overcome sorafenib resistance and synergized with sorafenib to induce tumor eradication in HCC cells and mouse models. Finally, immunostaining showed a positive correlation between TET1 and YAP1 in clinical samples. Our findings have identified a previously unrecognized molecular pathway underlying HCC sorafenib resistance, thus revealing a promising strategy for cancer therapy.Graphical

Hepatocellular carcinoma (HCC) is a prevalent and aggressive tumor. Sorafenib is the first-line treatment for patients with advanced HCC, but resistance to sorafenib has become a significant challenge in this therapy. Cancer stem cells play a crucial role in sorafenib resistance in HCC. Our previous study revealed that the long non-coding RNA (lncRNA) KIF9-AS1 is an oncogenic gene in HCC. However, the role of KIF9-AS1 in drug resistance and cancer stemness in HCC remains unclear. Herein, we aimed to investigate the function and mechanism of the lncRNA KIF9-AS1 in cancer stemness and drug resistance in HCC. To describe the role of the lncRNA KIF9-AS1 in cancer stemness and drug resistance in HCC and elucidate the underlying mechanism. Tumor tissue and adjacent non-cancerous tissue samples were collected from HCC patients. Sphere formation was quantified via a tumor sphere assay. Cell viability, proliferation, and apoptosis were evaluated via Cell Counting Kit-8, flow cytometry, and colony formation assays, respectively. The interactions between the lncRNA KIF9-AS1 and its downstream targets were confirmed via RNA immunoprecipitation and coimmunoprecipitation. The tumorigenic role of KIF9-AS1 was validated in a mouse model. Compared with that in normal controls, the expression of the lncRNA KIF9-AS1 was upregulated in HCC tissues. Knockdown of KIF9-AS1 inhibited stemness and attenuated sorafenib resistance in HCC cells. Mechanistically, N6-methyladenosine modification mediated by methyltransferase-like 3/insulin-like growth factor 2 mRNA-binding protein 1 stabilized and increased the expression of KIF9-AS1. Additionally, KIF9-AS1 increased the stability and expression of short stature homeobox 2 by promoting ubiquitin-specific peptidase 1-induced deubiquitination. Furthermore, depletion of KIF9-AS1 alleviated sorafenib resistance in a xenograft mouse model of HCC. The N6-methyladenosine-modified lncRNA KIF9-AS1 promoted stemness and sorafenib resistance in HCC by upregulating short stature homeobox 2 expression.

Drug resistance and immune escape of tumor cells severely compromise the treatment efficiency of hepatocellular carcinoma (HCC). Long non-coding RNA KCNQ1 overlapping transcript 1 (lncRNA KCNQ1OT1) has been shown to be involved in drug resistance in several cancers. The aim of the present study was to investigate the role of KCNQ1OT1 in sorafenib resistance and immune escape of HCC cells. Reverse transcription-quantitative PCR analysis, western blotting and immunohistochemistry were performed to detect the expression of KCNQ1OT1, miR-506 and programmed death-ligand-1 (PD-L1). Cell Counting Kit-8 assay, flow cytometry and Transwell assays were used to evaluate IC50 value, cell apoptosis and metastasis. ELISA was performed to detect the secretion of cytokines. Dual-luciferase reporter assay was conducted to verify the targeting relationships between miR-506 and KCNQ1OT1 or PD-L1. KCNQ1OT1 and PD-L1 were found to be upregulated and miR-506 was downregulated in sorafenib-resistant HCC tissues and cells. Furthermore, KCNQ1OT1 knockdown reduced the IC50 value of sorafenib, suppressed cell metastasis and promoted apoptosis in sorafenib-resistant HCC cells. Moreover, KCNQ1OT1 knockdown changed the tumor microenvironment and T-cell apoptosis in a sorafenib-resistant HCC/T-cell co-culture model. In addition, it was demonstrated that KCNQ1OT1 functioned as a competing endogenous RNA of miR-506 and increased PD-L1 expression in sorafenib-resistant HCC cells. miR-506 inhibition abolished the effects of KCNQ1OT1 knockdown on sorafenib sensitivity, tumor growth, the tumor microenvironment and T-cell apoptosis. In conclusion, KCNQ1OT1 knockdown inhibited sorafenib resistance and PD-L1-mediated immune escape by sponging miR-506 in sorafenib-resistant HCC cells.