Sorafenib-resistant Hepatocellular Carcinoma Research Articles

LARP4B promotes hepatocellular carcinoma progression and impairs sorafenib efficacy by activating SPINK1-mediated EGFR pathway.

La-related proteins (LARPs) regulate gene expression by binding to RNAs and exhibit critical effects on disease progression, including tumors. However, the role of LARP4B and its underlying mechanisms in the progression of hepatocellular carcinoma (HCC) remain largely unclear. In this study, we found that LARP4B expression is upregulated and correlates with poor prognosis in patients with HCC. Gain- and loss-of-function assays showed that LARP4B promotes stemness, proliferation, metastasis, and angiogenesis in vitro and in vivo. Furthermore, LARP4B inhibition enhances the antitumor effects of sorafenib and blocks the metastasis-enhancing effects of low sorafenib concentrations in HCC. Mechanistically, LARP4B expression is upregulated by METTL3-mediated N6-methyladenosine (m6A)-IGF2BP3-dependent modification in HCC. RNA- and RNA immunoprecipitation (RIP)- sequencing uncovered that LARP4B upregulates SPINK1 by binding to SPINK1 mRNA via the La motif and maintaining mRNA stability. LARP4B activates the SPINK1-mediated EGFR signaling pathway, which supports stemness, progression and sorafenib resistance in HCC. Additionally, a positive feedback loop with the LARP4B/SPINK1/p-AKT/C/EBP-β axis is responsible for the sorafenib-therapeutic benefit of LARP4B depletion. Overall, this study demonstrated that LARP4B facilitates HCC progression, and LARP4B inhibition provides benefits to sorafenib treatment in HCC, suggesting that LARP4B might be a potential therapeutic target for HCC.

Novel STAT3 oligonucleotide compounds suppress tumor growth and overcome the acquired resistance to sorafenib in hepatocellular carcinoma.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and progression of tumors, leading to resistance and poor prognosis. Activation of STAT3 signaling is frequently detected in hepatocellular carcinoma (HCC), but potent and less toxic STAT3 inhibitors have not been discovered. Here, based on antisense technology, we designed a series of stabilized modified antisense oligonucleotides targeting STAT3 mRNA (STAT3 ASOs). Treatment with STAT3 ASOs decreased the STAT3 mRNA and protein levels in HCC cells. STAT3 ASOs significantly inhibited the proliferation, survival, migration, and invasion of cancer cells by specifically perturbing STAT3 signaling. Treatment with STAT3 ASOs decreased the tumor burden in an HCC xenograft model. Moreover, aberrant STAT3 signaling activation is one of multiple signaling pathways involved in sorafenib resistance in HCC. STAT3 ASOs effectively sensitized resistant HCC cell lines to sorafenib in vitro and improved the inhibitory potency of sorafenib in a resistant HCC xenograft model. The developed STAT3 ASOs enrich the tools capable of targeting STAT3 and modulating STAT3 activity, serve as a promising strategy for treating HCC and other STAT3-addicted tumors, and alleviate the acquired resistance to sorafenib in HCC patients. A series of novel STAT3 antisense oligonucleotide were designed and showed potent anti-cancer efficacy in hepatocellular carcinoma in vitro and in vivo by targeting STAT3 signaling. Moreover, the selected STAT3 ASOs enhance sorafenib sensitivity in resistant cell model and xenograft model.

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.

Metallothionein-3 is a multifunctional driver that modulates the development of sorafenib-resistant phenotype in hepatocellular carcinoma cells

Background & aimsMetallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib.MethodsIntrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics.ResultsThe phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib.ConclusionshMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.

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.

Deciphering the role of non-coding RNAs involved in sorafenib resistance

Sorafenib is an important treatment strategy for advanced hepatocellular carcinoma (HCC). Unfortunately, drug resistance has become a major obstacle in sorafenib application. In this study, whole transcriptome sequencing (WTS) was conducted to compare the paired differences between non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and mRNAs, in sorafenib-resistant and parental cells. The overlap of differentially expressed ncRNAs (DENs) between the SMMC7721/S and Huh7/S cells and their parental cells was determined. 2 upregulated and 3 downregulated lncRNAs, 2 upregulated and 1 downregulated circRNAs, as well as 10 upregulated and 2 downregulated miRNAs, in both SMMC7721/S and Huh7/S cells, attracted more attention. The target genes of these DENs were then identified as the overlaps between the differentially expressed mRNAs achieved using the WTS analysis and the predicted genes of DENs obtained using the “co-localization” or “co-expression,” miRanda, and RNAhybrid analysis. Consequently, the potential regulatory network between overlapping DENs and their target genes in both SMMC7721/S and Huh7/S cells was explored. The “lncRNA-miRNA-mRNA” and “circRNA-miRNA-mRNA” networks were constructed based on the competitive endogenous RNA (ceRNA) theory using the Cytoscape software. In particular, lncRNA MED17-203-miRNA (miR-193a-5p, miR-197-3p, miR-27a-5p, miR-320b, miR-767-3p, miR-767-5p, miR-92a-3p, let-7c-5p)-mRNA,” “circ_0002874-miR-27a-5p-mRNA” and “circ_0078607-miR-320b-mRNA” networks were first introduced in sorafenib-resistant HCC. Furthermore, these networks were most probably connected to the process of metabolic reprogramming, where the activation of the PPAR, HIF-1, Hippo, and TGF-β signaling pathways is governed. Alternatively, the network “circ_0002874-miR-27a-5p-mRNA” was also involved in the regulation of the activation of TGF-β signaling pathways, thus advancing Epithelial-mesenchymal transition (EMT). These findings provide a theoretical basis for exploring the mechanisms underlying sorafenib resistance mediated by metabolic reprogramming and EMT 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.

Abstract 4741: Targeting sorafenib-resistant HCCs by protein X-armed T cell immunotherapy

Abstract Hepatocellular carcinoma (HCC) is the most common type of liver cancer globally, ranking fourth according to the World Health Organization's GLOBOCAN database. Sorafenib and lenvatinib are single-drug therapies used for advanced HCC, but only around 30% of patients benefit from them, and drug resistance typically develops within a few months. Our previous study has demonstrated the specific IL-6-IGF-1R-YAP signaling in regulating the stemness expressions, tumor metastasis, and drug-resistance of the sorafenib resistant HCC (2015 Clinical Cancer Res, 2019 J Exp Clinical Cancer Research, 2021 Cancers, 2023 Cancers). In advance our previous study, this study aims to develop a novel immune therapeutic strategy for sorafenib-resistant HCC treatment. We found, when compared to peri-tumor tissues, the immunohistochemical staining showed a high co-expression of both protein X and PD-L1 in liver tumor tissues (6.25%, n=32 versus 28.125%, n = 64, respectively). To further confirm this observation, we used in vitro cell line models with sorafenib-naïve HCCs (HepG2215_N, Hep3B_N, Huh7_N, and PLC5_N) and sorafenib-resistant HCCs (HepG2215_R, Hep3B_R, Huh7_R, and PLC5_R) in this study. As PD-L1 on tumor cells can dampen T cell cytotoxicity, we generated CD3/protein X bispecific antibodies, which can bind to both CD3 and protein X, and coupled them to T cells (Arm-T cells) and normal T cells (N-T cells). We tested their cytotoxic effects on sorafenib-resistant HCCs using the sorafenib-resistant HepG2215_R cells, co-cultured with Arm-T cells and N-T cells for 24 hours. Our results showed that Arm-T cells exhibited a significantly stronger cytotoxic effect on sorafenib-resistant HepG2215_R cells, with several notable findings: (1) The binding time of Arm-T cells to HepG2215_R cells was significantly longer than that of N-T cells (248.9 ± 146.6 minutes, n=23 versus 28.3 ± 13.49 minutes, n=24); (2) The cytotoxicity of Arm-T cells was significantly higher than that of N-T cells (29.7 ± 2.3% versus 13.6 ± 3.3%); and (3) The secreted levels of INFγ and TNFα by Arm-T cells were significantly higher than those of N-T cells (138.1 ± 7.2 ng/ml and 34.6 ± 0.7 ng/ml versus 31.4 ± 2.5 ng/ml and 3.6 ± 1.1 ng/ml, respectively). These findings were also consistent when using Hep3B-R cells (data not shown). To summarize, our study shows that Arm-T cells possess strong cytotoxic abilities against sorafenib-resistant HCC. These findings have implications for developing effective immuno-therapeutic strategies for patients with sorafenib-resistant HCCs. Citation Format: Mai-Huong Thi Ngo, Michael Chen, Pei-Chi Lan, Kuo-Hsiang Chuang, Yen-Hua Huang. Targeting sorafenib-resistant HCCs by protein X-armed T cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4741.

Abstract 4324: IL6ST increases sorafenib-resistance in hepatocellular carcinoma by regulating BECN1-mediated mitophagic cell death

Abstract Introduction: Sorafenib resistance is often seen in patients with late-stage hepatocellular carcinoma (HCC) and it is a serious concern for the treatment of these patients. Lethal mitophagy plays an important role in anti-tumorigenic mechanisms by inducing mitochondrial dysfunction and mitophagic cell death. Sorafenib resistance is associated with the activation of IL6ST signaling, inhibiting mitochondrial quality. However, how lethal mitophagy regulates sorafenib resistance through IL6ST and mitochondrial dysfunction in HCC is not fully understood. Experimental procedures: Hepatocyte-specific TAK1 deleted (TAK1ΔHep) and TAK1/IL6STΔHep were used for in vivo study. Histology, immunoblots, qPCR, microscopy, FACS, and seahorse bioanalyzer were used for analyzing liver and tumor tissues and Hep3B cells. Results: We identified that sorafenib suppressed IL6ST signaling and that IL6ST signaling was activated in sorafenib-resistant HCC. The number and maximum size of spontaneous HCC in 9-month-old TAK1ΔHep mice were decreased when hepatocyte IL6ST was additionally deleted. Expression of pro-carcinogenic H-19, c-SRC, and MDM2 was increased in 1-month-old TAK1/IL6STΔHep mice compared to TAK1ΔHep mice. An additional IL6ST deletion induced decreases in mitochondrial membrane potential and glycolysis, suggesting that IL6ST protects against mitochondrial dysfunction. Interestingly, IL6ST deletion increases PRKN and BECN1 interaction and translocation to normal mitochondria and subsequent degradation, hereafter referred to as lethal mitophagy. Furthermore, inhibition of BECN1 restores IL6ST deletion-induced lethal mitophagy in HCC, suggesting that IL6ST-mediated lethal mitophagy is dependent on BECN1. Mechanistically, IL6ST inhibited BECN1-lethal mitophagy through STAT3(S705) phosphorylation independently of mTORC1 activation. Notably, enhanced BECN1 through a combination of sorafenib and inhibition of IL6ST increased mitophagic cell death, reducing sorafenib resistance and tumor growth. In addition, enhanced lethal mitophagy through inhibition of GP130 effectively killed sorafenib-resistant HCCs by silencing sorafenib resistance. We further defined the relationship between IL6ST, BECN1, and PRKN expression and sorafenib resistance in terms of HCC patients, RNA sequencing datasets. Conclusion:  These results provide the importance of IL6ST in the development of sorafenib resistance and could be an innovative treatment strategy for sorafenib-resistant patients by regulating the IL6ST/STAT3/BECN1 axis-regulated lethal mitophagy. Citation Format: Hwan Ma, Yoon-Seok Roh. IL6ST increases sorafenib-resistance in hepatocellular carcinoma by regulating BECN1-mediated mitophagic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4324.

Abstract 3284: Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma cell lines

Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer which accounts for more than 90% of occurrences of liver cancer cases. Surgical therapies such as liver transplantation and hepatectomy may help cure hepatocellular carcinoma diagnosed in its early stages. Advanced stages of the disease have a poor prognosis and a high refractoriness because of drug resistance. Sorafenib is a tyrosine kinase inhibitor that has been approved for the systemic treatment of patients with advanced HCC. However, the efficacy of this medication has shown a limited prolongation of the overall survival in patients with advanced HCC. The mechanism of resistance to sorafenib in advanced HCC is poorly understood. The TCGA analysis of the HCC patient cohort shows an elevated expression of YBX1 in tumors as compared to normal. The expression is also correlated with progression, metastasis, and poor survival of HCC patients. Transcription factor YBX1 is involved in the regulation of drug resistance in different cancers, especially breast and colorectal cancers. Our lab is interested in understanding if YBX1 is involved in sorafenib resistance in HCC. Different HCC cell lines procured from ATCC SK-Hep1, Huh-7luc, and C3A, were analyzed for their IC50 values for sorafenib using MTT assays, at different time points. SK-Hep1 showed 8.780µM and 8.174µM, HepG2luc 4.659µM and 3.596µM at 48 and 72 hrs respectively. Overexpression of YBX1 in the SK-Hep1 cell line increased the cell survival and IC50 value of sorafenib at 48 and 72 hrs at 13.19µM and 10.64µM respectively. This supports the hypothesis which demonstrates the role of YBX1 in mediating resistance to sorafenib in advanced HCC. YBX1 could be a major therapeutic target and by inhibiting its action could improve the sensitivity of advanced HCC to sorafenib. We have developed YBX1 overexpressing and knockdown (GFP expressing) cell lines, FACS sorted and Puromycin selected for further mechanistic studies. We are also further developing the sorafenib-resistant HCC cell lines to understand the mechanism underlying sorafenib resistance in HCC. Protein and RNA analyses revealed a relative increase in YBX1 overexpression in sorafenib-resistant cell lines as compared to its parental cell lines. Understanding the mechanism of YBX1-induced drug resistance to sorafenib and its associated signaling pathways could help improve the sensitivity of advanced hepatocellular carcinoma to treatment by sorafenib and thus improve the livelihood of its patients. Citation Format: Dennis Kwabiah, Ana AyalaPazzi, Kyle Doxtater, Sophia Leslie, Manish K. Tripathi. Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3284.

Retracted: Studying the Role and Molecular Mechanisms of MAP4K3 in Sorafenib Resistance of Hepatocellular Carcinoma.

[This retracts the article DOI: 10.1155/2020/4965670.].

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.

Role of sirtuin 1 (SIRT1) in regulation of autophagy and nuclear factor-kappa Beta (NF-ĸβ) pathways in sorafenib-resistant hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) remains a major global health problem with high incidence and mortality. Diagnosis of HCC at late stages and tumour heterogeneity in patients with different genetic profiles are known factors that complicate the disease treatment. HCC therapy becomes even more challenging in patients with drug resistance such as resistance to sorafenib, which is a common drug used in HCC patients. Sorafenib resistance can further aggravate HCC by regulating various oncogenic pathways such as autophagy and nuclear factor-kappa Beta (NF-ĸβ) signalling. Sirtuin 1 (SIRT1), is a nicotinamide adenosine dinucleotide (NAD)-dependent histone deacetylases that regulates various metabolic and oncogenic events such as cell survival, apoptosis, autophagy, tumourigenesis, metastasis and drug resistance in various cancers, but its role in HCC, particularly in sorafenib resistance is underexplored. In this study, we generated sorafenib-resistant HepG2 and Huh-7 liver cancer cell models to investigate the role of SIRT1 and its effect on autophagy and nuclear factor-kappa Beta (NF-ĸβ) signalling pathways. Western blot analysis showed increased SIRT1, altered autophagy pathway and activated NF-ĸβ signalling in sorafenib-resistant cells. SIRT1-silenced HCC cells demonstrated down-regulated autophagy in both parental and chemoresistant cells. This may occur through the deacetylation of key autophagy molecules such as FOXO3, beclin 1, ATGs and LC3 by SIRT1, highlighting the role of SIRT1 in autophagy induction. Silencing of SIRT1 also resulted in activated NF-ĸβ signalling. This is because SIRT1 failed to deacetylate p65 subunit of NF-κB, translocate the NF-κB from nucleus to cytoplasm, and suppress NF-κB activity due to the silencing. Hence, the NF-κB transcriptional activity was restored. These findings summarize the role of SIRT1 in autophagy/NF-ĸβ regulatory axis, with a similar trend observed in both parental and sorafenib-resistant cells. The present work promotes a better understanding of the role of SIRT1 in autophagy and NF-ĸβ signalling in HCC and sorafenib-resistant HCC. As some key proteins in these pathways are potential therapeutic targets, a better understanding of SIRT1/autophagy/NF-ĸβ axis could further improve the therapeutic strategies against 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.

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.

L-Methionine potentiates anticancer activity of Sorafenib by epigenetically altering DUSP3/ERK pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most common cancer-related cause of death worldwide. Although Sorafenib is the standard systemic therapy for treating HCC, but it develops resistance very quickly, leading to poor prognosis. The current study was planned to explore the effect of l-methionine on the anticancer activity of Sorafenib in HCC. Ten millimolarof l-methionine treatment significantly reduced the IC50 of Sorafenib from 5.513 ± 0.171to 0.8095 ± 0.0465 µM in HepG2 cell line. It also resulted in concomitant increase in oxidative stress and deactivation of ERK/AMPK/AKT pathway. Additionally, it also resulted in the increased expression of dual specificity phosphatase 3 (DUSP3). In a rat model of sorafenib-resistant HCC induced by diethylnitrosamine (DEN) (100 mg/L/day) and Sorafenib (10 mg/kg), l-methionine (300 and 500 mg/kg/day) supplementation overcame the drug resistance, as indicated by the reduced formation of surface tumor nodules, prevention of cellular hypertrophy, hyperplasia and inflammation, and improved animal survival. Furthermore, l-methionine in combination with Sorafenib also inhibited AMPK/AKT and ERK pathway. At chromatin level, l-methionine supplementation prevented global methylation of H3K27me3, an inactivation mark,and demethylation of H3K36me2, an activation mark. Interestingly, our findings suggest that inhibition of theERK pathway via increased activity of DUSP3 is epigenetically regulated. Besides, chromatin immunoprecipitation data exhibited augmented H3K36me2 (an activation mark) levels on the DUSP3 promoter region. To the best of our knowledge, we are the first to report that l-methionine supplementation improves the chemosensitivity in Sorafenib-resistant HCC via modulating the epigenetic landscape and can be a potential therapeutic strategy.

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.