Sensitivity Of Hepatocellular Carcinoma Cells Research Articles

Calpain 2 promotes Lenvatinib resistance and cancer stem cell traits via both proteolysis-dependent and independent approach in hepatocellular carcinoma

Lenvatinib, an approved first-line regimen, has been widely applied in hepatocellular carcinoma (HCC). However, clinical response towards Lenvatinib was limited, emphasizing the importance of understanding the underlying mechanism of its resistance. Herein, we employed integrated bioinformatic analysis to identify calpain-2 (CAPN2) as a novel key regulator for Lenvatinib resistance in HCC, and its expression greatly increased in both Lenvatinib-resistant HCC cell lines and clinical samples. Further in vitro and in vivo experiments indicated that knocking down CAPN2 greatly sensitized HCC cells to Lenvatinib treatment, while overexpression of CAPN2 achieved opposite effects in a Lenvatinib-sensitive HCC cell line. Interestingly, we observed a close relationship between CAPN2 expression and cancer stem cell (CSC) traits in HCC cells, evidenced by impaired sphere-forming and CSC-related marker expressions after CAPN2 knockdown, and verse vice. Mechanistically, we strikingly discovered that CAPN2 exerted its function by both enzyme-dependent and enzyme-independent manner simultaneously: activating β-Catenin signaling through its enzyme activity, and preventing GLI1/GLI2 degradation through direct binding to YWHAE in an enzyme-independent manner, which disrupting the association between YWHAE and GLI1/GLI2 to inhibit YWHAE-induced degradation of GLIs. Notably, further co-immunoprecipitation assays revealed that YWHAE could promote the protein stability of CAPN2 via recruiting a deubiquitinase COPS5 to prevent ubiquitination-induced degradation of CAPN2. In summary, our data demonstrated that CAPN2 promoted Lenvatinib resistance via both catalytic activity-dependent and -independent approaches. Reducing CAPN2 protein rather than inhibiting its activity might be a promising strategy to improve Lenvatinib treatment efficiency in HCC.

Targeting Deltex E3 Ubiquitin Ligase 2 Inhibits Tumor-associated Neutrophils and Sensitizes Hepatocellular Carcinoma Cells to Immunotherapy.

Several E3 ligases have been found to affect the immune microenvironment of hepatocellular carcinoma (HCC) and lead to the resistance of immunotherapy. In this study, genes of E3 ligases are screened based on The Cancer Genome Atlas (TCGA) dataset. Through cytometry by time of flight (CyTOF), flow cytometry, and further experiments, Deltex E3 ubiquitin ligase 2 (DTX2) in HCC cells is identified to promote the infiltration and polarization of tumor-associated neutrophils (TANs) with a protumor phenotype, thus attenuating the infiltration and cytotoxicity of CD8+ T cells partially through C-X-C motif chemokine 2 (CXCL2) and C-X-C motif chemokine 6 (CXCL6). Mechanistically, DTX2 can interact with histone H2B and promote its monoubiquitination at lysine120 (H2BK120ub1), thereby increasing CXCL2 and CXCL6 transcription through histone epigenetic regulation. Different tumor models in vivo demonstrated that DTX2 inhibitor treatment inhibited tumor growth and sensitized HCC cells to the therapeutic effects of programmed cell death protein 1 (PD-1) antibody. In summary, this study identifies DTX2 as a potential target for HCC immunotherapy.

Pralatrexate represses the resistance of HCC cells to molecular targeted agents via the miRNA-34a/Notch pathway

Metabolism-related pathways are important targets for intervention in the treatment of hepatocellular carcinoma (HCC), but few studies have reported on the combination of inhibitors of folate metabolism-related enzymes and molecularly targeted drugs for HCC. The results of the present work are the first to reveal the effects of an inhibitor of dihydrofolate reductase (DHFR), pralatrexate, on the sensitivity of HCC cells to molecularly targeted agents examined using multiple assays. In HCC cells, knockdown of DHFR or treatment with pralatrexate enhanced the sensitivity of HCC cells to molecularly targeted agents, such as sorafenib, regorafenib, lenvatinib, cabozantinib, or anlotinib. Mechanically, pralatrexate decreased the methylation rates of miRNA-34a’s promoter region to enhance the expression of miRNA-34a. Treatment with pralatrexate inhibited the expression of Notch and its downstream factors by enhancing the expression of miRNA-34a in HCC cells. In clinical specimens, the expression of miRNA-34a was negatively correlated with DHFR expression, while DHFR expression was positively correlated with the Notch intracellular domain (NICD) and downstream factors of the Notch pathway. The expression of miRNA-34a was negatively correlated with DHFR expression, while the methylation rates of miRNA-34a’s promoter were positively related to DHFR. The effect of pralatrexate on the metabolic profile of HCC cells is very different from that of small molecule inhibitors related to glycolipid metabolism. Therefore, pralatrexate upregulates the sensitivity of HCC cells to molecularly targeted drugs. These results expand our understanding of folate metabolism and HCC and can help provide more options for HCC treatment.

Abstract A009: Increasing the Sensitivity to sorafenib of Resistant Hepatocellular Carcinoma Cells with Bovine Lactoferrin through Ferroptosis

Abstract Hepatocellular carcinoma (HCC) remains one of the most lethal cancers worldwide, with a high mortality rate largely due to late-stage diagnosis and the limited efficacy of current therapeutic options. Sorafenib is one of the first-line therapies for advanced HCC. However, its effectiveness is often compromised by the development of drug resistance, significantly limiting its long-term therapeutic potential. This limitation has driven extensive research into alternative therapeutic strategies that can target this resistance, thereby improving the clinical outcomes of patients. Bovine Lf (bLf), which is functionally similar to human Lf (hLf), is safe for human use and has demonstrated anticancer properties, including synergistic effects with chemotherapy. In our study, we explored the role of bovine lactoferrin (bLf) as a potential agent to increase the sensitivity of sorafenib-resistant HCC cells. We developed sorafenib-resistant HCC cell lines through repeated exposure to the drug, with IC50 calculations confirming their increased drug tolerance. These resistant cells were subsequently treated with a combination of sorafenib and bLf. This combination significantly reduced cell viability, with a sorafenib IC50 below the level of the parental sensitive cell line. RNA-seq data revealed significant changes in gene expression profiles, highlighting the differential expression of both mRNAs and long noncoding RNAs (lncRNAs) among resistant, nonresistant, and resistant bLf-treated cells. Further insights were gained through gene set enrichment analysis (GSEA), which identified ferroptosis as a critical pathway significantly enriched in resistant cells treated with bLf and sorafenib vs sorafenib treatment only. Among the differentially expressed lncRNAs, LINC02015 was the most upregulated in resistant cells, and the most downregulated in resistant cells treated with bLf and sorafenib. This lncRNA has emerged as a potentially influencing ferroptosis in other pathologies. Notably, LINC02015 was found to be correlated with HMOX1, a gene with established roles in oxidative stress and iron metabolism. Based on these findings, we conducted a more focused analysis of the ATF3/HMOX1/GPX4 axis, a signaling pathway previously implicated in the regulation of ferroptosis and cellular responses to oxidative stress. Our data suggest that this axis could play a role in modulating lactoferrin-induced ferroptosis in the context of sorafenib resistance. In conclusion, our study provides compelling evidence that bovine lactoferrin (bLf) has the potential to serve as a novel therapeutic agent for overcoming sorafenib resistance in hepatocellular carcinoma by inducing ferroptosis. By potentially targeting the LINC02015/ATF3/HMOX1/GPX4 axis, bLf not only enhances the efficacy of sorafenib but also opens new avenues for the development of combinatory treatment strategies aimed at improving outcomes for HCC patients. Citation Format: Hury V Hernandez-Galdamez, Mireya De la Garza, Karla Rubio, Ivonne Ramirez-Diaz, Josue Guzman-Linares, Theresa Azcarraga Acosta, Guillermo Barreto, stefan günther, Teresita del niño Jesus Flores-Tellez, Mario A Aguilar-Chaparro, Pablo Muriel de la torre, Magda Reyes-López, Saul Villa-Treviño, Carolina Piña-Vazquez. Increasing the Sensitivity to sorafenib of Resistant Hepatocellular Carcinoma Cells with Bovine Lactoferrin through Ferroptosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A009.

EGFR bypass activation mediates acquired resistance to regorafenib in hepatocellular carcinoma.

Regorafenib, a tyrosine kinase inhibitor (TKI), is used in the treatment of unresectable hepatocellular carcinoma (HCC). However, the occurrence of acquired resistance limits its antitumor efficacy. While multiple studies have highlighted the crucial role of bypass activation in acquired TKI resistance, few have focused on bypass activation in regorafenib resistance in HCC. High-throughput proteomics was used to identify differential proteins associated with bypass activation between acquired regorafenib-resistant cells and parental cells. The ability of epidermal growth factor receptor (EGFR) bypass inhibition to reverse resistance was evaluated both in vitro and in vivo using direct microscopic observation, the CCK-8 assay, colony formation assay, Annexin V-FITC/propidium iodide double staining, cell cycle analysis, western blotting, and a xenograft model. The expression of EGFR, a member of the receptor tyrosine kinase (RTK) family, was significantly increased in acquired regorafenib-resistant HCC cells compared with parental cells. Pharmacological inhibition of EGFR with gefitinib restored the sensitivity of regorafenib-resistant HCC cells to regorafenib. In a xenograft mouse model, gefitinib sensitized resistant tumors to regorafenib. Additionally, levels of RAS, RAF, and P-ERK1/2, components of the downstream EGFR signaling pathway, were positively associated with EGFR expression. EGFR overexpression promotes acquired resistance to regorafenib through RAS/RAF/ERK bypass activation in HCC. Inhibition of EGFR restores sensitivity to regorafenib, and the combination of gefitinib and regorafenib demonstrates significant antitumor efficacy both in vivo and in vitro. These findings suggest that this combination could be a potential strategy for patients with advanced HCC.

Iberverin Downregulates GPX4 and SLC7A11 to Induce Ferroptotic Cell Death in Hepatocellular Carcinoma Cells.

Ferroptosis, a recently elucidated style of regulated cell death, has emerged as a significant area of investigation in cancer biology. Natural active compounds that have anti-cancer effects are promising candidates for cancer prevention. Iberverin, a natural compound derived from Brassica oleracea var. capitata, has been shown to exert anti-tumor activities in some cancers. However, its role in hepatocellular carcinoma (HCC) cells and the molecular mechanisms are still poorly understood. In this study, we proved that iberverin can induce intracellular reactive oxygen species (ROS) generation to inhibit cell proliferation and initiate ferroptotic cell death in HCC cells, which can be eradicated by the ferroptosis inhibitor ferrostatin-1 (Fer-1) or deferoxamine mesylate (DFO) and ROS scavenger (GSH or NAC). Mechanistically, iberverin treatment can simultaneously downregulate SLC7A11 mRNA level and degrade GPX4 through the ubiquitination pathway, leading to lipid peroxidation and ferroptotic cell death in HCC cells. Significantly, a low dose of iberverin can remarkably increase the sensitivity of HCC cells to ferroptosis induced by canonical ferroptosis inducers RSL3 and imidazole ketone erastin (IKE). This study uncovers a critical function of iberverin in preventing HCC through ferroptosis and provides a promising strategy for HCC treatment either via iberverin alone or in combination with canonical ferroptosis inducers in the future.

N6-Methyladenosine modification activates the serine synthesis pathway to mediate therapeutic resistance in liver cancer

Metabolic adaptation serves as a significant driving force for cancer growth and poses a substantial obstacle for cancer therapies. Herein, we unravelled the role of m6A -mediated serine synthesis pathway regulation in both hepatocellular carcinoma (HCC) development and therapeutic resistance. We demonstrated that the highly specific m6A inhibitor (STM2457) treatment effectively inhibited HCC cell line growth and suppressed spontaneous HCC formation in mice driven by liver specific Tp53 knockout and Myc overexpression. Using GLORI-seq, we delineated a single-base resolution m6A landscape in human HCC cell lines. Interestingly, we identified three core enzymes in the serine synthesis pathway (PHGDH, PSAT1, and PSPH) as novel targets of METTL3-mediated m6A modification. In these SSP genes, m6A modification recruited IGF2BP3 reader to stabilize their mRNA transcripts, thereby enhancing their mRNA and protein expression in HCC cells. Most importantly, our GLORI-seq data revealed that sorafenib-resistant HCC cells elevated m6A modification in SSP genes to promote protein expression and antioxidant production. STM2457 treatment attenuated the serine synthesis pathway, induced oxidative stress, and sensitized HCC cells to sorafenib and lenvatinib treatments. In conclusion, our findings suggest that targeting m6A could be a potential therapeutic strategy for HCC treatment.

Intracellular Magnetic Hyperthermia Sensitizes Sorafenib to Orthotopic Hepatocellular Carcinoma Via Amplified Ferroptosis.

Sorafenib (SRF) is recognized as the primary treatment for hepatocellular carcinoma (HCC), yet the emergence of SRF resistance in many HCC patients results in unfavorable outcomes. Enhancing the efficacy of SRF in HCC remains a significant challenge. SRF works in inducing ferroptosis, a form of cell death, in cancer cells through the inhibition of glutathione peroxidase 4 (GPX4). The effectiveness of this process is limited by the low levels of cellular iron and reactive oxygen species (ROS). A promising approach to circumvent this limitation is the use of intracellular magnetic hyperthermia (MH) mediated by magnetic iron oxide nanomaterials (MIONs). When MIONs are subjected to an alternating magnetic field (AMF), they heat up, enhancing the Fenton reaction, which in turn significantly increases the production of ROS within cells. In this study, we explore the capability of MH facilitated by high-performance ferrimagnetic vortex-domain iron oxide nanoring (FVIO) to enhance the effectiveness of SRF treatment in HCC. The increased iron uptake facilitated by FVIO significantly enhances the sensitivity of HCC cells to SRF-induced ferroptosis. Moreover, the nanoheat generated by FVIO in response to an AMF further elevates ROS levels and stimulates lipid hydroperoxide (LPO) production and GPX4 inactivation, thereby intensifying ferroptosis. Both in vitro and in vivo animal studies demonstrate that combining FVIO-mediated MH with SRF significantly reduces cell viability and inhibits tumor growth, primarily through enhanced ferroptosis, with minimal side effects. The effectiveness of this combination therapy is affected by the ferroptosis inhibitor ferrostatin-1 (Fer-1) and the iron chelator deferoxamine (DFO). The combination treatment of FVIO-mediated MH and SRF offers a strategy for HCC treatment by promoting accelerated ferroptosis, presenting a different perspective for the development of ferroptosis-based anticancer therapies.

RABIF promotes hepatocellular carcinoma progression through regulation of mitophagy and glycolysis

The RAB interacting factor (RABIF) is a putative guanine nucleotide exchange factor that also functions as a RAB-stabilizing holdase chaperone. It has been implicated in pathogenesis of several cancers. However, the functional role and molecular mechanism of RABIF in hepatocellular carcinoma (HCC) are not entirely known. Here, we demonstrate an upregulation of RABIF in patients with HCC, correlating with a poor prognosis. RABIF inhibition results in decreased HCC cell growth both in vitro and in vivo. Our study reveals that depleting RABIF attenuates the STOML2-PARL-PGAM5 axis-mediated mitophagy. Consequently, this reduction in mitophagy results in diminished mitochondrial reactive oxygen species (mitoROS) production, thereby alleviating the HIF1α-mediated downregulation of glycolytic genes HK1, HKDC1, and LDHB. Additionally, we illustrate that RABIF regulates glucose uptake by controlling RAB10 expression. Importantly, the knockout of RABIF or blockade of mitophagy sensitizes HCC cells to sorafenib. This study uncovers a previously unrecognized role of RABIF crucial for HCC growth and identifies it as a potential therapeutic target.

Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

AbstractChemotherapeutic drugs, such as cisplatin (cis-dichlorodiamineplatinum [II], cDDP) and 5-fluorouracil (5Fu), are widely used in transarterial chemoembolization (TACE), which is a standard therapy for patients with hepatocellular carcinoma (HCC). Chemoresistance is a major cause of TACE treatment failure in HCC patients. Our previous studies have identified the expression levels of miR-101 responsive genes, such as EED, EZH2, STMN1 and JUNB, exhibit significant correlation with the occurrence and progression of HCC, while the role of miR-101 responsive gene signatures in the chemoresistance of HCC treatment remains unclear. In this study, we identified ubiquitin-coupled enzyme E2D1 (UBE2D1) as a crucial regulatory factor in the chemoresistance of HCC, which is a direct target of miR-101 and exhibits significant correlation with miR-101-responsive gene signatures. The bioinformatics analysis showed the expression of UBE2D1 was significantly increased in HCC tissues and was closely correlated with the poor prognosis. In addition, we analyzed the role of miR-101/UBE2D1 axis in regulating chemo-sensitive of HCC cells. Our results showed that miR-101 increases the DNA damage and apoptosis of HCC cells by inhibiting the expression of UBE2D1, which in turn increases the sensitivity of HCC cells to cDDP and 5Fu both in vitro and in vivo. Therefore, simultaneous assessment of miR-101 and UBE2D1 expression levels might provide an effective approach in preselecting HCC patients with survival benefit from TACE treatment. Moreover, further elucidation of the underlying molecular mechanisms of the miR-101/UBE2D1 axis could provide novel insight for targeted therapy of HCC.

Knockdown of SLC16A3 decreases extracellular lactate concentration in hepatocellular carcinoma, alleviates hypoxia and induces ferroptosis

SLC16A3/monocarboxylate transporter 4 (MCT4) regulates intracellular lactate transport and is highly expressed in many tumors, indicating poor prognosis. It may be related to inducing hypoxia, apoptosis and other mechanisms, but the study of MCT4 in HCC is far from complete. In this study, we first analyzed the expression of SLC16A3 in HCC tumor and non-tumor tissue samples based on TCGA data and immunohistochemistry. Subsequently, the effects of SLC16A3 expression on cell proliferation and invasion were analyzed using hepatocellular carcinoma (HCC) lines, and Western blot (WB) analysis was performed to explore the changes in pathway proteins and ferroptosis proteins. Finally, the drug sensitivity was tested by CCK8 kit. We found that SLC16A3 was significantly upregulated in tumor tissues, and was significantly correlated with TNM stage, histological grade, and macrovascular invasion. TCGA data and WB analysis showed that the high expression of SLC16A3 induced hypoxia, and knockdown could reverse hypoxia and inhibit ERK phosphorylation, thus limiting the malignant behavior of HCC cells. Moreover, knockdown of SLC16A3 significantly increased the level of lipid peroxidation and reactive oxygen species (ROS), while the expressions of GPX4, DHODH and SLC7A11 were inhibited. The expression of SLC16A3 affected the sensitivity of HCC cells to chemotherapy and targeted drugs, and RNA sequencing data suggested that the expression level influenced tumor microenvironment and response to immunotherapy. So, we draw a conclude that SLC16A3 is associated with poor prognosis of HCC. Inhibition of SLC16A3 expression is a potential therapeutic target for HCC.

TFCP2L1 drives stemness and enhances their resistance to Sorafenib treatment by modulating the NANOG/STAT3 pathway in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent and aggressive malignancy associated with high risks of recurrence and metastasis. Liver cancer stem cells (CSCs) are increasingly recognized as pivotal drivers of these processes. In our previous research, we demonstrated the involvement of TFCP2L1 in maintaining the pluripotency of embryonic stem cells. However, its relevance to liver CSCs remains unexplored. In this study, we report an inverse correlation between TFCP2L1 protein levels in HCC tissue and patient outcomes. The knockdown of TFCP2L1 significantly reduced HCC cell proliferation, invasion, metastasis, clonal formation, and sphere-forming capacity, while its overexpression enhanced these functions. In addition, experiments using a nude mouse model confirmed TFCP2L1’s essential role in liver CSCs’ function and tumorigenic potential. Mechanistically, we showed that TFCP2L1 promotes the stemness of CSCs by upregulating NANOG, which subsequently activates the JAK/STAT3 pathway, thereby contributing to HCC pathogenesis. Importantly, we identified a specific small molecule targeting TFCP2L1’s active domain, which, in combination with Sorafenib, sensitizes hepatoma cells to treatment. Together, these findings underscore TFCP2L1’s pathological significance in HCC progression, supporting its potential as a prognostic biomarker and therapeutic target in this disease.

CCT3/ACTN4/TFRC axis protects hepatocellular carcinoma cells from ferroptosis by inhibiting iron endocytosis

Sorafenib is widely used in treating advanced hepatocellular carcinoma (HCC). However, its effectiveness in prolonging patient survival is limited by the development of drug resistance. To systematically investigate the resistance mechanisms of Sorafenib, an integrative analysis combining posttranslational modification (PTM) omics and CRISPR/Cas9 knockout library screening was conducted. This analysis identified ubiquitination at lysine 21 (K21) on chaperonin-containing TCP1 subunit 3 (CCT3) as being associated with Sorafenib resistance. Transcriptomic data from HCC patients treated with Sorafenib revealed that CCT3 expression was lower in responders compared to non-responders. Experimentally, inhibiting the expression of CCT3 sensitized HCC cells to Sorafenib and enhanced Sorafenib-induced ferroptosis. Additionally, CCT3 was found to interact with ACTN4, hindering the recycling of transferrin receptor protein 1 (TFRC) to the cell membrane, thus obstructing iron endocytosis. Mechanistically, the inhibition of ferroptosis by CCT3 depends on the deubiquitination of K6-linked non-degradative ubiquitination at its K21, which occurs upon Sorafenib treatment. Moreover, CCT3 knockdown enhanced the anti-tumor effects of Sorafenib in nude mice. In summary, we have identified a novel function of the chaperone protein. Targeting the CCT3/ACTN4/TFRC axis offers a promising strategy to enhance ferroptosis and overcome Sorafenib resistance in HCC.

Natural Product Auraptene Targets SLC7A11 for Degradation and Induces Hepatocellular Carcinoma Ferroptosis.

The natural product auraptene can influence tumor cell proliferation and invasion, but its effect on hepatocellular carcinoma (HCC) cells is unknown. Here, we report that auraptene can exert anti-tumor effects in HCC cells via inhibition of cell proliferation and ferroptosis induction. Auraptene treatment induces total ROS and lipid ROS production in HCC cells to initiate ferroptosis. The cell death or cell growth inhibition of HCC cells induced by auraptene can be eliminated by the ROS scavenger NAC or GSH and ferroptosis inhibitor ferrostatin-1 or Deferoxamine Mesylate (DFO). Mechanistically, the key ferroptosis defense protein SLC7A11 is targeted for ubiquitin-proteasomal degradation by auraptene, resulting in ferroptosis of HCC cells. Importantly, low doses of auraptene can sensitize HCC cells to ferroptosis induced by RSL3 and cystine deprivation. These findings demonstrate a critical mechanism by which auraptene exhibits anti-HCC effects via ferroptosis induction and provides a possible therapeutic strategy for HCC by using auraptene or in combination with other ferroptosis inducers.

Sensitization of hepatocellular carcinoma cells to HDACi is regulated through hsa-miR-342-5p/CFL1

BackgroundIncreased prevalence of hepatocellular carcinoma (HCC) remains a global health challenge. HCC chemoresistance is a clinical obstacle for its management. Aberrant miRNA expression is a hallmark for both cancer progression and drug resistance. However, it is unclear which miRNAs are involved in HCC chemoresistance.MethodsMicroRNA microarray analysis revealed a differential expression profile of microRNAs between the hepatocellular carcinoma HA22T cell line and the HDACi-R cell line, which was validated by quantitative real-time PCR (qRT-PCR). To determine the biological function of miR-342-5p and the mechanism of the microRNA-342-5p/CFL1 axis in hepatocellular carcinoma HDACi resistance, loss- and gain-of-function studies were conducted in vitro.ResultsHere we demonstrated the molecular mechanism of histone deacetylase inhibitor (HDACi) resistance in HCC. Differential miRNA expression analysis showed significant down regulation of miR-342-5p in HDACi-R cells than in parental HA22T cells. Mimics of miR-342-5p enhanced apoptosis through upregulation of Bax, cyto-C, cleaved-caspase-3 expressions with concomitant decline in anti-apoptotic protein (Bcl-2) in HDACi-R cells. Although HDACi did not increase cell viability of HDACi-R, overexpression of miR-342-5p decreased cofilin-1 expression, upregulated reactive oxygen species (ROS) mediated apoptosis, and sensitized HDACi-R to HDACi in a dose-dependent manner.ConclusionOur findings demonstrated the critical role of miR-342-5p in HDACi resistance of HCC and that this mechanism might be attributed to miR-342-5p/cofilin-1 regulation.

Aqueous Extracts of Ocimum gratissimum Sensitize Hepatocellular Carcinoma Cells to Cisplatin through BRCA1 Inhibition.

Ocimum gratissimum (O. gratissimum), a medicinal herb with antifungal and antiviral activities, has been found to prevent liver injury and liver fibrosis and induce apoptosis in hepatocellular carcinoma (HCC) cells. In this study, we evaluated the effect of aqueous extracts of O. gratissimum (OGE) on improving the efficacy of chemotherapeutic drugs in HCC cells. Proteomic identification and functional assays were used to uncover the critical molecules responsible for OGE-induced sensitization mechanisms. The antitumor activity of OGE in combination with a chemotherapeutic drug was evaluated in a mouse orthotopic tumor model, and serum biochemical tests were further utilized to validate liver function. OGE sensitized HCC cells to the chemotherapeutic drug cisplatin. Proteomic analysis and Western blotting validation revealed the sensitization effect of OGE, likely achieved through the inhibition of breast cancer type 1 susceptibility protein (BRCA1). Mechanically, OGE treatment resulted in BRCA1 protein instability and increased proteasomal degradation, thereby synergistically increasing cisplatin-induced DNA damage. Moreover, OGE effectively inhibited cell migration and invasion, modulated epithelial-to-mesenchymal transition (EMT), and impaired stemness properties in HCC cells. The combinatorial use of OGE enhanced the efficacy of cisplatin and potentially restored liver function in a mouse orthotopic tumor model. Our findings may provide an alternate approach to improving chemotherapy efficacy in HCC.

Matrix stiffness-dependent PD-L2 deficiency improves SMYD3/xCT-mediated ferroptosis and the efficacy of anti-PD-1 in HCC

IntroductionHeterogeneous tissue stiffening promotes tumor progression and resistance, and predicts a poor clinical outcome in patients with hepatocellular carcinoma (HCC). Ferroptosis, a congenital tumor suppressive mechanism, mediates the anticancer activity of various tumor suppressors, including immune checkpoint inhibitors, and its induction is currently considered a promising treatment strategy. However, the role of extracellular matrix (ECM) stiffness in regulating ferroptosis and ferroptosis-targeted resistance in HCC remains unclear. ObjectivesThis research aimed to explore how extracellular matrix stiffness affects ferroptosis and its treatment efficacy in HCC. MethodsFerroptosis analysis was confirmed via cell activity, intracellular ferrous irons, and mitochondrial pathology assays. Baseline PD-L2, SMYD3, and SLC7A11 (xCT) were evaluated in 67 sorafenib-treated patients with HCC (46 for non-responder and 21 for responder) from public data. The combined efficacy of shPD-L2, sorafenib, and anti-PD-1 antibody in HCC was investigated in vivo. ResultsHere, we revealed that matrix stiffness-induced PD-L2 functions as a suppressor of xCT-mediated ferroptosis to promote cancer growth and sorafenib resistance in patients with HCC. Mechanically, matrix stiffening induced the expression of PD-L2 by activating SMYD3/H3K4me3, which acts as an RNA binding protein to enhance the mRNA stability of FTL and elevate its protein level. Knockdown of PD-L2 significantly promoted xCT-mediated ferroptosis induced by RSL3 or sorafenib on stiff substrate via FTL, whereas its overexpression abolished these upward trends. Notably, PD-L2 deletion in combination with sorafenib and anti-PD-1 antibody significantly sensitized HCC cells and blunted cancer growth in vivo. Additionally, we found the ferroptosis- and immune checkpoint-related prognostic genes that combined PD-L2, SLC7A11 and SYMD3 well predict the clinical efficacy of sorafenib in patients with HCC. ConclusionThese findings expand our understanding of the mechanics-dependent PD-L2 role in ferroptosis, cancer progression and resistance, providing a basis for the clinical translation of PD-L2 as a therapeutic target or diagnostic biomarker.

Chemotherapy-induced acetylation of ACLY by NAT10 promotes its nuclear accumulation and acetyl-CoA production to drive chemoresistance in hepatocellular carcinoma

Chemotherapeutic efficacy is seriously impeded by chemoresistance in more than half of hepatocellular carcinoma (HCC) patients. However, the mechanisms involved in chemotherapy-induced upregulation of chemoresistant genes are not fully understood. Here, this study unravels a novel mechanism controlling nuclear acetyl-CoA production to activate the transcription of chemoresistant genes in HCC. NAT10 is upregulated in HCC tissues and its upregulation is correlated with poor prognosis of HCC patients. NAT10 is also upregulated in chemoresistant HCC cells. Targeting NAT10 increases the cytotoxicity of chemotherapy in HCC cells and mouse xenografts. Upon chemotherapy, NAT10 translocates from the nucleolus to the nucleus to activate the transcription of CYP2C9 and PIK3R1. Additionally, nuclear acetyl-CoA is specifically upregulated by NAT10. Mechanistically, NAT10 binds with ACLY in the nucleus and acetylates ACLY at K468 to counteract the SQSTM1-mediated degradation upon chemotherapy. ACLY K468-Ac specifically accumulates in the nucleus and increases nuclear acetyl-CoA production to activate the transcription of CYP2C9 and PIK3R1 through enhancing H3K27ac. Importantly, K468 is required for nuclear localization of ACLY. Significantly, ACLY K468-Ac is upregulated in HCC tissues, and ablation of ACLY K468-Ac sensitizes HCC cells and mouse xenografts to chemotherapy. Collectively, these findings identify NAT10 as a novel chemoresistant driver and the blockage of NAT10-mediated ACLY K468-Ac possesses the potential to attenuate HCC chemoresistance.

Epigallocatechin Gallate Derivative Y6 Reverses Oxaliplatin Resistance in Hepatocellular Carcinoma via Targeting the MiR-338-3p/HIF-1α/TWIST Axis to Inhibit EMT.

The emergence of drug resistance to oxaliplatin (OXA) is one of the critical obstacles in the therapy of advanced Hepatocellular Carcinoma (HCC). As an ethyl derivative of the natural compound epigallocatechin gallate (epigallocatechin-3-gallate, EGCG), Y6 was found to be able to enhance the sensitivity of HCC cells to doxorubicin. This study aimed to investigate the effect of Y6 on oxaliplatin resistance in HCC. MTT was used to determine the reversal effect of Y6 on OXA resistance. To further explore the reversal mechanism, we treated OXA alone or in combination with Y6 or EGCG in drugresistant cells and observed the morphological changes of the cells. At the same time, transwell assay was used to detect the invasion and migration ability of cells. Moreover, Real-time PCR and Western blot analysis were performed to determine the expression levels of the miR-338-3p gene, HIF-1α/Twist proteins, and EMT-related proteins. We found that Y6 could inhibit the proliferation of HCC cells and effectively reverse the drug resistance of oxaliplatin-resistant human liver cancer cells (SMMC-7721/OXA) to OXA, and the reversal effect was more significant than that of its lead drug EGCG. Most of the cells in the control group and OXA group showed typical mesenchymal-like cell morphology, while most of the cells in co-administration groups showed typical epithelioid cell morphology, and the ability of the cells to invade and migrate decreased dramatically, particularly in Y6 plus OXA group. At the same time, Y6 could up-regulate the EMT epithelial marker protein E-cadherin and down-regulate the interstitial marker protein Vimentin. In addition, in co-administration groups, the expression of miR-338-3p was up-regulated, while the expression of HIF-1α and Twist was down-regulated. Y6 significantly enhanced the susceptibility of drug-resistant cells to OXA, and the process may be related to the regulation of miR-338-3p/HIF-1α / TWIST pathway to inhibit EMT. Therefore, Y6 could be considered an effective medication resistance reversal agent, which could improve the therapeutic effect for hepatocellular cancer patients.

BNIP3-mediated mitophagy boosts the competitive growth of Lenvatinib-resistant cells via energy metabolism reprogramming in HCC

An increasing evidence supports that cell competition, a vital selection and quality control mechanism in multicellular organisms, is involved in tumorigenesis and development; however, the mechanistic contributions to the association between cell competition and tumor drug resistance remain ill-defined. In our study, based on a contructed lenvitinib-resistant hepatocellular carcinoma (HCC) cells display obvious competitive growth dominance over sensitive cells through reprogramming energy metabolism. Mechanistically, the hyperactivation of BCL2 interacting protein3 (BNIP3) -mediated mitophagy in lenvatinib-resistant HCC cells promotes glycolytic flux via shifting energy production from mitochondrial oxidative phosphorylation to glycolysis, by regulating AMP-activated protein kinase (AMPK) -enolase 2 (ENO2) signaling, which perpetually maintaining lenvatinib-resistant HCC cells’ competitive advantage over sensitive HCC cells. Of note, BNIP3 inhibition significantly sensitized the anti-tumor efficacy of lenvatinib in HCC. Our findings emphasize a vital role for BNIP3-AMPK-ENO2 signaling in maintaining the competitive outcome of lenvitinib-resistant HCC cells via regulating energy metabolism reprogramming; meanwhile, this work recognizes BNIP3 as a promising target to overcome HCC drug resistance.