Mitochondrial Plasticity Promotes Resistance to Sorafenib and Vulnerability to STAT3 Inhibition in Human Hepatocellular Carcinoma

The multi-kinase inhibitor sorafenib is a primary treatment modality for advanced-stage hepatocellular carcinoma (HCC). However, the therapeutic benefits are short-lived due to innate and acquired resistance. Here, we examined how HCC cells respond to sorafenib and adapt to continuous and prolonged exposure to the drug. Sorafenib-adapted HCC cells show a profound reprogramming of mitochondria function and marked activation of genes required for mitochondrial protein translation and biogenesis. Mitochondrial ribosomal proteins and components of translation and import machinery are increased in sorafenib-resistant cells and sorafenib-refractory HCC patients show similar alterations. Sorafenib-adapted cells also exhibited increased serine 727 phosphorylated (pSer727) STAT3, the prevalent form in mitochondria, suggesting that STAT3 might be an actionable target to counteract resistance. Consistently, a small-molecule STAT3 inhibitor reduces pSer727, reverts mitochondrial alterations, and enhances the response to sorafenib in resistant cells. These results sustain the importance of mitochondria plasticity in response to sorafenib and identify a clinically actionable strategy for improving the treatment efficacy in HCC patients.

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USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Oncogenesis ◽

10.1038/s41389-021-00338-7 ◽

2021 ◽

Vol 10 (7) ◽

Author(s):

Ruize Gao ◽

David Buechel ◽

Ravi K. R. Kalathur ◽

Marco F. Morini ◽

Mairene Coto-Llerena ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Transcriptional Activity ◽

Kinase Inhibitor ◽

Aerobic Glycolysis ◽

Hypoxia Inducible Factor ◽

Aberrant Expression ◽

Key Enzyme ◽

Hcc Cells

AbstractUnderstanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

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Mitochondrial Protein Translation: Emerging Roles and Clinical Significance in Disease

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.675465 ◽

2021 ◽

Vol 9 ◽

Author(s):

Fei Wang ◽

Deyu Zhang ◽

Dejiu Zhang ◽

Peifeng Li ◽

Yanyan Gao

Keyword(s):

Ribosomal Proteins ◽

Large Fraction ◽

Mitochondrial Protein ◽

Mitochondrial Diseases ◽

Protein Translation ◽

Genetic System ◽

Mitochondrial Rna ◽

Trna Synthetases ◽

Translation Factors ◽

Genes Encoding

Mitochondria are one of the most important organelles in cells. Mitochondria are semi-autonomous organelles with their own genetic system, and can independently replicate, transcribe, and translate mitochondrial DNA. Translation initiation, elongation, termination, and recycling of the ribosome are four stages in the process of mitochondrial protein translation. In this process, mitochondrial protein translation factors and translation activators, mitochondrial RNA, and other regulatory factors regulate mitochondrial protein translation. Mitochondrial protein translation abnormalities are associated with a variety of diseases, including cancer, cardiovascular diseases, and nervous system diseases. Mutation or deletion of various mitochondrial protein translation factors and translation activators leads to abnormal mitochondrial protein translation. Mitochondrial tRNAs and mitochondrial ribosomal proteins are essential players during translation and mutations in genes encoding them represent a large fraction of mitochondrial diseases. Moreover, there is crosstalk between mitochondrial protein translation and cytoplasmic translation, and the imbalance between mitochondrial protein translation and cytoplasmic translation can affect some physiological and pathological processes. This review summarizes the regulation of mitochondrial protein translation factors, mitochondrial ribosomal proteins, mitochondrial tRNAs, and mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in the mitochondrial protein translation process and its relationship with diseases. The regulation of mitochondrial protein translation and cytoplasmic translation in multiple diseases is also summarized.

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

10.21203/rs.3.rs-97867/v1 ◽

2020 ◽

Author(s):

Tong-tong Li ◽

Jie Mou ◽

Yao-jie Pan ◽

Fu-chun Huo ◽

Wen-qi Du ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Kinase Inhibitor ◽

Flow Cytometric Analysis ◽

Luciferase Reporter ◽

Advanced Hepatocellular Carcinoma ◽

Cell Viability Assay ◽

Resistant Cells

Abstract Background: Kinase inhibitor sorafenib is the first-line targeted drug for advanced hepatocellular carcinoma (HCC) patients. However, the appearance of anti-cancer agents’ resistance has limited its therapeutic effect. Methods: In this study, quantitative real-time PCR (qPCR) and Western Blot were utilized to detect the levels of PAK5 in HCC sorafenib-resistant cells and their parental cells. The biological functions of miR-138-1-3p and PAK5 in sorafenib-resistant cells and their parental cells were explored by cell viability assay, plate colony formation assay and flow cytometric analysis. The potential mechanisms of PAK5 were evaluated via co-immunoprecipitation (co-IP), immunofluorescence, dual luciferase reporter assay and chromatin immunoprecipitation (ChIP). The effects of miR-138-1-3p and PAK5 on HCC sorafenib chemoresistant characteristics were investigated by a xenotransplantation model. Results: We detected significant down-regulation of miR-138-1-3p and up-regulation of PAK5 in HCC sorafenib resistance cell lines. Mechanical studies revealed that miR-138-1-3p reduced the protein expression of PAK5 by directly targeting the 3′-UTR of PAK5 mRNA. In addition, we verified that PAK5 elevated the phosphorylation and nuclear translocation of β-catenin that enhanced the transcriptional activity of multidrug resistance protein ABCB1. Conclusions: PAK5 contributed to the sorafenib chemoresistant characteristics of HCC by activity β-catenin/ABCB1 signaling pathway. Our findings identified the correlation between miR-138-1-3p and PAK5 and the molecular mechanisms of PAK5-mediated HCC sorafenib resistance, which provided a potential therapeutic target for advanced HCC patients.

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HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis

eLife ◽

10.7554/elife.46976 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 17

Author(s):

Shuping Zhang ◽

Alejandra Macias-Garcia ◽

Jacob C Ulirsch ◽

Jason Velazquez ◽

Vincent L Butty ◽

...

Keyword(s):

Iron Deficiency ◽

Mitochondrial Function ◽

Ribosomal Proteins ◽

Target Genes ◽

Mitochondrial Protein ◽

Erythroid Differentiation ◽

Protein Translation ◽

Ribosome Profiling ◽

Underlying Mechanisms

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.

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eIF6 Promotes the Malignant Progression of Human Hepatocellular Carcinoma via the mTOR Signaling Pathway

10.21203/rs.3.rs-78737/v1 ◽

2020 ◽

Author(s):

Liping Sun ◽

Shuguang Liu ◽

Xiaopai Wang ◽

Xuefeng Zheng ◽

Ya Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathway ◽

Prognostic Value ◽

Molecular Mechanisms ◽

Protein Translation ◽

Translation Initiation Factor ◽

Gene Set Enrichment Analysis ◽

The Cancer Genome Atlas ◽

Initiation Factor ◽

Hcc Cells

Abstract Background Eukaryotic translation initiation factor 6 (eIF6) has a crucial function in the maturation of 60S ribosomal subunits, and it controls the initiation of protein translation. Although emerging studies indicate that eIF6 is aberrantly expressed in various types of cancers, the functions and underlying molecular mechanisms of eIF6 in the pathological progression of hepatocellular carcinoma (HCC) remain unclear. This study aimed to evaluate the potential diagnostic and prognostic value of eIF6 in patients with HCC. Methods HCC samples enrolled from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and our cohort were used to explore the role and mechanism of eIF6 in HCC. The diagnostic power of eIF6 was verified by receiver operating characteristic curve (ROC) analysis and its prognostic value was assessed by Kaplan-Meier analysis, and then related biological functions of eIF6 were determined in vitro and in vivo cancer models. In addition, potential molecular mechanism of eIF6 in HCC was unveiled by the gene set enrichment analysis and western blot assay. Results We demonstrated that eIF6 expression was markedly increased in HCC, and elevated eIF6 expression correlated with pathological progression of HCC. Besides, eIF6 served as not only a new diagnostic biomarker but also an independent risk factor for OS in HCC patients. Functional studies indicated that the deletion of eIF6 displayed tumor-suppressor activity in HCC cells. Furthermore, we found that eIF6 could activate the mTOR-related signaling pathway and regulate the expression level of its target genes, such as CCND1, CDK4, CDK6, MYC, CASP3 and CTNNBL1, and these activities promoted proliferation and invasion of HCC cells. Conclusions The findings of this study provided a novel basis for understanding the potential role of eIF6 in promoting tumor growth and invasion, and exploited a promising strategy for improving diagnosis and prognosis of HCC.

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PERK/ATF4-Dependent ZFAS1 Upregulation Is Associated with Sorafenib Resistance in Hepatocellular Carcinoma Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22115848 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5848

Author(s):

Jiunn-Chang Lin ◽

Pei-Ming Yang ◽

Tsang-Pai Liu

Keyword(s):

Hepatocellular Carcinoma ◽

Kinase Inhibitor ◽

Advanced Hepatocellular Carcinoma ◽

Sorafenib Therapy ◽

Activating Transcription Factor 4 ◽

Signaling Axis ◽

Cancer Phenotypes ◽

The Unfolded Protein Response ◽

Hcc Cells

Sorafenib, a multi-kinase inhibitor, is the first-line treatment for advanced hepatocellular carcinoma (HCC) patients. However, this drug only provides a short improvement of patients’ overall survival, and drug resistance is commonly developed. Thus, the identification of resistant factor(s) or biomarker(s) is needed to develop more efficient therapeutic strategies. Long, non-coding RNAs (lncRNAs) have recently been viewed as attractive cancer biomarkers and drive many important cancer phenotypes. A lncRNA, ZFAS1 (ZNFX1 antisense RNA 1) has been found to promote HCC metastasis. This study found that sorafenib induced ZFAS1 expression specifically in sorafenib-resistant HCC cells. Although ZFAS1 knockdown did not restore the sensitivity of HCC cells to sorafenib, its expression may act as a resistant biomarker for sorafenib therapy. Bioinformatics analysis predicted that sorafenib tended to induce pathways related to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in sorafenib-resistant HCC cells. In vitro experimental evidence suggested that sorafenib induced protein kinase RNA-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-dependent ZFAS1 expression, and sorafenib resistance could be overcome by PERK/ATF inhibitors. Therefore, PERK/ATF4/ZFAS1 signaling axis might be an attractive therapeutic and prognostic biomarker for sorafenib therapy in HCC.

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Bis-benzylidine Piperidone RA190 Treatment of Hepatocellular Carcinoma via Binding RPN13 and Inhibiting NF-κB Signaling

10.21203/rs.2.18702/v3 ◽

2020 ◽

Author(s):

Ruey-Shyang Soong ◽

Ravi K. Anchoori ◽

Richard B. S. Roden ◽

Rou-Ling Cho ◽

Yi-Chan Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Proteasome Inhibitor ◽

Time Course ◽

Kinase Inhibitor ◽

Xenograft Model ◽

Therapeutic Activity ◽

Rapid Accumulation ◽

Hcc Cells

Abstract Background According to GLOBOSCAN, hepatocellular carcinoma (HCC) claimed 782,000 lives in 2018. The tyrosine kinase inhibitor sofafenib is used to treat HCC, but new anticancer agents targeting different pathways are urgently needed to improve outcomes for patients with advanced disease. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as demonstrated by bortezomib treatment of multiple myeloma. However, resistance does emerge, and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, an ubiquitin receptor subunit of the proteasome’s 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Considerable evidence suggests that nuclear factor κB (NF-κB) signaling, which is dependent upon the proteasome, is a major driver of inflammation-associated cancers, including HCC. Methods Human HCC cell lines were treated with titrations of RA190. The time course of endoplasmic reticulum stress and NF-κB-related mechanisms by which RA190 may trigger apoptosis were assessed. The therapeutic activity of RA190 was also determined in an orthotopic HCC xenograft mouse model. Results RA190 is toxic to HCC cells and synergizes with sofafenib. RA190 triggers rapid accumulation of polyubiquitinated proteins, unresolved endoplasmic reticulum stress, and cell death via apoptosis. RA190 blocks proteasomal degradation of IκBα and consequent release of NF-κB into the nuclei of HCC cells. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. Conclusions RA190 has therapeutic activity in a xenograft model, and with sorafenib exhibited synergetic killing of HCC cells in vitro, suggesting further exploration of such a combination treatment of HCC is warranted.

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Flt3/ITD Confers Resistance to FLT3 Inhibitor AC220 By up-Regulating Runx1

Blood ◽

10.1182/blood.v124.21.2223.2223 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2223-2223

Author(s):

Tomohiro Hirade ◽

Mariko Abe ◽

Chie Onishi ◽

Seiji Yamaguchi ◽

Seiji Fukuda

Keyword(s):

Tyrosine Kinase ◽

Prolonged Exposure ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Specific Inhibitor ◽

Hematopoietic Stem ◽

Runx1 Expression ◽

Cells Cultured ◽

Resistant Cells

Abstract FMS-like tyrosine kinase 3 (FLT3) is a membrane type tyrosine kinase and has important roles for the proliferation and differentiation of the hematopoietic cells. The Internal Tandem Duplications of FLT3 (FLT3/ITD) is detected in approximately 30 % of patients with acute myeloid leukemia (AML) and the prognoses of FLT3/ITD+ AML are very poor. While a number of inhibitors targeting FLT3 tyrosine kinase have been developed, few drugs are effective for the FLT3/ITD+ AML because of emergence of resistant cells against the drugs. Recently, AC220 (Quizartinib), a second generation class III tyrosine kinase inhibitor (TKI) for FLT3/ITD+ AML was developed and used in clinical trials. Although AC220 is a more potent and specific inhibitor for FLT3/ITD compared to the other TKIs, report demonstrates that prolonged exposure to AC220 can generate resistant clones to AC220 in FLT3/ITD+ cells (Smith et al. Nature 2012). These findings underscore the need to develop additional therapeutic strategies to overcome the resistance of FLT3/ITD+ AML to TKIs. However, the mechanism responsible for drug resistance of FLT3/ITD+ AML cells remains to be investigated. We previously reported that mRNA expression of RUNX1, a core-binding transcription factor that regulates the differentiation and proliferation of hematopoietic stem cells, is significantly higher in FLT3/ITD+AML cells compared to FLT3/ITD-AML cells (Hirade et al. ASH 2013). Although loss of RUNX1 function (i.e. RUNX1/ETO fusion gene) contributes to the development of AML, RUNX1 also promotes survival of AML cells (Goyama et al. JCI 2013) and can function as an oncogene in cancer cells (Kilbey et al. Cancer Research 2010). These findings lead us hypothesize that RUNX1 may confer resistance of AML cells to TKIs. In the present study, we investigated if Runx1 is involved in the refractory phenotype of Flt3/ITD+cells to AC220. Transduction of Flt3/ITD into IL3-dependent mouse 32D cells allowed the cells proliferate in a growth factor independent fashion, concomitant with up-regulation of Runx1 mRNA level, similar to the patients’ samples with FLT3/ITD+AML. Silencing Runx1 expression using shRNA resulted in 70% reduction of Flt3/ITD+32D cells that proliferated in the absence of growth factors. Similarly, incubating the Flt3/ITD+32D cells with 0.5nM AC220 inhibited their factor independent proliferation by 95%, which was further accentuated up to 99% by the combination with shRNA mediated silencing of Runx1. Although the number of factor independent Flt3/ITD+32D cells cultured in the presence of 2nM AC220 rapidly declined within 96 hours, the residual cells subsequently re-proliferate within 14 days and became no longer sensitive to AC220. Surprisingly, the expression of Runx1 mRNA in the resistant cells to AC220 was 5.0±0.2 fold higher (P<0.05) compared to control Flt3/ITD+32D cells sensitive to AC220. Silencing Runx1 using shRNA abrogated the proliferation of AC220-resistant Flt3/ITD+32D cells cultured in the presence of 2nM AC220, leading to 99.5% reduction in the viable cells. Our data indicates that knocking down Runx1 expression enhances the cytotoxic effect of AC220 on Flt3/ITD+32D cells and that Runx1 expression is significantly up-regulated by the AC220 resistance cells. Moreover, Runx1 knockdown recovered the cytotoxicity of AC220 in the refractory Flt3/ITD+32D cells, demonstrating that Flt3/ITD confers resistance to AC220 by up-regulating the expression of Runx1. These findings demonstrate that antagonizing RUNX1 may represent potential therapeutic strategy in the patients with FLT3/ITD+ AML that become refractory to AC220. Disclosures No relevant conflicts of interest to declare.

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CISD2 Promotes Resistance to Sorafenib-Induced Ferroptosis by Regulating Autophagy in Hepatocellular Carcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.657723 ◽

2021 ◽

Vol 11 ◽

Author(s):

Bowen Li ◽

Shibo Wei ◽

Liang Yang ◽

Xueqiang Peng ◽

Yingbo Ma ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Poor Prognosis ◽

Kinase Inhibitor ◽

Bioinformatic Analysis ◽

Advanced Hepatocellular Carcinoma ◽

Iron Ions ◽

Sorafenib Treatment ◽

Effective Therapeutic Strategy ◽

Detect Gene Expression ◽

Hcc Cells

PurposeSorafenib is a multi-kinase inhibitor that is used as a standard treatment for advanced hepatocellular carcinoma (HCC). However, the mechanism of sorafenib resistance in HCC is still unclear. It has been shown that CISD2 expression is related to the progression and poor prognosis of HCC. Here, we show a new role for CISD2 in sorafenib resistance in HCC.MethodsBioinformatic analysis was used to detect the expression of negative regulatory genes of ferroptosis in sorafenib-resistant samples. The concentration gradient method was used to establish sorafenib-resistant HCC cells. Western blot was used to detect the protein expression of CISD2, LC3, ERK, PI3K, AKT, mTOR, and Beclin1 in HCC samples. Quantitative real-time PCR (qPCR) was used to detect gene expression. CISD2 shRNA and Beclin1 shRNA were transfected to knock down the expression of the corresponding genes. Cell viability was detected by a CCK-8 assay. ROS were detected by DCFH-DA staining, and MDA and GSH were detected with a Lipid Peroxidation MDA Assay Kit and Micro Reduced Glutathione (GSH) Assay Kit, respectively. Flow cytometry was used to detect apoptosis and the levels of ROS and iron ions.ResultsCISD2 was highly expressed in HCC cells compared with normal cells and was associated with poor prognosis in patients. Knockdown of CISD2 promoted a decrease in the viability of drug-resistant HCC cells. CISD2 knockdown promoted sorafenib-induced ferroptosis in resistant HCC cells. The levels of ROS, MDA, and iron ions increased, but the change in GSH was not obvious. Knockdown of CISD2 promoted uncontrolled autophagy in resistant HCC cells. Inhibition of autophagy attenuated CISD2 knockdown-induced ferroptosis. The autophagy promoted by CISD2 knockdown was related to Beclin1. When CISD2 and Beclin1 were inhibited, the effect on ferroptosis was correspondingly weakened.ConclusionInhibition of CISD2 promoted sorafenib-induced ferroptosis in resistant cells, and this process promoted excessive iron ion accumulation through autophagy, leading to ferroptosis. The combination of CISD2 inhibition and sorafenib treatment is an effective therapeutic strategy for resistant HCC.

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Bis-benzylidine Piperidone RA190 Treatment of Hepatocellular Carcinoma via Binding RPN13 and Inhibiting NF-κB Signaling

10.21203/rs.2.18702/v1 ◽

2019 ◽

Author(s):

Yu-Chiau Shyu ◽

Ruey-Shyang Soong ◽

Anchoori Ravi ◽

Richard Roden ◽

Yi-Chan Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Anticancer Agents ◽

Proteasome Inhibitor ◽

Kinase Inhibitor ◽

Nuclear Factor Κb ◽

Time Frame ◽

Killing Effect ◽

Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third leading cause of cancer mortality worldwide. The development of new anticancer agents targeting different pathways is imperative to improve the advanced HCC. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as first demonstrated by the success of bortezomib treatment for multiple myeloma. However, resistance does emerge and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, a ubiquitin receptor subunit of the proteasome’s 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Methods Human HCC cell lines were treated by RA190 in vitro in different concentration and time frame. We checked the killing effect and the possible mechanisms that lead the tumor apoptosis. We also performed the orthotopic HCC animal model to show the RA190 had significant killing effect in vivo. Results We showed RA190 is also toxic to HCC cells by triggering the rapid build-up of polyubiquitinated proteins, resulting in endoplasmic reticulum stress and the induction of cell death via apoptosis. Considerable evidence suggests that nuclear factor κB (NF-κB) signal is essential for promoting inflammation-associated cancer. Here, we showed that RA190 inhibited the NF-κB pathway in HCC by preventing the degradation of IκBα via the proteasome. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. We therefore explored combining RA190 with a tyrosine kinase inhibitor currently used to the treat HCC, Sorafenib. Conclusions RA190 and Sorafenib exhibited synergetic killing of HCC cells in vitro, suggesting further exploration of such a combination treatment of HCC is warranted.

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