Resistance Of Hepatocellular Carcinoma Cells Research Articles

NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma.

Solid evidence shows that tumor-initiating cells (T-ICs) are the root of tumor relapse and drug resistance, which lead to a poor prognosis in patients with hepatocellular carcinoma (HCC). Through an in vitro liver T-IC enrichment approach, we identified nuclear factor (erythroid-derived 2)-like 2 (NRF2) as a transcription regulator that is significantly activated in enriched liver T-IC populations. In human HCCs, NRF2 was found to be overexpressed, which was associated with poor patient survival. Through a lentiviral based knockdown approach, NRF2 was found to be critical for regulating liver T-IC properties, including self-renewal, tumorigenicity, drug resistance and expression of liver T-IC markers. Furthermore, we found that ROS-induced NRF2 activation regulates sorafenib resistance in HCC cells. Mechanistically, NRF2 was found to physically bind to the promoter of sonic hedgehog homolog (SHH), which triggers activation of the sonic hedgehog pathway. The effect of NRF2 knockdown was eliminated upon administration of recombinant SHH, demonstrating that NRF2 mediated T-IC function via upregulation of SHH expression. Our study suggests a novel regulatory mechanism for the canonical sonic hedgehog pathway that may function through the NRF2/SHH/GLI signaling axis, thus mediating T-IC phenotypes.

LncRNA DANCR Promotes Sorafenib Resistance via Activation of IL-6/STAT3 Signaling in Hepatocellular Carcinoma Cells.

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

Low‑intensity ultrasound enhances the antitumor effects of doxorubicin on hepatocellular carcinoma cells through the ROS‑miR‑21‑PTEN axis.

Hepatocellular carcinoma (HCC) is a type of liver cancer and is a leading cause of cancer-associated mortality. In China, ~466,000 patients are diagnosed with HCC and it is responsible for ~422,000 cases of mortality each year. Surgery is the most effective treatment available; however it is only suitable for patients with early-stage HCC. Chemotherapy has been confirmed as a necessary treatment for patients with advanced HCC, although drug resistance may limit its clinical outcome. Low intensity ultrasound (LIUS) represents a novel therapeutic approach to treat patients with HCC; however, its underlying molecular mechanism remains unclear. In the present study, cell viability, apoptosis and reactive oxygen species (ROS) generation were determined via Cell Counting Kit-8, flow cytometry and 2′,7′-dichlorofluorescein diacetate assays, respectively. The expression of miRNA in HCC cells following exposure to LIUS and doxorubicin (Dox) was analyzed using a microarray and reverse transcription-quantitative polymerase chain reaction analysis. It was revealed treatment with LIUS in combination with Dox was able to induce apoptosis of Huh7 cells, increasing the intracellular levels of reactive oxygen species (ROS) and malondialdehyde. Glutathione peroxidase and superoxide dismutase 1 are ROS-scavenging enzymes, which serve important roles in the oxidative balance, preventing oxidative stress. The protein expression levels of these two enzymes were significantly decreased following treatment with LIUS combined with Dox. The present results suggested that LIUS may decrease Dox resistance in HCC cells and that LIUS may be combined with chemotherapy to treat HCC. By performing microarray analysis, the expression levels of microRNA-21 (miR-21) were decreased following treatment with LIUS combined with Dox. Functional experiments showed that knockdown of miR-21 enhanced the antitumor activity of Dox, whereas overexpression of miR-21 reversed these effects. Phosphatase and tensin homolog (PTEN), a well-known tumor suppressor, was revealed to be a direct target of miR-21, and its translation was suppressed by miR-21. Finally, it was determined that combined treatment of LIUS and Dox induced anticancer effects by blocking the activation of the AKT/mTOR pathway, as demonstrated by the downregulation of phosphorylated (p-)AKT and p-mTOR; N-acetylcysteine, a general ROS inhibitor reversed the suppressive effects on the AKT/mTOR pathway mediated by LIUS and Dox. Collectively, the present results suggested that LIUS increased cell sensitivity to Dox via the ROS/miR-21/PTEN pathway. Chemotherapy combined with LIUS may represent a novel effective therapeutic strategy to treat patients with advanced HCC.

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

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

Global transcriptomic study of circRNAs expression profile in sorafenib resistant hepatocellular carcinoma cells.

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

Acetylshikonin Sensitizes Hepatocellular Carcinoma Cells to Apoptosis through ROS-Mediated Caspase Activation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown strong and explicit cancer cell-selectivity, which results in little toxicity toward normal tissues, and has been recognized as a potential, relatively safe anticancer agent. However, several cancers are resistant to the apoptosis induced by TRAIL. A recent study found that shikonin b (alkannin, 5,8-dihydroxy-2-[(1S)-1-hydroxy-4-methylpent-3-en-1-yl]naphthalene-1,4-dione) might induce apoptosis in TRAIL-resistant cholangiocarcinoma cells through reactive oxygen species (ROS)-mediated caspases activation. However, the strong cytotoxic activity has limited its potential as an anticancer drug. Thus, the current study intends to discover novel shikonin derivatives which can sensitize the liver cancer cell to TRAIL-induced apoptosis while exhibiting little toxicity toward the normal hepatic cell. The trypan blue exclusion assay, western blot assay, 4′,6-diamidino-2-phenylindole (DAPI) staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as well as the ‘comet’ assay, were used to study the underlying mechanisms of cell death and to search for any mechanisms of an enhancement of TRAIL-mediated apoptosis in the presence of ASH. Herein, we demonstrated that non-cytotoxic doses of acetylshikonin (ASH), one of the shikonin derivatives, in combination with TRAIL, could promote apoptosis in HepG2 cells. Further studies showed that application of ASH in a non-cytotoxic dose (2.5 μM) could increase intracellular ROS production and induce DNA damage, which might trigger a cell intrinsic apoptosis pathway in the TRAIL-resistant HepG2 cell. Combination treatment with a non-cytotoxic dose of ASH and TRAIL activated caspase and increased the cleavage of PARP-1 in the HepG2 cell. However, when intracellular ROS production was suppressed by N-acetyl-l-cysteine (NAC), the synergistic effects of ASH and TRAIL on hepatocellular carcinoma (HCC) cell apoptosis was abolished. Furthermore, NAC could alleviate p53 and the p53 upregulated modulator of apoptosis (PUMA) expression induced by TRAIL and ASH. Small (or short) interfering RNA (siRNA) targeting PUMA or p53 significantly reversed ASH-mediated sensitization to TRAIL-induced apoptosis. In addition, Bax gene deficiency also abolished ASH-induced TRAIL sensitization. An orthotopical HCC implantation mice model further confirmed that co-treated ASH overcomes TRAIL resistance in HCC cells without exhibiting potent toxicity in vivo. In conclusion, the above data suggested that ROS could induce DNA damage and activating p53/PUMA/Bax signaling, and thus, this resulted in the permeabilization of mitochondrial outer membrane and activating caspases as well as sensitizing the HCC cell to apoptosis induced by TRAIL and ASH treatment.

MiR-541 potentiates the response of human hepatocellular carcinoma to sorafenib treatment by inhibiting autophagy

ObjectiveAutophagy participates in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib. We investigated the feasibility of sensitising HCC cells to sorafenib by modulating miR-541-initiated...

Downregulation of LncRNA GAS5 promotes liver cancer proliferation and drug resistance by decreasing PTEN expression.

Accumulating evidence has shown that the long noncoding RNAs (lncRNAs) play a crucial role in the regulation of hepatocellular carcinoma (HCC) progression and drug resistance. In this study, we aimed to investigate the biological function roles of lncRNAs growth arrest-specific 5 (GAS5) and its underlying molecular mechanism in the development of HCC. qRT-PCR was used to detect GAS5, miR-21, and PTEN levels. MTT, cell counting assays, and xenograft mouse model were applied to measure cell proliferation rate in vitro and in vivo. The luciferase reporter assay and RNA immune-precipitation assay were introduced to evaluate the relationship between GAS5 and miR-21. We found that GAS5 was downregulated in HCC cell lines and tumor tissues. Knockdown of GAS5 enhanced HCC cell proliferation in vitro and in vivo and increased HCC cell resistance to doxorubicin. GAS5 acted as a sponge for miR-21 silencing and consequently led to the elevation of PTEN expression. Our data demonstrated that GAS5 functioned as a tumor suppressor role in HCC through regulation of miR-21-PTEN singling pathways, suggesting a potential application of GAS5 in HCC therapy.

Circ_0003418 Inhibits Tumorigenesis And Cisplatin Chemoresistance Through Wnt/β-Catenin Pathway In Hepatocellular Carcinoma.

BackgroundAccumulating evidences indicate that circRNAs play important roles in the progression and chemoresistance of human cancers. The present study is designated for researching the roles of circ_0003418 in hepatocellular carcinoma (HCC).MethodsWe detected the expression profile of circ_0003418 in human HCC tissues and cell lines by quantitative real-time-PCR assays. CCK-8 assay, transwell migration assay, transwell invasion assay and drug-sensitivity analysis were carried out to estimate the effects of circ_0003418 on HCC cells' proliferation, migration, invasion and resistance to cisplatin, respectively. Mouse xenograft model was conducted to monitor the role of circ_0003418 in cisplatin resistance in vivo. Western blotting was performed to explore the changes of the Wnt/β-catenin pathway after knockdown of circ_0003418. The rescue experiment was carried out to explore circ_0003418-activated biological functions through Wnt/β-catenin pathway.ResultsThe expression level of circ_0003418 was downregulated in HCC tissues and cell lines, and the level correlated with tumor size, TNM stage and HBsAg level in HCC patients. circ_0003418 knockdown promoted HCC cells' proliferation, migration, and invasion. Additionally, suppression of circ_0003418 enhanced cisplatin resistance of HCC cells in vivo and vitro. Knockdown of circ_0003418 activated the Wnt/β-catenin signalling pathway in HCC cells. The effect of circ-0003418 on sensitivity of HCC cells to cisplatin was reversed after inhibition of Wnt/β-catenin pathway.Conclusioncirc-0003418 exerts an antitumorigenic role in HCC and advances the sensitivity of HCC cells to cisplatin by restraining the Wnt/β-catenin pathway. Thus, circ-0003418 may represent a novel biomarker and provide us a new strategy for the treatment of HCC.

Histone Deacetylase Expressions in Hepatocellular Carcinoma and Functional Effects of Histone Deacetylase Inhibitors on Liver Cancer Cells In Vitro.

Hepatocellular carcinoma (HCC) is a leading cause for deaths worldwide. Histone deacetylase (HDAC) inhibition (HDACi) is emerging as a promising therapeutic strategy. However, most pharmacological HDACi unselectively block different HDAC classes and their molecular mechanisms of action are only incompletely understood. The aim of this study was to systematically analyze expressions of different HDAC classes in HCC cells and tissues and to functionally analyze the effect of the HDACi suberanilohydroxamic acid (SAHA) and trichostatin A (TSA) on the tumorigenicity of HCC cells. The gene expression of all HDAC classes was significantly increased in human HCC cell lines (Hep3B, HepG2, PLC, HuH7) compared to primary human hepatocytes (PHH). The analysis of HCC patient data showed the increased expression of several HDACs in HCC tissues compared to non-tumorous liver. However, there was no unified picture of regulation in three different HCC patient datasets and we observed a strong variation in the gene expression of different HDACs in tumorous as well as non-tumorous liver. Still, there was a strong correlation in the expression of HDAC class IIa (HDAC4, 5, 7, 9) as well as HDAC2 and 8 (class I) and HDAC10 (class IIb) and HDAC11 (class IV) in HCC tissues of individual patients. This might indicate a common mechanism of the regulation of these HDACs in HCC. The Cancer Genome Atlas (TCGA) dataset analysis revealed that HDAC4, HDAC7 and HDAC9 as well as HDAC class I members HDAC1 and HDAC2 is significantly correlated with patient survival. Furthermore, we observed that SAHA and TSA reduced the proliferation, clonogenicity and migratory potential of HCC cells. SAHA but not TSA induced features of senescence in HCC cells. Additionally, HDACi enhanced the efficacy of sorafenib in killing sorafenib-susceptible cells. Moreover, HDACi reestablished sorafenib sensitivity in resistant HCC cells. In summary, HDACs are significantly but differently increased in HCC, which may be exploited to develop more targeted therapeutic approaches. HDACi affect different facets of the tumorigenicity of HCC cells and appears to be a promising therapeutic approach alone or in combination with sorafenib.

Overexpression of CASC2 Improves Cisplatin Sensitivity in Hepatocellular Carcinoma Through Sponging miR-222.

The long noncoding RNA cancer susceptibility candidate 2 (CASC2) has been shown to play a crucial role in cancer cell chemoresistance. However, its function and underlying molecular mechanism in hepatocellular carcinoma (HCC) chemoresistance remain unknown. In this study, we used cisplatin (DDP)-resistant HCC cells to investigate CASC2 function and its underlying mechanism. The results demonstrated that CASC2 expression was significantly reduced in HCC tissues and cells, especially in DDP-resistant HCC tissues and cells. Lower CASC2 expression was strongly correlated with shorter survival times in patients with HCC. Functionally, CASC2 overexpression sensitized DDP-resistant Huh7/DDP and SMMC-7721/DDP cells to DDP. Mechanically, CASC2 improved the sensitivity of HCC cells to DDP through sponging miR-222. Taken together, these findings suggested that overexpression of CASC2 overcame DDP resistance in HCC by regulating miR-222 expression, thereby providing a potential therapeutic strategy for overcoming HCC cell chemoresistance.

H1, a derivative of tetrandrine, enhances the efficacy of 5-FU in Bel7402/5-FU cells via suppressing STAT3/MCL-1 and inducing PUMA

Currently, 5-fluorouracil (5-FU) resistance became a major obstacle to its clinical use for patients with hepatocellular carcinoma (HCC). It’s urgent to develop a novel strategy for enhancing the therapeutic efficacy of 5-FU. Herein, we found that H1 (a derivative of Tetrandrine) exerted a potent anti-MDR effect on growth of 5-FU resistant HCC cells (Bel7402/5FU). The resistant fold (RF) of 5-FU is over 160-fold, while the RF of H1 is only 4.8-fold in Bel7402/5-FU cells. Further studies demonstrated that blockage of STAT3/MCL-1 signaling and induction of PUMA is responsible to anti-MDR activity of H1. Moreover, combination of H1 and 5-FU (Ratio = 1:2) could synergistically induce apoptosis of Bel7402/5-FU cells. Co-treatment of H1 enhances the suppression of p-STAT3 and MCL-1, and significantly increases PUMA expression. Finally, the combination of H1 and 5-FU results in an increase of cleaved PARP. Taken together, H1 effectively improve the cytotoxic effect of 5-FU against Bel7402/5-FU cells via blocking STAT3/MCL-1 pathway and inducing PUMA. Our findings suggested that combination 5-FU with anti-MDR agents might present a novel strategy to enhance the therapeutic efficacy of 5-FU in resistant HCC.

CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma.

Regorafenib is used for hepatocellular carcinoma (HCC), but its response does not last long, partly due to chemoresistance acquisition. We performed a clustered regularly interspaced short palindromic repeats (CRISPR)-based loss-of-function genetic screen and aimed to discover molecules involved in regorafenib resistance in HCC. Xenograft tumors established from Cas9-expressing HCC cells with pooled CRISPR kinome libraries were treated with regorafenib or a vehicle. Sequencing analysis identified 31 genes, with the abundance of multiple guide RNAs increased in regorafenib-treated tumors compared to that in vehicle-treated tumors, including 2 paralogues, LATS2 and LATS1, core components of the Hippo signaling pathway. Notably, all eight designed guide RNAs targeting LATS2 increased in regorafenib-treated tumors, suggesting that LATS2 deletion confers regorafenib resistance in HCC cells. LATS2 knockdown significantly mitigated the cytotoxic and proapoptotic effects of regorafenib on HCC cells. LATS2 inhibition stabilized the Hippo signaling mediator YAP, leading to the upregulation of antiapoptotic Bcl-xL and the multidrug resistance transporter ABCB1. Among 12 hepatoma cell lines, the half maximal inhibitory concentration (IC50) values of regorafenib were positively correlated with any of YAP, Bcl-xL and ABCB1 levels. The inhibition of YAP or Bcl-xL in regorafenib-insensitive HCC cells restored their susceptibility to regorafenib. In conclusion, our screen identified the Hippo signaling pathway as the mediator of regorafenib efficacy in HCC.

E26 transformation-specific variant 4 promotes sorafenib and cisplatin resistance in hepatocellular carcinoma cells in vitro

To investigate the role of E26 transformation-specific variant 4 (ETV4) in sorafenib and cisplatin resistance in hepatocellular carcinoma (HCC). HCC cell lines SMMC-7721 and HCC-LM3 were transfected with an ETV4- overexpressing plasmid or small interfering RNAs (siRNAs) targeting ETV4. The cells with ETV4 overexpression or ETV4 interference were treated with DMSO, sorafenib (5 μmol/L) or cisplatin (5 μmol/L) for 48 h, and the total protein and total RNA were collected. Western blotting, flow cytometry, EdU proliferation assay were used to analyze the apoptosis and proliferation of the cells. We also obtained clinical specimens of HCC tissues and paired adjacent tissues from 11 patients for detecting ETV4 mRNA expression levels using real-time fluorescence quantitative PCR (q-PCR). The effect of ETV4 interference on the mRNA expression levels of immediate early response gene 3 (IER3) was examined in HCC cells that were treated with DMSO, sorafenib or cisplatin for 48 h. The expression of ETV4 mRNA was significantly higher in HCC tissues than in the paired adjacent tissues. Overexpression of ETV4 in the HCC cell lines obviously inhibited cell apoptosis induced by sorafenib or cisplatin. Conversely, ETV4 interference significantly enhanced the apoptosis and inhibited the proliferation of the HCC cells following treatments with sorafenib or cisplatin. In addition, ETV4 regulated the mRNA expression levels of IER3 in the cells treatmed with sorafenib and cisplatin. ETV4 promotes resistance of HCC cells to sorafenib or cisplatin in vitro.

Enhancer of zeste 2 polycomb repressive complex 2 subunit promotes sorafenib resistance of hepatocellular carcinoma though insulin-like growth factor 1 receptor.

Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) is the core component of polycomb repressive complex 2 and is overexpressed in several types of solid malignancies. It has been reported that EZH2 contributes to sorafenib resistance of hepatocellular carcinoma (HCC). However, its underlying molecular mechanisms remain unknown. In this study, we demonstrated that EZH2 induced sorafenib resistance of HCC cells in vitro. Mechanistically, EZH2 was a potent regulator of insulin-like growth factor 1 receptor (IGF1R) and EZH2-modulated IGF1R expression by directly transcriptionally repressing a set of microRNAs (miRNAs) including miR-101, miR-122, miR-125b, and miR-139. These miRNAs were required for EZH2-mediated sorafenib resistance by promoting IGF1R expression. Surprisingly, IGF1R inhibitors significantly reversed EZH2-induced sorafenib resistance. Collectively, we proposed a novel model for an EZH2 - miRNAs - IGF1R regulatory axis, which might provide insights into how EZH2 contributes to sorafenib resistance in HCC.

Down-regulation of Survivin by BIX-01294 Pretreatment Overcomes Resistance of Hepatocellular Carcinoma Cells to TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cancer-selective, cell-death-inducing agent with little toxicity to normal cells. However, various human cancers and cancer cell lines have been reported to be resistant to TRAIL. Molecular clarification of resistance mechanism is needed. Compound screening, proliferation assays, western blotting, and flow cytometry were used to examine the sensitizer activity of methyl transferase inhibitor BIX-01294 in combination with TRAIL, in hepatocellular carcinoma (HCC) cells. RNA sequencing analysis and single guide (sg)RNA-mediated gene deletion were used to investigate the role of survivin in sensitization. In HCC cells, BIX-01294 enhanced TRAIL sensitivity by reducing survivin expression at the RNA level. Small interference RNA-mediated gene knockdown demonstrated the mechanism of sensitization to be via the reduction of survivin. Euchromatin histone methyltransferase 2 (EHMT2) inhibition by BIX-01294 may be a potent anti-tumor therapeutic strategy for human HCC.

Abstract 2914: Hexokinase 2-mediated metabolic reprogramming and apoptosis inhibition supports hepatocellular carcinoma radiation resistance

Abstract Purpose: Hepatocellular carcinoma (HCC) cells are metabolically distinct from normal hepatocytes by selectively upregulating hexokinase 2 (HK2), a key enzyme that catalyzes the first committed step of aerobic glycolysis, which provides genetic proof of concept that HK2 can be inhibited to treat HCC without adverse physiological consequences. The present study was designed to investigate the role of HK2-mediated metabolic reprogramming and apoptosis inhibition in radiation resistance of HCC cells. Experimental design: Acquired radiation-resistant cells were generated by sequential irradiation and recovery, and proteomics approach was used to illustrate the biological changes and molecular mechanisms involved. qRT-PCR was performed to evaluate the expression of glycolysis genes including HK2. TCGA and GEO analysis were used to investigate the prognostic value of HK2. Intrinsic radiation responsive and resistant HCC cells were also used to further identify the resistant role of HK2. Either lentivirus-mediated HK2 knockdown or pharmacological blockade by 3-bromopyruvate (BrPA) was used to inhibit HK2. Radiation resistance was evaluated by DNA damage response, in vitro cytotoxicity and colony formation assay. Apoptosis and ROS levels were measured with Annexin V and DC-FDA. Cytochrome c release and HK2 mitochondrial translocation were detected by immunofluorescence and western blots. 2-NBDG uptake, HK2 activity, lactate production, and 13C isotopomer tracing experiment were conducted to illustrate the glycolytic activities. HK2 translation efficiency was measured by polyribosome analysis. The upstream AKT-mTOR signaling were detected by western blots and rescued by pharmacological inhibitors. Xenografts were treated with radiation, BrPA, or a combination of radiation and BrPA. Results: Aerobic glycolysis was significantly activated and was indispensable for cell survival in both acquired and intrinsic radiation-resistant HCC cells. Mechanically, both up-regulation of mRNA expression of hexokinase (HK2) after sequential irradiation and increased translation of HK2 mRNA after one-fractional irradiation were mediated by the activation of AKT-mTOR axis. In addition, high HK2 expression was significantly associated with poor overall survival of HCC patients. Inhibition of HK2 expression or its mitochondrial translocation re-sensitized HCC cells to radiation therapy by promoting mitochondrial cytochrome c release and inhibiting of glycolysis both in vitro and in vivo. Conclusion: AKT-mTOR-HK2 axis plays crucial roles in both acquired and intrinsic radiation resistance of HCC cells by facilitating aerobic glycolysis and inhibiting cytochrome c release. Targeting HK2 could be an attractive and beneficial approach to improve the therapeutic effects of radiotherapy in HCC patients. Citation Format: Yuan Fang, Yizhi Zhan, Yiyi Li, Wei Wang, Dehua Wu, Yi Ding. Hexokinase 2-mediated metabolic reprogramming and apoptosis inhibition supports hepatocellular carcinoma radiation resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2914.

Abstract 4679: NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The long-term prognosis of HCC remains unsatisfactory due to high recurrence rates and chemoresistance. Solid evidence shows that tumor-initiating cells (T-ICs) are the root of tumor relapse and drug resistance, which led to poor prognosis of patients with hepatocellular carcinoma (HCC). Therefore, identification of crucial transcriptional regulator in liver T-IC may improve the prognosis of these patients. Through in vitro liver T-IC enrichment approach, we identified Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2) as transcriptions regulator be significantly upregulated in enriched liver T-IC populations. NRF2 regulated liver T-IC properties including self-renewal, tumorigenicity, and liver T-IC marker expression and significantly affected patients’ clinical outcome. Furthermore, NRF2 repression led to sensitization to doxorubicin and 5-FU treatment. Furthermore, we found that ROS-induced NRF2 activation regulates sorafenib resistance of HCC cells. Mechanistically, NRF2 was found to physically bind to promoter SHH, which triggers activation of sonic hedgehog pathway. NRF2 was significantly correlated with SHH expression, which was associated with poor patient’s survival. The effect of NRF2 knockdown was eliminated upon administration of recombinant SHH, demonstrating that NRF2 mediated the TIC function via upregulation of SHH expression. Our study suggested a novel regulatory mechanism for the canonical sonic hedgehog pathway that may function through NRF2/SHH/GLI signaling axis, thus mediating T-IC phenotypes. Targeting NRF2 mediated signaling cascade alone or in combination with other treatment modalities may be a new a potential therapeutic approach for treatment of HCC. Citation Format: Hoi Wing Leung, Eunice Yuen Ting Lau, Kin Wah Lee. NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4679.

LncRNA FOXD2-AS1 as a competitive endogenous RNA against miR-150-5p reverses resistance to sorafenib in hepatocellular carcinoma.

The current study elucidated the role of a long non‐coding RNA (lncRNA), FOXD2‐AS1, in the pathogenesis of hepatocellular carcinoma (HCC) and the regulatory mechanism underlying FOXD2‐AS1/miR‐150‐5p/transmembrane protein 9 (TMEM9) signalling in HCC. Microarray analysis was used for preliminary screening of candidate lncRNAs in HCC tissues. qRT‐PCR and Western blot analyses were used to detect the expression of FOXD2‐AS1. Cell proliferation assays, luciferase assay and RNA immunoprecipitation were performed to examine the mechanism by which FOXD2‐AS1 mediates sorafenib resistance in HCC cells. FOXD2‐AS1 and TMEM9 were significantly decreased and miR‐150‐5p was increased in SR‐HepG2 and SR‐HUH7 cells compared with control parental cells. Overexpression of FOXD2‐AS1 increased TMEM9 expression and overcame the resistance of SR‐HepG2 and SR‐HUH7 cells. Conversely, knockdown of FOXD2‐AS1 decreased TMEM9 expression and increased the sensitivity of HepG2 and Huh7 cells to sorafenib. Our data also demonstrated that FOXD2‐AS1 functioned as a sponge for miR‐150‐5p to modulate TMEM9 expression. Taken together, our findings revealed that FOXD2‐AS1 is an important regulator of TMEM9 and contributed to sorafenib resistance. Thus, FOXD2‐AS1 may serve as a therapeutic target against sorafenib resistance in HCC.

Simultaneous knockdown of YAP and TAZ increases apoptosis of hepatocellular carcinoma cells under hypoxic condition

Hepatocellular carcinoma (HCC) is one of the common malignant tumors of the digestive system and its five-year survival rate is low. Hypoxia is an important feature of HCC, which can promote cell death resistance. However, the key regulator of HCC cell survival remains elusive in the hypoxic condition. Emerging researches have showed that the Hippo signaling pathway is involved in the initiation and progression of HCC. Here, we provide evidence that key downstream effectors YAP and its paralog TAZ play vital role in apoptosis resistance of HCC cells under hypoxia. Knockdown of YAP or TAZ does not affect the survival of HCC cells in normoxic and hypoxic microenvironment. In addition, the rate of apoptosis by knockdown or inhibition of both YAP and TAZ under hypoxic condition is largely higher than which under normoxia. In conclusion, simultaneous knockdown or inhibition of YAP and TAZ promote apoptosis of HCC cells dramatically. To our knowledge, this is the first research to explore the role of both YAP and TAZ in HCC hypoxic microenvironment in vitro and in vivo. Therefore, it may be useful for establishing novel targeted therapies of HCC, especially subtypes with plenty of hypoxic areas.