Treatment Of Hepatocellular Carcinoma Cells Research Articles

Methylation in MIRLET7A3 Gene Induces the Expression of IGF-II and Its mRNA Binding Proteins IGF2BP-2 and 3 in Hepatocellular Carcinoma.

miR-let-7a is a tumor suppressor miRNA with reduced expression in most cancers. Methylation of MIRLET7A3 gene was reported to be the cause of this suppression in several cancers; however, it was not explicitly investigated in hepatocellular carcinoma (HCC). We aimed at investigating miR-let-7a expression and molecular mode in HCC, identifying drug-targetable networks, which might be affected by its abundance. Our results illustrated a significant repression of miR-let-7a, which correlated with hypermethylation of its gene of origin MIRLRT7A3. This was further supported by the induction of miR-let-7a expression upon treatment of HCC cells with a DNA-methyltransferase inhibitor. Using a computational approach, insulin-like growth factor (IGF)-II and IGF-2 mRNA binding proteins (IGF2BP)-2/-3 were identified as potential targets for miR-let-7a that was further confirmed experimentally. Indeed, miR-let-7a mimics diminished IGF-II as well as IGF2BP-2/-3 expression. Direct binding of miR-let-7a to each respective transcript was confirmed using a luciferase reporter assay. In conclusion, this study suggests that DNA hypermethylation leads to epigenetic repression of miR-let-7a in HCC cells, which induces the oncogenic IGF-signaling pathway.

Novel role for fatty acid binding protein-4 (FABP4) in hepatocellular carcinoma (HCC) progression

Introduction: FABP4 is normally expressed in adipocytes and macrophages and normally regulates intracellular lipid trafficking. Recent data suggests FABP4 is elevated in non-alcoholic liver disease, and may be released into the circulation to act as a paracrine/endocrine signaling molecule. This study sought to determine FABP4 expression and correlation with NAFLD activity score (NAS) in patients with liver disease, and identify mechanism(s) by which FABP4 may affect disease progression. Methods: FABP4 was measured in serum from patients with and without liver disease by ELISA, and compared to NAS from biopsies. To examine the role of FABP4 in HCC cell function, human HCC cells (HepG2 and HuH7) were treated with exogenous FABP4 (0-100ng/mL) and analyzed for p38-MAPK, Erk1/2, and JNK1/2 activity. In parallel, proliferation and migration assays were performed using cells pretreated with inhibitors of p38MAPK (SB203580), Erk1/2 (U0126), or JNK1/2 (SP600125), followed by exogenous FABP4 treatment. Results: Patients with NAFLD/ALD and HCC exhibited significantly elevated FABP4 serum-levels compared to healthy subjects, and FABP4 levels correlated with increased NAS. Treatment of HCC cells with FABP4 resulted in increased phospho-Erk1/2 activity, but not p38MAPK or JNK1/2 activity. Pretreatment with U0126 abrogated FABP4-mediated Erk1/2 activation and significantly inhibited FABP4-mediated HCC cell proliferation and migration. Conclusions: Serum FABP4 represents a novel circulating marker of underlying hepatosteatosis, and FABP4 promotes HCC proliferation/migration via an Erk1/2-dependent pathway. These data suggest a potential role for FABP4 as a non-invasive biomarker for liver disease progression, and suggest elevated FABP4 levels in hepatosteatosis may play a role in HCC development/progression.

Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of mTOR

Purpose: Hepatocellular carcinoma (HCC) ranks second in cancer mortality and has limited therapeutic options. We recently described the synergistic effect of allosteric and ATP-site competitive inhibitors against the mTOR for the treatment of HCC. However, such inhibitors induce hyperglycemia and increase mitochondrial efficiency. Here we determined whether the mitochondrial complex I inhibitor phenformin could reverse both side effects, impose an energetic stress on cancer cells, and suppress the growth of HCC.Experimental Design: Human HCC cell lines were used in vitro to access the signaling and energetic impact of mTOR inhibitors and phenformin, either alone or in combination. Next, the therapeutic utility of these drugs alone or in combination was investigated preclinically in human orthotopic tumors implanted in mice, by analyzing their impact on the tumor burden and overall survival.Results: We found phenformin caused mitochondrial dysfunction and fragmentation, inducing a compensatory shift to glycolysis. In contrast, dual inhibition of mTOR impaired cell growth and glycolysis, while increasing mitochondrial fusion and efficiency. In a mouse model of human HCC, dual inhibition of mTOR, together with phenformin, was highly efficacious in controlling tumor burden. However, more strikingly, pretreatment with phenformin sensitized tumors to dual inhibition of mTOR, leading to a dramatic improvement in survival.Conclusions: Treatment of HCC cells in vitro with the biguanide phenformin causes a metabolic shift to glycolysis, mitochondrial dysfunction and fragmentation, and dramatically sensitizes orthotopic liver tumors to dual inhibition of mTOR. We therefore propose this therapeutic approach should be tested clinically in HCC. Clin Cancer Res; 24(15); 3767-80. ©2018 AACR.

Abstract 1334: Withaferin A induces cytoprotective autophagy in hepatocellular cancer cells and concomitant autophagic inhibition augments the efficacy of Withaferin A in hepatocellular carcinoma

Abstract Background and Aim: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most common cause of cancer-related deaths. While multiple chemotherapeutic and targeted agents have been developed for other cancers, Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced HCC. Clearly, it is imperative to develop novel effective therapeutic strategies for HCC to improve long-term survival of HCC patients. We found Withaferin A (WFA), a bioactive alkaloid derived from Withania somnifera, as an effective agent for HCC inhibition. Interestingly, we observed that WFA also induces autophagy in HCC cells. Autophagy can be cancer inducing or cancer inhibiting at the functional level. We aim to systemically investigate the anti-HCC efficacy of WFA, investigate the functional impact of autophagy and explore the efficacy of combined regimens of WFA and autophagic inhibitors. Methods: Using in vitro and in vivo models, we established that WFA inhibits liver tumorigenesis. tfLC3B, LC3B-GFP/Lysotracker and LC3B/Rab7 were used to examine the fusion of autophagosomes with lysosomes. Cathepsin-D activation and DQ-BSA assays were conducted to analyze the protein degradation activity in lysosomes. Multiple cell based assays and Chou-Talalay synergy quantification assays were conducted to analyze the synergistic effect of WFA with autophagic inhibitors. IHC staining was used to study autophagic markers in vivo. Results: WFA inhibited anchorage dependent cell growth in vitro and reduced the HCC tumor burden in vivo. In addition to inducing apoptotic cell death, WFA induced cleavage of Light Chain 3B (LC3B) and increased the expression of autophagic proteins (ATG5, ATG7 and Beclin1) both in vitro and in vivo. The redistribution of LC3B-GFP from cytosol to autophagosome demonstrated enhanced autophagosome formation in WFA treated cells. The fusion of autophagosome with lysosome was observed in WFA-treated HCC cells indicating autolysosome formation. WFA treated HCC cells exhibited elevated lysosomal degradation activity. Inhibiting autophagy either by blocking autophagosome formation or elevating lysosomal pH (CQ and BAF) enhanced WFA-induced growth inhibition and apoptosis indicating the presence of cytoprotective autophagy. Combination index analysis of WFA and CQ showed synergism and increased efficacy as compared to either monotherapy. These in vitro findings were corroborated with in vivo experiments. Conclusion: Our results reveal that WFA inhibits hepatocellular carcinoma by apoptotic induction but the efficacy of WFA is somewhat abrogated by the concomitant induction of cytoprotective autophagy. Our preclinical findings present evidence to support WFA and chloroquine as an effective combination treatment regimen for HCC. Citation Format: Sumit Siddharth, Nethaji Muniraj, Neeraj K. Saxena, Dipali Sharma. Withaferin A induces cytoprotective autophagy in hepatocellular cancer cells and concomitant autophagic inhibition augments the efficacy of Withaferin A in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1334.

Abstract 133: Synergistic effect between natural killer cells and anysomycin against hepatocellular carcinoma cells

Abstract Although advances in the clinical management of hepatocellular carcinoma (HCC) have been made, HCC is the second leading cause of cancer-related death worldwide. Currently, there are limited treatment options for advanced HCC. Therefore, novel treatment strategies for HCC are required. Here, we described a promising anti-tumor effect of anisomycin, which not only had direct killing effects but also enhanced a natural killer cell-mediated immunotherapeutic effect in HCC. To better elucidate the anti-tumor mechanisms of anisomycin, we performed a genome-scale analysis of gene expression profiles. We found that anisomycin treatment of HCC differentially regulated a broad range of immune regulation-associated genes; among them, we found that CD58, which was significantly increased on anisomycin treated HCC cells was one of the critical components for natural killer cell-mediated immunotherapeutic effect enhanced by anisomycin. Notably, this is the first report for synergistic effect of anisomycin in combination with NK cell therapy as a potential therapeutic option for HCC. Citation Format: Chang Hoon Lee, Miok Kim, Kyung Won Lee. Synergistic effect between natural killer cells and anysomycin against hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 133.

P-324 - Phenformin-induced mitochondrial dysfunction sensitizes hepatocellular carcinoma for dual inhibition of mTOR

Purpose: Hepatocellular carcinoma (HCC) ranks second in cancer mortality and has limited therapeutic options. We recently described the synergistic effect of allosteric and ATP-site competitive inhibitors against the mTOR for the treatment of HCC. However, such inhibitors induce hyperglycemia and increase mitochondrial efficiency. Here we determined whether the mitochondrial complex I inhibitor phenformin could reverse both side effects, impose an energetic stress on cancer cells, and suppress the growth of HCC. Experimental Design: Human HCC cell lines were used in vitro to access the signaling and energetic impact of mTOR inhibitors and phenformin, either alone or in combination. Next, the therapeutic utility of these drugs alone or in combination was investigated preclinically in human orthotopic tumors implanted in mice, by analyzing their impact on the tumor burden and overall survival. Results: We found phenformin caused mitochondrial dysfunction and fragmentation, inducing a compensatory shift to glycolysis. In contrast, dual inhibition of mTOR impaired cell growth and glycolysis, while increasing mitochondrial fusion and efficiency. In a mouse model of human HCC, dual inhibition of mTOR, together with phenformin, was highly efficacious in controlling tumor burden. However, more strikingly, pretreatment with phenformin sensitized tumors to dual inhibition of mTOR, leading to a dramatic improvement in survival. Conclusions: Treatment of HCC cells in vitro with the biguanide phenformin causes a metabolic shift to glycolysis, mitochondrial dysfunction and fragmentation, and dramatically sensitizes orthotopic liver tumors to dual inhibition of mTOR. We therefore propose this therapeutic approach should be tested clinically in HCC. Clin Cancer Res; 24(15); 3767–80. ©2018 AACR.

MiR-221 suppression through nanoparticle-based miRNA delivery system for hepatocellular carcinoma therapy and its diagnosis as a potential biomarker.

BackgroundMicroRNA-221(miR-221) is frequently dysregulated in cancer. The purpose of this study was to explore whether miR-221 can be used as a potential diagnostic marker or therapeutic target for hepatocellular carcinoma (HCC).MethodsIn this study, we investigated whether miR-221 expression was associated with clini-copathological characteristics and prognosis in HCC patients, and we developed a nanoparticle-based miRNA delivery system and detected its therapeutic efficacy in vitro and in vivo.ResultsWe found that miR-221 was upregulated in HCC tissues, cell lines and blood of HCC patients. Upregulated miR-221 was associated with clinical TNM stage and tumor capsular infiltration, and showed poor prognosis, suggesting that its suppression could serve as an effective approach for hepatocellular carcinoma therapy. Treatment of HCC cells with nanoparticle/miR-221 inhibitor complexes suppressed their growth, colony formation ability, migration and invasion. In vivo, the growth of the tumors treated by the nanoparticle/miR-221 inhibitor complexes were significantly less than those treated by the nanoparticle/miRNA scramble complexes. In addition, circulating miR-221 may act as a potential tumor biomarker for early diagnosis of HCC, and combined serum miR-221 and AFP detection gave a better performance than individual detection in early diagnosis of HCC.ConclusionThese findings suggest that a nanoparticle-based miRNA delivery system could potentially serve as a safe and effective treatment and miR-221 could also be a potential diagnostic marker for HCC.

Novel combination of thymoquinone and resveratrol enhances anticancer effect on hepatocellular carcinoma cell line

Hepatocellular carcinoma remains one of the most dominant malignancies worldwide. Neutraceuticals have become under focus in anticancer treatment. Resveratrol is one of the major components of Polygonum Cuspidatum and is known as chemo-preventive agent. Thymoquinone is one of the most potent constituents in Nigella Sativa and has many medicinal effects. The aim of the present study is to investigate the combined effect of thymoquinone and resveratrol on treatment of hepatocellular carcinoma cells (HepG2). We evaluated the effect of thymoquinone and resveratrol separately and in combination on HepG2. Cell viability, caspase-3 activity, glutathione and malondialdehyde content were determined. The IC50 values of thymoquinone and resveratrol were (46 μM and 64.5 μM) respectively, where each showed potent anti-tumor activity on HepG2. The cell viability was 47.2% and 49.9% respectively. Comparing to the control group, treatment with thymoquinone and resveratrol increased caspase-3 enzyme by 77% and 98.5% respectively, while content of glutathione decreased by 22.8% and 35.6% while malondialdehyde content decreased by 18% and 29.6% correspondingly. The combination (thymoquinone + resveratrol) affected the cell viability leading to further decrease by 9.9% and 12.6%. The content of caspase-3 increased by 89% and 67.5% while the glutathione content had further decrease by 25.6% and 12.8%. Malondialdehyde content decreased by 32.3% and 20.7% all are comparing to thymoquinone and resveratrol separate treatment.Thymoquinone and resveratrol combination showed significant cell inhibition and increase in caspase-3 indicating cell apoptosis. Both drugs raised reactive oxygen species leading to decrease of glutathione and minor effect on lipid peroxidation, all these results give a new promising combination with enhanced anticancer effect.

Pharmacological or transcriptional inhibition of both HDAC1 and 2 leads to cell cycle blockage and apoptosis via p21Waf1/Cip1 and p19INK4d upregulation in hepatocellular carcinoma.

Histone deacetylases (HDACs) are commonly dysregulated in cancer and represent promising therapeutic targets. However, global HDAC inhibitors have shown limited efficacy in the treatment of solid tumours, including hepatocellular carcinoma (HCC). In this study, we investigated the therapeutic effect of selectively inhibiting HDAC1 and 2 in HCC. HDAC1 inhibitor Tacedinaline (CI994), HDAC2 inhibitor Santacruzamate A (CAY10683), HDAC1/2 common inhibitor Romidepsin (FK228) and global HDAC inhibitor Vorinostat (SAHA) were used to treat HCC cells. Cell cycle, apoptosis and the protein levels of CDKs and CDKNs were performed to evaluate HCC cell growth. Inhibition of HDAC1/2 by RNAi was further investigated. Combined inhibition of HDAC1/2 led to HCC cell morphology changes, growth inhibition, cell cycle blockage and apoptosis in vitro and suppressed the growth of subcutaneous HCC xenograft tumours in vivo. p21Waf1/Cip1 and p19INK4d , which play roles in cell cycle blockage and apoptosis induction, were upregulated. Inhibition of HDAC1/2 by siRNA further demonstrated that HDAC1 and 2 cooperate in blocking the cell cycle and inducing apoptosis via p19INK4d and p21Waf1/Cip1 upregulation. Finally, H3K18, H3K56 and H4K12 in the p19INK4d and p21Waf1/Cip1 promoter regions were found to be targets of HDAC1/2. Pharmacological or transcriptional inhibition of HDAC1/2 increases p19INK4d and p21Waf1/Cip1 expression, decreases CDK expression and arrests HCC growth. These results indicated a potential pharmacological mechanism of selective HDAC1/2 inhibitors in HCC therapy.

Shufeng Jiedu Capsule and its active ingredients induce apoptosis, inhibit migration and invasion, and enhances doxorubicin therapeutic efficacy in hepatocellular carcinoma

BackgroundShufeng Jiedu Capsule (SFJDC), a traditional Chinese medicine, has been used widely as antiviral, antibacterial, antitumor, and anti-inflammatory drugs. Previous studies indicated that some active ingredients of Shufeng Jiedu Capsule, such as resveratrol and quercetin, could suppress hepatocellular carcinoma (HCC) cells through various signaling pathways. However, anti-HCC activity of SFJDC as a complementary medicine remains unexplored. Here, we use a combination of Shufeng Jiedu Capsule and doxorubicin to treat HCC cells and investigated the effects and mechanisms of SFJDC and its ingredientsin vitro. MethodsIn this study, two HCC cell lines, HepG2 and HepG2.2.15, were employed and all cells were separated into seven groups: doxorubicin group, SFJDC group, combination of doxorubicin and SFJDC group, resveratrol group, quercetin group, resveratrol and quercetin group, and control group. Through this research, the cellular functional experiments, such as MTT assay, Hoechst 33,258 staining, would healing assay, and transwell assay, were took to observe the effects of those agents on proliferation, apoptosis, migration and invasion of cells. Then, apoptosis and invasion related genes and proteins were detected by real-time PCR and western blot to illuminate the signaling pathways. ResultsThe combination group induced more significant apoptosis and inhibition of migration and invasion by affecting proteins and mRNA of apoptosis, migration, and invasion related elements, such as Bcl-2, Bax, mTOR, and NF-κB. Furthermore, the research suggested SFJDC, as a mixture of a number of ingredients, had stronger activities than particular component or simple mixture of a few components. ConclusionsSFJDC and its active ingredients could play a role as complementary medicine to increase antitumor effect of doxorubicin by targeting mitochondrial, Akt/mTOR, and NF-κB signaling pathways.

Altered fatty acid‐binding protein 4 (FABP4) expression and function in human and animal models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality. Risk factors for developing HCC include viral hepatitis, alcohol and obesity. Fatty acid-binding proteins (FABPs) bind long-chain free fatty acids (FFAs) and are expressed in a tissue-specific pattern; FABP1 being the predominant hepatic form, and FABP4 the predominant adipocyte form. The aims of this study were to investigate the expression and function of FABPs1-9 in human and animal models of obesity-related HCC. FABP1-9 expression was determined in a mouse model of obesity-promoted HCC. Based on these data, expression and function of FABP4 was determined in human HCC cells (HepG2 and HuH7) in vitro. Serum from patients with different underlying hepatic pathologies was analysed for circulating FABP4 levels. Livers from obese mice, independent of tumour status, exhibited increased FABP4 mRNA and protein expression concomitant with elevated serum FABP4. In vitro, FABP4 expression was induced in human HCC cells by FFA treatment, and led to FABP4 release into culture medium. Treatment of HCC cells with exogenous FABP4 significantly increased proliferation and migration of human HCC cells. Patient serum analysis demonstrated significantly increased FABP4 in those with underlying liver disease, particularly non-alcoholic fatty liver disease (NAFLD) and HCC. These data suggest FABP4, an FABP not normally expressed in the liver, can be synthesized and secreted by hepatocytes and HCC cells, and that FABP4 may play a role in regulating tumour progression in the underlying setting of obesity.

Metformin Promotes 2-Deoxy-2-[18F]Fluoro-D-Glucose Uptake in Hepatocellular Carcinoma Cells Through FoxO1-Mediated Downregulation of Glucose-6-Phosphatase.

The early diagnosis of primary hepatocellular carcinoma (HCC) has the potential to lead to significant improvements for the treatment and survival rates of cancer patients. 2-Deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is often used as a tracer for positive emission tomography (PET) and X-ray computed tomography (CT) imaging of cancer cells; however, [18F]FDG PET/CT cannot currently be used as an early diagnostic technique for HCC. This is because these cancer cells express high levels of glucose-6-phosphatase (G6Pase) that is responsible for poor cellular retention of [18F]FDG. Here, we sought to investigate the feasibility of metformin treatment to promote [18F]FDG uptake in HCC and the mechanism involved. Human SMMC-7721 HCC cells were treated with metformin (up to 10mM) or FoxO1 siRNA. The transcriptional and expression levels of FoxO1 and G6Pase were determined by quantitative RT-PCR and Western blotting, respectively. The feasibility of using metformin to promote [18F]FDG uptake was investigated by both in vitro cell uptake analysis and in vivo microPET/CT imaging. Stable doxycycline-inducible cell lines with FoxO1-overexpression (FoxO1-OE) and FoxO1-knockdown (FoxO1-KD) were constructed to evaluate the impact of FoxO1 on G6Pase expression in vitro and [18F]FDG uptake in vivo. Treatment of HCC cells with metformin (Met) leads to a dose-dependent reduction in the expression levels of FoxO1 at the protein level, but not at the mRNA level. Met-induced phosphorylation of FoxO1 initiates a reduction in the expression levels of G6Pase mRNA, which results in an overall increase in the uptake of [18F]FDG into HCC cells and tumors. We propose that treatment of HCC cells with Met may be a useful strategy for improving the efficacy of [18F]FDG as a tracer for PET/CT imaging of HCC tumors in patients.

Cell-surface major vault protein promotes cancer progression through harboring mesenchymal and intermediate circulating tumor cells in hepatocellular carcinomas

Circulating tumor cells (CTCs) play a major role in the metastasis and recurrence of hepatocellular carcinoma (HCC). Here, we found that major vault protein (MVP) is expressed on the surface of HCC cells and further induced under stressful environments. MVP knockdown reduces cell proliferation and induces apoptosis in HCC cells. Treatment of HCC cells with anti-MVP antibody (α-MVP) recognizing cell-surface MVP (csMVP) inhibits cell proliferation, migration, and invasion. csMVP-positive HCC cells have a higher clonogenic survival than csMVP-negative HCC cells, and treatment of HCC cells with α-MVP inhibits clonogenic survival, suggesting that csMVP contributes to HCC cell survival, migration, and invasion. The function of csMVP is mediated through mTOR, FAK, ERK and Akt signaling pathways. csMVP-positive CTCs are detected in HCC patients (89.7%) but not in healthy donors, and the number of csMVP-positive CTCs is further increased in patients with metastatic cancers. csMVP is exclusively detectable in CTCs with mesenchymal phenotype or intermediate phenotype with neither epithelial nor mesenchymal markers, suggesting that csMVP-associated survival and metastatic potential harbor CTCs with nonepithelial phenotypes. The results suggest that csMVP promotes cancer progression and serves as a surface marker for mesenchymal and intermediate CTCs in patients with HCC and metastatic cancers.

Abstract 3315: Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis

Abstract The existence of a cancer stem cell (CSC) subset is well established in hepatocellular carcinoma (HCC). This subset can be characterized, among others, by its CD133 expression. Since the discovery of this marker, studies have revealed deregulated pathways and epigenetic alterations associated with this subset, but few have focused on changes in DNA methylation. This study aims to study the aberrant DNA methylation patterns governing CD133+ CSC-driven HCC. Results from Infinium HumanMethylation450 BeadChip (HuMet450 BeadChip) have led to the discovery of differential methylation patterns between CD133+ liver CSCs and matched CD133- differentiated samples. ZFP42/REX1 is one of the top-ranking differentially methylated candidates. Probes corresponding to its promoter are extensively hypermethylated in the CD133+ samples. We validated the down-regulation of REX1 mRNA levels in sorted CD133+ HCC cell lines (n=4) and HCC clinical samples (n=46, p<0.05), as compared to CD133- counterparts and non-tumor liver samples. REX1 and CD133 expressions are also significantly negatively correlated in HCC cell lines (n=8, R2=0.65). To explore the hypothesis that promoter DNA hypermethylation modulates REX1 expression, we treated HCC cells with 5-aza. Only low-REX1-expressing cells re-expressed REX1. Results of pyrosequencing of a CG island of interest located in the REX1 promoter show that REX1 expression appears to be negatively correlated with the degree of DNA methylation in HCC cell lines (n=4); and that HCC samples (n=15) exhibit a predominantly hypermethylated state compared to non-tumor samples (n=16). To add onto the clinical relevance of REX1, HuMet450 BeadChip datasets of HCC sample cohorts were obtained from Gene Expression Omnibus and the Cancer Genome Atlas. In these cohorts, the REX1 promoter region is significantly hypermethylated in HCC samples and this methylation is correlated to poor prognosis as well as increased expression of stemness markers. In addition to DNA methylation, we also discovered other regulatory mechanisms for REX1 expression involving histone methylation and acetylation. Functional studies with stable REX1 over-expressing and knock-down HCC cells suggest REX1 expression to negatively correlate with the cells' abilities to migrate, invade, and form foci in vitro; and initiate tumor formation, metastasize and self-renew in vivo. In summary, REX1 is a tumor-suppressor gene that is frequently down-regulated in human HCCs. The multiplicity of REX1 deactivating mechanisms together facilitates REX1 down-regulation to mediate various cancer and stem-like properties in the disease. Citation Format: Steve Tin Chi Luk, Man Tong, Kai Yu Ng, Kevin Yuk-Lap Yip, Xin Yuan Guan, Stephanie Ma. Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3315.

MicroRNA-493 suppresses hepatocellular carcinoma tumorigenesis through down-regulation of anthrax toxin receptor 1 (ANTXR1) and R-Spondin 2 (RSPO2)

Hepatocellular carcinoma (HCC) is known as a highly prevalent cancer with a poor prognosis and short survival time, despite intensive research and clinical efforts. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of hepatocellular carcinoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we report that the expression of microRNA-493 (miR-493) is decreased in HCC cell lines and in tumor tissues. Overexpression of miR-493 in HCC cells dramatically inhibited cell proliferation and colony-formation in vitro and inhibited tumor formation of HCC cell xenografts in vivo. miR-493 also suppressed cell migration and invasion in HCC cell lines. Novel targets ANTXR1 and RSPO2 were confirmed to be suppressed by miR-493 directly, and overexpression of ANTXR1 and RSPO2 could restore tumorigenesis in miR-493 treated HCC cell. Moreover, Wnt/β-catenin signaling pathway, which was reported to be activated by ANTXR1 and RSPO2, was also inhibited by miR-493 overexpression and might be involved in anti-tumor function of miR-493. These findings suggest that miR-493 acts as a negative regulator in hepatocellular carcinoma progression and may be a potential therapeutic target for HCC.

Inhibiting histone deacetylases suppresses glucose metabolism and hepatocellular carcinoma growth by restoring FBP1 expression

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers in the world. Elevated glucose metabolism in the availability of oxygen, a phenomenon called the Warburg effect, is important for cancer cell growth. Fructose-1,6-bisphosphatase (FBP1) is a rate-limiting enzyme in gluconeogenesis and is frequently lost in various types of cancer. Here, we demonstrated that expression of FBP1 was downregulated in HCC patient specimens and decreased expression of FBP1 associated with poor prognosis. Low expression of FBP1 correlated with high levels of histone deacetylase 1 (HDAC1) and HDAC2 proteins in HCC patient tissues. Treatment of HCC cells with HDAC inhibitors or knockdown of HDAC1 and/or HDAC2 restored FBP1 expression and inhibited HCC cell growth. HDAC-mediated suppression of FBP1 expression correlated with decreased histone H3 lysine 27 acetylation (H3K27Ac) in the FBP1 enhancer. Restored expression of FBP1 decreased glucose reduction and lactate secretion and inhibited HCC cell growth in vitro and tumor growth in mice. Our data reveal that loss of FBP1 due to histone deacetylation associates with poor prognosis of HCC and restored FBP1 expression by HDAC inhibitors suppresses HCC growth. Our findings suggest that repression of FBP1 by HDACs has important implications for HCC prognosis and treatment.

Targeting MUC1 and JNK by RNA interference and inhibitor inhibit the development of hepatocellular carcinoma.

Mucin 1 (MUC1), as an oncogene, is overexpressed in hepatocellular carcinoma (HCC) cells and promotes the progression and tumorigenesis of HCC through JNK/TGF‐β signaling pathway. In the present study, RNA interference (RNAi) and JNK inhibitor SP600125, which target MUC1 and/or JNK, were used to treat HCC cells in vitro, and the results showed that both silencing the expression of MUC1 and blocking the activity of JNK inhibited the proliferation of HCC cells. In addition, MUC1‐stable‐knockdown and SP600125 significantly inhibited the growth of tumors in the subcutaneous transplant tumor models that established in BALB/c nude mice rather than MUC1 or JNK siRNAs transiently transfection. Furthermore, the results from immunohistochemical staining assays showed that the inhibitory effects of MUC1 gene silencing and SP600125 on the proliferation of HCC cells in vivo were through the JNK/TGF‐β signaling pathway. These results indicate that MUC1 and JNK are attractive targets for HCC therapy and may provide new therapeutic strategies for the treatment of HCC.

Sorafenib and FH535 in combination act synergistically on hepatocellular carcinoma by targeting cell bioenergetics and mitochondrial function

Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the β-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways.

Reanalysis of microRNA expression profiles identifies novel biomarkers for hepatocellular carcinoma prognosis.

The aim of our study is to identify microRNAs (miRNAs) that have significance in the prognosis and pathogenesis of hepatocellular carcinoma (HCC). The miRNAs differentially expressed in HCC were examined by using a human miRNA microarray dataset, and then the acquired candidates were screened by another microarray dataset. As a result, we got 25 miRNAs which were aberrantly expressed in cancer and meanwhile predicated distinct prognosis. Among them, miR-139-5p was down-regulated in HCC and its low expression in cancer tissue meant poor prognosis. Additionally, we demonstrated that its low expression was also related to several clinicopathologic characteristics such as vein invasion, BCLC stage, p-AKT expression, and pIGFR1 expression. In vitro, it has been discovered that treatment of HCC cells with a miR-139-5p mimic lead to inhibition of cell growth and migration. Moreover, luciferase assay showed that KPNA4 was not the direct target of miR-139-5p. Ectopic expression of miR-139-5p has not repressed the expression of KPNA4, but inhibited the nuclear import of NF-κB and phosphorylation of Akt. In conclusion, for the first time, we identify 25 deregulated miRNAs that are associated with prognosis and prove that miR-139-5p functions as a tumor suppressor in HCC and its low expression predicts poor prognosis.

Increased NEK2 in hepatocellular carcinoma promotes cancer progression and drug resistance by promoting PP1/Akt and Wnt activation.

NIMA-related expressed kinase2(NEK2) participates in the carcinogenesis and progression of certain types of cancer, however, its expression and roles in the development of hepatocellular carcinoma(HCC) remains unknown. Here, we found that NEK2 expression was significantly upregulated in both human HCC tissues and cell lines, and increased NEK2 expression in HCC was significantly correlated with clinical progression of HCC in patients. Knockdown of NEK2 in HCC cells inhibited HCC progression, as determined by the suppressed cell proliferation, invasion and metastasis. Furthermore, knockdown of NEK2 inhibited drug resistance of HCC cells, as shown by the promoted suppression of cell viability in 5-fluorouracil(5‑FU)‑treated HCC cells. Mechanistically, protein phosphatase1(PP1)/Akt and Wnt signaling activation are significantly inhibited by NEK2 knockdown, which is responsible for the HCC progression and involved in NEK2‑induced cancer cell abnormal biological behavior. Thus, enhanced NEK2 expression in HCC promotes HCC progression and drug resistance by promoting PP1/Akt and Wnt pathway activation, which may represent a new therapeutic target for HCC.