Molecular Target For Hepatocellular Carcinoma Therapy Research Articles

MiR-516a-3p is a Novel Mediator of Hepatocellular Carcinoma Oncogenic Activity and Cellular Metabolism

Hepatocellular carcinoma (HCC) remains one of the most lethal malignancies. We previously demonstrated that the chromosome 19 microRNA cluster (C19MC) was associated with tumor burden and prognosis in patients with HCC. In the current study, we aim to explore the role of miR-516a-3p—an identical mature microRNA (miRNA) co-spliced by four oncogenic pre-miRNAs of C19MC (i.e., mir-516a-1, mir-516a-2, mir-516b-1, and mir-516b-2)—in HCC. In our cohort of HCC patients, miR-516a-3p was highly expressed in HCC tissues in comparison with adjacent non-tumor tissues. High expression of tumor miR-516a-3p significantly correlated with advanced tumor stages, distinguished high HCC recurrence and mortality, and independently predicted poor prognosis. We further found that miR-516a-3p enhanced the proliferation, migration, and invasiveness of HCC cells in vitro and promoted tumor growth and metastasis in vivo . Among cancer cells, miR-516a-3p could be delivered via exosomes or extracellular vesicles and increased the oncogenic activity of recipient cells. Moreover, we performed comprehensive transcriptomics, proteomics, and metabolomics analysis on the potential mechanism underlying miR-516a-3p-promoted oncogenicity. MixOmic DIABLO analysis showed a close correlation and strong cluster consistency between the proteomics and metabolomics datasets. We further confirmed six proteins (i.e., LMBR1, CHST9, RBM3, SLC7A6, PTGFRN, and NOL12) as the direct targets of miR-516a-3p and as central players in miR-516a-3p-mediated metabolism regulation. The integrated multi-omics and co-enriched pathway analysis showed that miR-516a-3p regulates the metabolic pathways of HCC cells, particularly purine and pyrimidine metabolism. In conclusion, our findings suggest that miR-516a-3p promotes malignant behaviors in HCC cells by regulating cellular metabolism and affecting neighboring cells via the exosome delivery system. Thus, we suggest miR-516a-3p as a novel molecular target for HCC therapy.

Enabled homolog (ENAH) regulated by RNA binding protein splicing factor 3b subunit 4 (SF3B4) exacerbates the proliferation, invasion and migration of hepatocellular carcinoma cells via Notch signaling pathway

ABSTRACT Enabled homolog (ENAH) is an actin-binding protein that implicated in multiple malignant tumors. High ENAH expression has been verified to be associated with poor prognosis in hepatocellular carcinoma (HCC). We aimed to reveal the role of ENAH in HCC and the potential mechanism. ENAH expression in HCC tissues and the prognostic correlation were analyzed by GEPIA2 database. RT-qPCR and Western blot were used to test ENAH expression in HCC cells. Following ENAH silencing, cell proliferation was estimated by CCK-8 and colony formation assays. Transwell and wound healing assays were to assess cell invasion and migration. ENCORI database was to analyze the correlation between ENAH and splicing factor 3b subunit 4 (SF3B4) in HCC tissues, which was then verified by RIP and actinomycin D assay. Then, the expression of Notch signaling-related proteins was detected by Western blotting after ENAH knockdown. Afterward, Notch1 was overexpressed to validate whether ENAH impacted the biological events of HCC cells through mediating Notch signaling. Results revealed that ENAH expression was elevated in HCC tissues and cells and associated with poor prognosis. ENAH deficiency mitigated proliferation, invasion and migration of HCC cells. Mechanistically, ENAH was positively correlated with SF3B4 in HCC tissues. SF3B4 could bind to ENAH mRNA and stabilized ENAH. Besides, ENAH activated Notch signaling. Notch1 up-regulation reversed the influence of ENAH knockdown on biological events of HCC cells. Collectively, ENAH regulated by SF3B4 promoted the development of HCC through activating Notch signaling, which identified ENAH as a potent molecular target for HCC therapy and prognosis.

A New CYP2E1 Inhibitor, 12-Imidazolyl-1-dodecanol, Represents a Potential Treatment for Hepatocellular Carcinoma.

Cytochrome P450 2E1 (CYP2E1) is a key target protein in the development of alcoholic and nonalcoholic fatty liver disease (FLD). The pathophysiological correlate is the massive production of reactive oxygen species. The role of CYP2E1 in the development of hepatocellular carcinoma (HCC), the final complication of FLD, remains controversial. Specifically, CYP2E1 has not yet been defined as a molecular target for HCC therapy. In addition, a CYP2E1-specific drug has not been developed. We have already shown that our newly developed CYP2E1 inhibitor 12-imidazolyl-1-dodecanol (I-ol) was therapeutically effective against alcoholic and nonalcoholic steatohepatitis. In this study, we investigated the effect of I-ol on HCC tumorigenesis and whether I-ol could serve as a possible treatment option for terminal-stage FLD. I-ol exerted a very highly significant antitumour effect against hepatocellular HepG2 cells. Cell viability was reduced in a dose-dependent manner, with only the highest doses causing a cytotoxic effect associated with caspase 3/7 activation. Comparable results were obtained for the model colorectal adenocarcinoma cell line, DLD-1, whose tumorigenesis is also associated with CYP2E1. Transcriptome analyses showed a clear effect of I-ol on apoptosis and cell-cycle regulation, with the increased expression of p27Kip1 being particularly noticeable. These observations were confirmed at the protein level for HepG2 and DLD-1 cells grafted on a chorioallantoic membrane. Cell-cycle analysis showed a complete loss of proliferating cells with a simultaneous increase in S-phase arrest beginning at a threshold dose of 30 μM. I-ol also reduced xenograft tumour growth in nude mice. This antitumour effect was not associated with tumour cachexia. I-ol was not toxic to healthy tissues or organs. This study demonstrates for the first time the therapeutic effect of the specific CYP2E1 inhibitor I-ol on the tumorigenesis of HCC. Our findings imply that I-ol can potentially be applied therapeutically on patients at the final stage of FLD.

Prognostic significance of miR-203 and ZEB1 expression in early-stage hepatocellular carcinoma.

Background: Approximately one-quarter of patients with early-stage hepatocellular carcinoma (HCC) suffer from tumor recurrence within the first year after hepatectomy. Identification of patients at high risk of recurrence and new therapeutic approaches are crucial to improve clinical outcome. This study aimed to assess the prognostic significance of miR-203 and Zinc finger E-box binding homeobox 1 (ZEB1) in early-stage HCC and explore the association between the expression of ZEB1 and miR-203 in HCC.Methods: Tissue microarray-based immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to investigate ZEB1 and miR-203 expression in 73 patients with early-stage HCC and their correlation with clinicopathological features and prognosis of patients were analyzed. The prognostic value of the two factors was also measured by public KM plotter database. Quantitative reverse transcription PCR (qRT-PCR) assays were conducted to study the relationship between miR-203 and ZEB1. Transwell assays, Cell Counting Kit-8 (CCK-8) assays were performed to detect the roles of miR-203 in migration, invasion and proliferation of HCC cells.Results: We found low expression of miR-203 was associated significantly with tumor recurrence (P<0.001) and poor survival (P=0.020) of patients with early-stage HCC. Multivariate analysis revealed that low miR-203 expression was a poor prognostic factor for both overall survival (OS) (P=0.036) and recurrence free survival (RFS) (P=0.017). ZEB1 did not show any prognostic significance in our cohort. Correlation analysis indicated that there was no significant correlation between miR-203 and ZEB1 on both mRNA and protein levels. Furthermore, functional studies indicated that miR-203 repressed migration, invasion and proliferation of HCC cells in vitro.Conclusion: Our study suggested that miR-203 could be a novel predictor in early-stage HCC and might also be a potential molecular target for HCC therapy.

SSH3 promotes malignant progression of HCC by activating FGF1-mediated FGF/FGFR pathway.

To investigate the impact of silencing SSH3 on the expression of FGF/FGFR pathway-related genes FGF1, FGFR1, and FGFR2 in hepatocellular carcinoma (HCC) cell line, so as to further understand the role of SSH3 in proliferation and apoptosis of HCC cells. TWe first detected SSH3 expression in 51 pairs of tumor tissue specimens and adjacent tissues collected from HCC patients through quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and analyzed the interplay between SSH3 expression and clinical characteristics of HCC patients. In vitro, after SSH3-silenced human HCC cell line was constructed by lentiviral transfection, Cell Counting Kit-8 (CCK-8), cell cloning assay, and flow apoptosis methods were conducted to explore the HCC cell functions. Finally, whether SSH3 exerts its biological characteristics through the FGF/FGFR pathway and the mutual regulation mechanism between SSH3 and FGF1 were further uncovered. It was found that SSH3 expression was remarkably higher in tumor tissues of HCC patients than that in normal tissues. Meanwhile, in comparison to patients with low expression of SSH3, patients with high expression of SSH3 had higher pathological grade and larger tumor size. In addition, after silencing SSH3, HCC cell proliferation ability was attenuated while the apoptosis ability was enhanced in comparison to the control group. Moreover, the protein levels of FGF1/FGFR pathway-related genes FGF1, FGFR1, and FGFR2 were markedly inhibited by the downregulation of SSH3. Meanwhile, cell recovery experiment demonstrated that the overexpression of FGF1 reversed the impact of SSH3 silencing on the proliferation and apoptosis of HCC cells. In summary, SSH3 is capable of accelerating the malignant progression of HCC by activating FGF1-mediated FGF/FGFR pathway, thus becoming a new molecular target for HCC therapy.

Glutamic-Pyruvic Transaminase 1 Facilitates Alternative Fuels for Hepatocellular Carcinoma Growth-A Small Molecule Inhibitor, Berberine.

Metabolic reprogramming is an essential hallmark of cancer. Besides the “Warburg effect”, cancer cells also actively reprogram amino acid metabolism to satisfy high nutritional demands in a nutrient-poor environment. In the glucose–alanine cycle, exogenous alanine taken up by hepatocytes is converted to pyruvate via glutamic-pyruvic transaminases (GPTs). However, the precise role of the glucose–alanine cycle in hepatocellular carcinoma (HCC) remains elusive. The current study revealed that alanine, as an alternative energy source, induced the metabolic reprogramming of HCC cells via activation of the downstream glucose–alanine cycle and thus promoted HCC growth in nutrient-depleted conditions. Further overexpression and loss-of-function studies indicated that GPT1 was an essential regulator for alanine-supplemented HCC growth. Combining molecular docking and metabolomics analyses, our study further identified a naturally occurring alkaloid, berberine (BBR), as the GPT1 inhibitor in HCC. Mechanically, BBR-mediated metabolic reprogramming of alanine-supplemented HCC via GPT1 suppression attenuated adenosine triphosphate (ATP) production and thus suppressed HCC growth. In conclusion, our study suggests that GPT1-mediated alanine–glucose conversion may be a potential molecular target for HCC therapy. Further demonstration of BBR-mediated metabolic reprogramming of HCC would contribute to the development of this Chinese medicine-derived compound as an adjuvant therapy for HCC.

The impact of MCM6 on hepatocellular carcinoma in a Southern Chinese Zhuang population

Minichromosome maintenance complex component 6 (MCM6) is involved in tumorigenesis of hepatocellular carcinoma (HCC). Because its effect on different populations remains unclear, this study investigated the impact of MCM6 on HCC in Southern Chinese Zhuang population. In addition to assessing the global mRNA levels of MCM6 based on The Cancer Genome Atlas database (TCGA) and The Gene Expression Omnibus database (GEO), associations between MCM6 mRNA levels and clinicopathological features were analyzed. High MCM6 levels were associated with high alpha-fetoprotein (AFP) (>20 ng/mL in serum) (P < 0.0001) and advanced clinical stage (III + IV) (P < 0.001). Higher MCM6 was associated with poorer outcomes (P < 0.01) in these databases. Furthermore, the mRNA and protein expression of MCM6 in the Guangxi Zhuang population was detected by quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry (IHC). The results showed that MCM6 levels were up-regulated in the Zhuang population with HCC. Higher MCM6 protein levels were correlated with larger tumor size (>5 cm) (P = 0.038) and advanced clinical stage (III + IV) (p = 0.023). Bioinformatic enrichment analysis of MCM6 and its interacting proteins (CDT1,WEE1,TRIM28 and MKI67) suggested that in addition to being involved in the cell cycle process, these complexes could also be involved in protein binding, pre-replication complex assemble, and nucleus metabolism. Based on the protein-protein interaction (PPI) network with module screen, the interactions between MCM6 and its potential interacting proteins were further studied through protein docking with hot spot analysis. Additionally, the results of the algorithms combining the ROC of MCM6 and its interacting proteins showed that combination biomarker analysis has better HCC diagnosis ability than the single MCM6 test. The combination of MCM6 and TRIM28 was more suitable for the Guangxi Zhuang population. Overall, our study suggests that MCM6 plays an important role in the growth of HCC. MCM6 could be an optimal biomarker for diagnosing HCC and a potential molecular target for HCC therapy in the Zhuang population.

The role of vesicular overexpressed in cancer pro-survival protein 1 in hepatocellular carcinoma proliferation.

Recently, hepatocellular carcinoma (HCC) has been ranked as the second leading cause of cancer-associated death. However, the underlying molecular mechanisms of HCC progression remain unclear. Vesicular overexpressed in cancer pro-survival protein 1 (VOPP1) could be upregulated in a quantity of human cancers, including squamous cell carcinoma (SCC), gastric cancer, and glioblastoma. However, the precise functional mechanism of VOPP1 in HCC remains poorly understood. The present study aimed to investigate the role of VOPP1 in HCC proliferation. Immunohistochemistry (IHC), Western blot and Reverse-transcription polymerase chain reaction (RT-PCR) were used to analyze the protein and mRNA expressions of VOPP1, mitogen-activated protein kinase (MAPK) 14, ribosomal protein S6 kinase β1 (RPS6KB1), cylindromatosis (CYLD) and Twist family bHLH transcription factor 1 (TWIST1). The cell proliferation and apoptosis were tested using Celigo cell imaging analyzer and annexin V-APC apoptosis detection kit respectively. Colony formation and tumor xenograft assays were performed to understand their roles in tumorigenicity. The expression of VOPP1 in HCC samples was higher than that in adjacent noncancerous tissues by immunohistochemistry. In addition, the down-regulation of VOPP1 using shRNA inhibited cell proliferation and tumour growth, and induced cell apoptosis in vitro and in vivo. Furthermore, VOPP1 silencing increased the expression of MAPK14 and RPS6KB1, indicating that the MAPK and mTOR signalling pathways might be involved in VOPP1-mediated cancer cell proliferation. The present data indicate that VOPP1 may play an important role in the progression of HCC by targeting the MAPK and mTOR signalling pathways, and that VOPP1 may potentially be a candidate as a novel molecular target for HCC therapy.

OGDHL silencing promotes hepatocellular carcinoma by reprogramming glutamine metabolism

Mitochondrial dysfunction and subsequent metabolic deregulation are commonly observed in cancers, including hepatocellular carcinoma (HCC). When mitochondrial function is impaired, reductive glutamine metabolism is a major cellular carbon source for de novo lipogenesis to support cancer cell growth. The underlying regulators of reductively metabolized glutamine in mitochondrial dysfunction are not completely understood in tumorigenesis. We systematically investigated the role of oxoglutarate dehydrogenase-like (OGDHL), one of the rate-limiting components of the key mitochondrial multi-enzyme OGDH complex (OGDHC), in the regulation of lipid metabolism in hepatoma cells and mouse xenograft models. Lower expression of OGDHL was associated with advanced tumor stage, significantly worse survival and more frequent tumor recurrence in 3 independent cohorts totaling 681postoperative HCC patients. Promoter hypermethylation and DNA copy deletion of OGDHL were independently correlated withreduced OGDHL expression in HCC specimens. Additionally, OGDHL overexpression significantly inhibited the growth of hepatoma cells in mouse xenografts, while knockdown of OGDHL promoted proliferation of hepatoma cells. Mechanistically, OGDHL downregulation upregulated the α-ketoglutarate (αKG):citrate ratio by reducing OGDHC activity, which subsequently drove reductive carboxylation of glutamine-derived αKG via retrograde tricarboxylic acid cycling in hepatoma cells. Notably, silencing of OGDHL activated the mTORC1 signaling pathway in anαKG-dependent manner, inducing transcription of enzymes with key roles in de novo lipogenesis. Meanwhile, metabolic reprogramming in OGDHL-negative hepatoma cells provided an abundant supply of NADPH and glutathione to support the cellular antioxidant system. The reduction of reductive glutamine metabolism through OGDHL overexpression or glutaminase inhibitors sensitized tumor cells to sorafenib, a molecular-targeted therapy for HCC. Our findings established that silencing of OGDHL contributed to HCC development and survival by regulating glutamine metabolic pathways. OGDHL is a promising prognostic biomarker and therapeutic target for HCC. Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and is correlated with a high mortality rate. In patients with HCC, lower expression of the enzyme OGDHL is significantly associated with worse survival. Herein, we show that silencing of OGDHL induces lipogenesis and influences the chemosensitization effect of sorafenib in liver cancer cells by reprogramming glutamine metabolism. OGDHL is a promising prognostic biomarker and potential therapeutic target in OGDHL-negative liver cancer.

A selective c-Met and Trks inhibitor Indo5 suppresses hepatocellular carcinoma growth

BackgroundHuman hepatocellular carcinoma (HCC) lacks effective curative therapy and there is an urgent need to develop a novel molecular-targeted therapy for HCC. Selective tyrosine kinase inhibitors have shown promise in treating cancers including HCC. Tyrosine kinases c-Met and Trks are potential therapeutic targets of HCC and strategies to interrupt c-Met and Trks cross-signaling may result in increased effects on HCC inhibition.MethodsThe effects of Indo5 on c-Met and Trks activity were determined with in vitro kinase activity assay, cell-based signaling pathway activation, and kinases-driven cell transformation. The in vivo anti-tumor activity was determined with xenograft mice and liver orthotopic mice models. The co-expression of c-Met and TrkB in 180 pairs of HCC and adjacent normal tissues were detected using immunohistochemical staining.ResultsIndo5, a novel lead compound displayed biochemical potency against both c-Met and Trks with selectivity over 13 human kinases. Indo5 abrogated HGF-induced c-Met signaling activation and BDNF/NGF-induced Trks signal activation, c-Met or TrkB-mediated cell transformation and migration. Furthermore, Indo5 significantly decreased the growth of HCC cells in xenograft mice and improved the survival of mice with liver orthotopic tumors. In addition, co-expression of c-Met and TrkB in HCC patients was a predictor of poor prognosis, and combined inhibition of c-Met and TrkB exerted a synergistic suppressive effect on HCC.ConclusionsThese findings indicate that Indo5 is associated with marked suppression of c-Met and Trks co-expressing HCC, supporting its clinical development as an antitumor treatment for HCC patients with co-active c-Met and Trks signaling.

Insulin-like Growth Factor I Receptor: A Novel Target for Hepatocellular Carcinoma Gene Therapy.

Human insulin-like growth factor (IGF) axis affects the molecular pathogenesis of hepatocellular carcinoma (HCC), especially in the abnormality of hepatic IGF-I receptor (IGF-IR) or IGF-II expression as a key molecule in hepatocarcinogenesis. However, the over-expression of hepatic IGFIR is associated with HCC progression with largely unknown mechanisms. The IGF-IR as one key molecule of the IGF signal pathway plays an important role in the hepatocyte malignant transformation. Attaching importance to IGF-IR might improve the prognostic or the therapeutic technique of HCC. This article reviews IGF-IR alteration during HCC development, and the effects of silencing IGF-IR gene by specific short hairpin RNA on the inhibition of cell proliferation in vitro or HCC xenograft growth in vivo to elucidate it as a novel molecular-targeted therapy for HCC.

PES1 is transcriptionally regulated by BRD4 and promotes cell proliferation and glycolysis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. However, the mechanism underlying the tumorigenesis of HCC is still unclear. Improper recruitment of Pescadillo homologue 1 (PES1) can lead to tumorigenesis in multiple cancer types, such as gastric cancer and colon cancer. Here, we reported that PES1 was upregulated and associated with a poor prognosis in HCC specimens. Next, we found that PES1 promoted the growth of HCC in vivo and in vitro. Furthermore, we showed that the knockdown of PES1 decreased glycolysis via altering the gene expression of GLUT1, PKM2, ENO1, FBP1, and PCK1, which are related to glucose metabolism in HCC cells. Moreover, we demonstrated that PES1 is regulated by bromodomain-containing protein 4 (BRD4) and is partially responsible for modulating the antitumor effect of BET inhibitors in HCC. Taken together, our results suggest that PES1 plays an important role in promoting the proliferation of human liver cancer cells, suggesting that PES1 may be an ideal molecular target for HCC therapy.

Four-and-a-half LIM protein 1 promotes paclitaxel resistance in hepatic carcinoma cells through the regulation of caspase-3 activation.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. To investigate the mechanisms of paclitaxel resistance in hepatocellular carcinoma cells and find promising molecular target for HCC therapy. To investigate the effects of FHL1 on chemo resistance in HCC cells, we generated FHL1 knock-down stable cell lines with HepG2 and SMMC7721 cells. Cell viability assay, colony formation and xenograft experiments assay were performed to detect effect of FHL1 on Paclitaxel or Oxaliplatin resistance in vitro and in vivo. Caspase activity assay was performed to explore the activation of caspase-3 and caspase-9 in paclitaxel treated FHL1-knockdown HepG2 cells. In the present study we have investigated that four-and-a-half LIM protein 1 (FHL1), which plays an important role in the development of cancer, is associated with both the chemo resistance of hepatocellular carcinomas cells in vitro and in vivo. Knockdown of FHL1 significantly enhanced the sensitivity of paclitaxel, but had no effects on sensitivity of oxaliplatin. Moreover, knockdown of FHL1 promoted the activation of caspase-3 and caspase-9, which were induced by paclitaxel. Interestingly, FHL1 negatively regulates the chemo resistance of HCC in xenografted nude mice. FHL1 promote paclitaxel resistance in hepatocellular carcinomas cells through regulating apoptosis induced by paclitaxel, suggesting that FHL1 may be a promising molecular target for HCC therapy.

Mcl-1 as a potential therapeutic target for human hepatocelluar carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.

SIRT1-mediated transcriptional regulation of SOX2 is important for self-renewal of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is a highly aggressive liver tumor containing cancer stem cells (CSCs), which participate in tumor invasion, therapeutic resistance, and tumor relapse leading to poor outcome and limited therapeutic options. Histone deacetylatase sirtuin 1 (SIRT1) has been shown to be up-regulated in human cancers; however, its role in liver CSCs is unknown. In this study, we explored the biological functions of SIRT1 in liver CSCs. Our data show that SIRT1 is highly expressed in liver CSCs and decreases during differentiation. In addition, high levels of SIRT1 predict a decreased probability of survival in patients with HCC. SIRT1 is responsible for the maintenance of self-renewal and tumorigenicity of liver CSCs, and overexpression of exogenous SIRT1 can restore self-renewal of non-CSCs. We demonstrated that SOX2 is a main downstream regulator of SIRT1-mediated self-renewal and tumorigenicity potential of liver CSCs. Mechanistically, SIRT1 regulates transcription of the SOX2 gene by way of chromatin-based epigenetic changes, which are dependent on DNA methylation. This effect is achieved by alternation of histone modification and interaction with DNA methyltransferase 3A, resulting in hypermethylation of SOX2 promoter. Furthermore, we demonstrated that insulin growth factor signaling plays an important role in maintaining SIRT1 expression through increased SIRT1 protein stability. These findings highlight the importance of SIRT1 in the biology of liver CSCs and suggest that SIRT1 may serve as a molecular target for HCC therapy. (Hepatology 2016;64:814-827).

RhoE/ROCK2 regulates chemoresistance through NF-κB/IL-6/ STAT3 signaling in hepatocellular carcinoma

Small Rho GTPase (Rho) and its immediate effector Rho kinase (ROCK) are reported to regulate cell survival, but the detailed molecular mechanism remains largely unknown. We had previously shown that Rho/ROCK signaling was highly activated in hepatocellular carcinoma (HCC). In this study, we further demonstrated that downregulation of RhoE, a RhoA antagonist, and upregulation of ROCK enhanced resistance to chemotherapy in HCC in both in vitro cell and in vivo murine xenograft models, whereas a ROCK inhibitor was able to profoundly sensitize HCC tumors to cisplatin treatment. Specifically, the ROCK2 isoform but not ROCK1 maintained the chemoresistance in HCC cells. Mechanistically, we demonstrated that activation of ROCK2 enhanced the phosphorylation of JAK2 and STAT3 through increased expression of IL-6 and the IL-6 receptor complex. We also identified IKKβ as the direct downstream target of Rho/ROCK, and activation of ROCK2 significantly augmented NF-κB transcription activity and induced IL-6 expression. These data indicate that Rho/ROCK signaling activates a positive feedback loop of IKKβ/NF-κB/IL-6/STAT3 which confers chemoresistance to HCC cells and is a potential molecular target for HCC therapy.

Downregulation and tumor-suppressive role of XPO5 in hepatocellular carcinoma.

XPO5 (Exp5, Exportin-5) is a transporter protein mainly mediating pre-microRNAs' nuclear export. Recent studies have demonstrated that XPO5 may play crucial roles in a few of cancers. However, little is known about XPO5 in hepatocellular carcinoma (HCC). In the present study, we elucidated the expression of XPO5 by quantitative real-time PCR (qRT-PCR) and immunohistochemical staining in HCC samples and conducted several functional analyses to address its effects on HCC development. The results demonstrated that both mRNA and protein levels of XPO5 were downregulated in HCC tissues compared to adjacent non-cancerous livers. Ectopic expression of XPO5 significantly suppressed cell proliferation, colony formation, growth in soft agar, and tumorigenicity in nude mice, whereas knockdown of XPO5 by RNA inference showed opposite phenotypes. Moreover, XPO5 knockdown promoted HCC cell migration and decreased the expression of E-cadherin and p53. Additionally, after treatment with DAC and TSA, the mRNA level of XPO5 was upregulated in HCC cells tested, implicating that epigenetic modulation may be involved in the transcription of XPO5. Collectively, our findings suggest that XPO5 functions as a potential tumor suppressor in the development and progression of HCC as well as a promising molecular target for HCC therapy.

A novel prognostic biomarker SPC24 up-regulated in hepatocellular carcinoma

SPC24 is an important component of the nuclear division cycle 80 (Ndc80) kinetochore complex, which plays an essential role in the coupling of kinetochore to spindle microtubules (MTs) and the accurate segregation of chromosomes during mitosis. However, the functional role of SPC24 in hepatocellular carcinoma (HCC) remains unknown. Here, we detected the expression of SPC24 in HCC and analyzed its association with clinicopathologic features and prognosis of HCC patients. The expression of SPC24 mRNA was investigated in 212 cases of paired HCC and adjacent liver tissues by quantitative real-time PCR (qRT-PCR) and in the tissues of 20 HCC patients by semi-quantitative RT-PCR. Additionally, the expression of SPC24 protein was detected in 69 cases of HCC by immunohistochemistry (IHC) or in 2 cases of HCC tissues by Western-blotting. Furthermore, small interfering RNA (siRNA)-mediated silencing of SPC24 was employed in SMMC7721 and HepG2 human HCC cells to investigate cell proliferation, invasion and apoptosis. Survival curves were plotted using the Kaplan-Meier method, and differences in survival probability were obtained using the log-rank test. Independent predictors associated with disease-free survival (DFS) and overall survival (OS) were analyzed using the Cox proportional-hazards regression model. In this study, we showed that SPC24 was noticeably increased in HCC tissues compared to normal adjacent noncancerous tissues, at both mRNA and protein levels. High expression of SPC24 was significantly correlated with alpha-fetoprotein (AFP) (p = 0.044), median size (p = 0.030), tumor number (p = 0.019), and Barcelona-Clinic Liver Cancer (BCLC) stage (p = 0.015). Kaplan-Meier analysis showed that the DFS and OS of high SPC24 expression group was significantly shorter than that of low SPC24 expression group (p < 0.001; p = 0.001; respectively). The prognostic impact of SPC24 was further confirmed by stratified survival analysis. Importantly, multivariate analysis identified SPC24 upregualtion (p = 0.001), PVTT (p = 0.007), size of tumor > 5 cm (p < 0.001) as independent risk factors of DFS after resection, and SPC24 upregualtion (p < 0.001), PVTT (p = 0.029), size of tumor > 5 cm (p = 0.002), recurrence (p < 0.001) as independent prognostic factors for the OS of HCC patients. Additionally, siRNA-mediated silencing of SPC24 dramatically suppressed cell growth, adhesion, invasion and increased apoptosis in HCC cells. In conclusion, these results showed for the first time that SPC24 expression was significantly up-regulated in HCC, which may act as a novel prognostic biomarker for patients suffering from this deadly disease. Additionally, silence of SPC24 inhibiting HCC cell growth indicated that SPC24 may be a promising molecular target for HCC therapy.

Down-regulating glypican-3 expression: molecular-targeted therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) prognosis is very poor, its early treatment is of the utmost importance. Glypican-3 (GPC-3), a membrane-associated heparan sulfate proteoglycan, plays a crucial role in cell proliferation and metastasis, particularly in progression. GPC-3 mediated oncogenesis involves signaling pathways during the malignant transformation of hepatocyte carcinogenesis, with an increasing expression of GPC-3 observed from non-cancerous- to cancerous-tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. GPC-3 expression in HCC tissues or circulating blood was associated with tumor size or HBV infection. Circulation of GPC-3-mRNA was detected in HCC patients with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis. After hepatoma, cells were transfected with shRNA, GPC-3 expression or proliferation was inhibited with promoting apoptosis, cell cycle arrested in G1 phase, alteration of hepatoma cell migration and invasion behaviors with down-regulation of β-catenin, IGF-II, and VEGF, and growth of nude mice xenograft tumors was significantly suppressed with the decreases in β-catenin, p-GSK3β, and cyclinD1 expression, suggesting that GPC-3 not only is a specific biomarker for HCC diagnosis, but also is a valuable molecular-target for HCC gene therapy.

Abstract 1047: The multikinase inhibitor K252a suppresses hepatocellular carcinoma tumor growth in a mouse xenograft model

Abstract There are several ongoing clinical trials of molecular-targeted therapy for hepatocellular carcinoma (HCC), but no clearly effective therapy has yet emerged. Under such conditions, we have been focusing on potentially powerful anticancer effects of the novel multikinase inhibitor K252a, since it targets unique kinases, including PKC, CaMKII, c-Met, TrkB, MLCK, and phosphorylase kinase. Indeed, the former five targets are expressed in HCC tissues and involved in cellular proliferation, invasion, and metastasis (Yoshiji et al., Cancer Res 1999, etc.). The broad spectrum of the targets may block escape pathways in HCC cells, that are resistant to conventional anticancer drugs and even sorafenib. The AIM of this study was to assess whether K252a suppressed cellular proliferation and altered malignant phenotypes such as epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Materials and Methods: The human HCC cell lines HAK-1A, HAK-1B (Yano et al., Hepatology 1993), KYN-2 (Yano et al., Pathol Int 1988), and Huh7 were used in this study. Effects of K252a on the proliferation and the cell cycle were assessed by a commercialized kit (Promega) and flow cytometry, respectively. Protein expression and localization was analyzed by Western blot and immunocytochemistry, respectively. Cellular mRNA level was evaluated by real-time PCR using TaqMan probes. HAK-1B-based xenograft model in nude mice was used to evaluate in-vivo efficacy of K252a. Results: K252a suppressed proliferation and clearly caused G2 cell-cycle arrest in all the cell lines studied. Additionally, the agent induced both polygonal transformation in cell shape and increase in cell size in several HCC cell lines having endogenous mesenchymal features. In Western blot analysis and immunocytochemistry, the expression levels of E2A, an EMT-regulating transcriptional factor, were universally decreased in the cells treated with K252a, in concert with increased expression levels of E-cadherin in some cell lines. In real-time PCR analyses, the decrease in E2A mRNA levels and the increase in E-cadherin mRNA levels were ubiquitously found. Of note, xenografted tumor tissues obtained from mice treated with K252a exhibited significant increase in E-cadherin protein expression, in comparison with those from control mice. Conclusions: Our findings suggest that K252a has robust antitumor effects on HCC through, at least in part, reversing EMT and inducing G2 cell-cycle arrest. This kind of multikinase inhibitor may help us establish the proof-of-concept for closing escape routes in drug-resistant HCC cells. Citation Format: Mitsuhiko Abe, Hironori Koga, Takafumi Yoshida, Hiroshi Masuda, Masahiro Sakata, Yu Ikezono, Toru Nakamura, Eitaro Taniguchi, Takumi Kawaguchi, Hirohisa Yano, Takuji Torimura, Michio Sata. The multikinase inhibitor K252a suppresses hepatocellular carcinoma tumor growth in a mouse xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1047. doi:10.1158/1538-7445.AM2014-1047