MicroRNA-125b attenuates epithelial-mesenchymal transitions and targets stem-like liver cancer cells through small mothers against decapentaplegic 2 and 4.

UNLABELLED: Emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving treatment of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play important roles in HCC; however, the mechanisms by which miRNAs target the EMT and their therapeutic potential remains largely unknown. To better explore the roles of miRNAs in the EMT process, we established an EMT model in HCC cells by transforming growth factor beta 1 treatment and found that several tumor-related miRNAs were significantly decreased. Among these miRNAs, miR-125b expression was most strongly suppressed. We also found down-regulation of miR-125b in most HCC cells and clinical specimens, which correlated with cellular differentiation in HCC patients. We then demonstrated that miR-125b overexpression attenuated EMT phenotype in HCC cancer cells, whereas knockdown of miR-125b promoted the EMT phenotype in vitro and in vivo. Moreover, we found that miR-125b attenuated EMT-associated traits, including chemoresistance, migration, and stemness in HCC cells, and negatively correlated with EMT and cancer stem cell (CSC) marker expressions in HCC specimens. miR-125b overexpression could inhibit CSC generation and decrease tumor incidence in the mouse xenograft model. Mechanistically, our data revealed that miR-125b suppressed EMT and EMT-associated traits of HCC cells by targeting small mothers against decapentaplegic (SMAD)2 and 4. Most important, the therapeutic delivery of synthetic miR-125b mimics decreased the target molecule of CSC and inhibited metastasis in the mice model. These findings suggest a potential therapeutic treatment of miR-125b for liver cancer. CONCLUSION: miR-125b exerts inhibitory effects on EMT and EMT-associated traits in HCC by SMAD2 and 4. Ectopic expression of miR-125b provides a promising strategy to treat HCC.

Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis.

UNLABELLED: Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. CONCLUSION: S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013).

Epimorphin promotes human hepatocellular carcinoma invasion and metastasis through activation of focal adhesion kinase/extracellular signal-regulated kinase/matrix metalloproteinase-9 axis.

UNLABELLED: The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin-2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM-mediated invasion and metastasis of cancer cells is found to require up-regulation of matrix metalloproteinase-9 (MMP-9) through the activation of focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) axis. CONCLUSION: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP-9 expression through the FAK-ERK pathway.

A Functional Screening Identifies a New Organic Selenium Compound Targeting Cancer Stem Cells: Role of c-Myc Transcription Activity Inhibition in Liver Cancer.

Cancer stem cells (CSCs) are reported to play essential roles in chemoresistance and metastasis. Pathways regulating CSC self-renewal and proliferation, such as Hedgehog, Notch, Wnt/ß-catenin, TGF-ß, and Myc, may be potential therapeutic targets. Here, a functional screening from the focused library with 365 compounds is performed by a step-by-step strategy. Among these candidate molecules, phenyl-2-pyrimidinyl ketone 4-allyl-3-amino selenourea (CU27) is chosen for further identification because it proves to be the most effective compound over others on CSC inhibition. Through ingenuity pathway analysis, it is shown CU27 may inhibit CSC through a well-known stemness-related transcription factor c-Myc. Gene set enrichment analysis, dual-luciferase reporter assays, expression levels of typical c-Myc targets, molecular docking, surface plasmon resonance, immunoprecipitation, and chromatin immunoprecipitation are conducted. These results together suggest CU27 binds c-Myc bHLH/LZ domains, inhibits c-Myc-Max complex formation, and prevents its occupancy on target gene promoters. In mouse models, CU27 significantly sensitizes sorafenib-resistant tumor to sorafenib, reduces the primary tumor size, and inhibits CSC generation, showing a dramatic anti-metastasis potential. Taken together, CU27 exerts inhibitory effects on CSC and CSC-associated traits in hepatocellular carcinoma (HCC) via c-Myc transcription activity inhibition. CU27 may be a promising therapeutic to treat sorafenib-resistant HCC.

Arsenic trioxide inhibits liver cancer stem cells and metastasis by targeting SRF/MCM7 complex.

Hepatocellular carcinoma (HCC) has a high mortality rate due to the lack of effective treatments and drugs. Arsenic trioxide (ATO), which has been proved to successfully treat acute promyelocytic leukemia (APL), was recently reported to show therapeutic potential in solid tumors including HCC. However, its anticancer mechanisms in HCC still need further investigation. In this study, we demonstrated that ATO inhibits tumorigenesis and distant metastasis in mouse models, corresponding with a prolonged mice survival time. Also, ATO was found to significantly decrease the cancer stem cell (CSC)-associated traits. Minichromosome maintenance protein (MCM) 7 was further identified to be a potential target suppressed dramatically by ATO, of which protein expression is increased in patients and significantly correlated with tumor size, cellular differentiation, portal venous emboli, and poor patient survival. Moreover, MCM7 knockdown recapitulates the effects of ATO on CSCs and metastasis, while ectopic expression of MCM7 abolishes them. Mechanistically, our results suggested that ATO suppresses MCM7 transcription by targeting serum response factor (SRF)/MCM7 complex, which functions as an important transcriptional regulator modulating MCM7 expression. Taken together, our findings highlight the importance of ATO in the treatment of solid tumors. The identification of SRF/MCM7 complex as a target of ATO provides new insights into ATO's mechanism, which may benefit the appropriate use of this agent in the treatment of HCC.

XB130 Knockdown Inhibits the Proliferation, Invasiveness, and Metastasis of Hepatocellular Carcinoma Cells and Sensitizes them to TRAIL-Induced Apoptosis.

BACKGROUND: XB130 is a recently discovered adaptor protein that is highly expressed in many malignant tumors, but few studies have investigated its role in hepatocellular carcinoma (HCC). Therefore, this study explored the relationship between this protein and liver cancer and investigated its molecular mechanism of action. METHODS: The expression of XB130 between HCC tissues and adjacent nontumor tissues was compared by real-time polymerase chain reaction, immunochemistry, and Western blotting. XB130 silencing was performed using small hairpin RNA. The effect of silencing XB130 was examined using Cell Counting Kit-8, colony assay, wound healing assay, and cell cycle analysis. RESULTS: We found that XB130 was highly expressed in HCC tissues (cancer tissues vs. adjacent tissues: 0.23 ± 0.02 vs. 0.17 ± 0.02, P < 0.05) and liver cancer cell lines, particularly MHCC97H and HepG2 (MHCC97H and HepG2 vs. normal liver cell line LO-2: 2.35 ± 0.26 and 2.04 ± 0.04 vs. 1.00 ± 0.04, respectively, all P < 0.05). The Cell Counting Kit-8 assay, colony formation assay, and xenograft model in nude mice showed that silencing XB130 inhibited cell proliferative ability both in vivo and in vitro, with flow cytometry demonstrating that the cells were arrested in the G0/G1 phase in HepG2 (HepG2 XB130-silenced group [shA] vs. HepG2 scramble group [NA]: 74.32 ± 5.86% vs. 60.21 ± 3.07%, P < 0.05) and that the number of G2/M phase cells was decreased (HepG2 shA vs. HepG2 NA: 8.06 ± 2.41% vs. 18.36 ± 4.42%, P < 0.05). Furthermore, the cell invasion and migration abilities were impaired, and the levels of the epithelial-mesenchymal transition-related indicators vimentin and N-cadherin were decreased, although the level of E-cadherin was increased after silencing XB130. Western blotting showed that the levels of phosphorylated phosphoinositide 3-kinase (PI3K) and phospho-protein kinase B (p-Akt) also increased, although the level of phosphorylated phosphatase and tensin homolog increased, indicating that XB130 activated the PI3K/Akt pathway. Furthermore, we found that a reduction in XB130 increased liver cancer cell sensitivity to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. CONCLUSIONS: Our findings suggest that XB130 might be used as a predictor of liver cancer as well as one of the targets for its treatment.

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1.

BACKGROUND: Decoy receptor 3 (DcR3) is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and the metastasis of the tumor. This in vitro study aimed to investigate the effect of downregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1. METHODS: Three different cell lines were cultured: human cholangiocarcinoma TFK-1, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. RESULTS: The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% (P < 0.01) and 48.03% (P < 0.05) of that of the control, respectively. It was found that the cell viability decreased to 61.87% of the control group (P < 0.01) after the downregulation of DcR3 in cholangiocarcinoma cell line TFK-1 by transfection with DcR3-siRNA, while the percentage of apoptotic cells was 2.98 times as compared with the control group (P < 0.05). Compared with the control group the ratio of G0/G1increased, and the ratio of G2/M decreased in the treatment group. However, the differences were not statistically significant. CONCLUSIONS: The effect of DcR3 on the growth and apoptosis of cholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma.

LncSHRG promotes hepatocellular carcinoma progression by activating HES6.

Hepatocellular carcinoma, one of the most common cancers, leads to mass mortality worldwide currently. However, the underlying mechanism of its oncogenesis remains to be elucidated. Here we identified that a long noncoding RNA, lncSHRG, was greatly upregulated in human hepatocellular carcinoma samples. We found that lncSHRG was essential for liver cancer cell proliferation and tumor propagation in mice. In mechanism, lncSHRG recruits SATB1 to bind to HES6 promoter and initiates HES6 expression. HES6, which is highly expressed in hepatocellular carcinoma, promotes tumor cell proliferation. High expression level of HES6 is positively correlated with clinical severity and poor prognosis of people with hepatocellular carcinoma. Altogether, our research provides a new insight on the mechanism of hepatocellular carcinoma progression.

Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2.

BACKGROUND: Decoy receptor 3 (DcR3) binds to Fas ligand (FasL) and inhibits FasL-induced apoptosis. The receptor is overexpressed in hepatocellular carcinoma (HCC), and it is associated with the growth and metastatic spread of tumors. DcR3 holds promises as a new target for the treatment of HCC, but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3. The present work, therefore, examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2. METHODS: HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3. After the knockdown of DcR3 was confirmed, cell proliferation, clone formation, ability of migrating across transwell membrane, and wound healing were assessed in vitro. Matrix metalloproteinase-9 (MMP 9) and vascular epithelial growth factor (VEGF)-C and D expressions of the DcR3 knockdown were also studied. Comparisons between multiple groups were done using one-way analysis of variance (ANOVA), while pairwise comparisons were performed using Student's t test. P< 0.05 was regarded statistically significant. RESULTS: DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2. Stable knockdown of DcR3 slowed down the growth of HepG2 (P < 0.05) and reduced the number of clones formed by 50% compared to those without DcR3 knockdown (P < 0.05). The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control (P < 0.05) and suppressed the closure of scratch wound (P < 0.05). In addition, the messenger RNA levels of MMP 9, VEGF-C, and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control (P < 0.05). CONCLUSIONS: Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2. Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC.

DcR3 regulates the growth and metastatic potential of SW480 colon cancer cells.

Decoy receptor 3 (DcR3) is considered to have anti­apoptotic and pro-metastatic functions, suggesting it might be a therapeutic target. We examined the role and mechanisms of DcR3 on growth and the metastatic ability of SW480 colon cancer cells to provide therapeutic information for targeting DcR3 by RNA interference (RNAi) technology. Growth and the metastatic ability were inhibited, apoptosis was induced and cell cycle profile was changed after decreasing DcR3 expression, with lower levels of vascular endothelial growth factors (VEGFs) and matrix metalloproteinases (MMPs) expression. Our results implied the therapeutic potential of silencing DcR3 expression by RNAi in colon cancer.