Jie Zuo

Cyclophilin A promotes human hepatocellular carcinoma cell metastasis via regulation of MMP3 and MMP9.

Cyclophilin A (CypA) is a member of peptidyl prolyl isomerases (PPIases), which catalyze the cis/trans isomerization of prolyl peptide bonds on the NH-terminal side of Pro residues in peptide chains. Altered expression of CypA has been reported in hepatocellular carcinoma (HCC), but the biological functions of CypA in HCC remain unknown. We found that the level of CypA expression correlated with the metastatic capability of two HCC cell lines, MHCC97-L and MHCC97-H. Stable expression of ectopic CypA in SK-Hep1 cells promotes cell adhesion, motility, chemotaxis, and in vivo lung metastasis, without affecting cell proliferation. We further analyzed microarray results to identify target genes controlled by CypA. Twenty-one genes related to metastasis were altered by CypA over-expression. A member of matrix metalloproteinase, MMP3, was identified by microarray analysis. The regulation of MMP3 and its homologue MMP9 by CypA were further confirmed by quantitative real-time RT-PCR and zymography assay. Taken together, our data suggest that CypA promotes HCC cell metastasis at least partially through up-regulation of MMP3 and MMP9.

Proapoptotic Function of Integrin β3 in Human Hepatocellular Carcinoma Cells

Purpose: This study evaluates the proapoptotic function of integrin β3 in human hepatocellular carcinoma (HCC). Experimental Design: The expression of integrin β3 in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin β3 in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin β3) was determined through colony formation, serum starvation, and anoikis assay. Results: Compared with neighboring pathologically normal liver tissue, ∼60% of the HCC specimens showed a significant down-regulated level of integrin β3 expression. Transient expression of integrin β3 in SMMC-7721 resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721 with stable expression of integrin β3 in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin αvβ3 in liver, was inhibited, which was correlated with the decreased integrin β3 expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin β3. Conclusions: Down-regulation of integrin β3 and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin β3 in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.

Proapoptotic function of integrin beta(3) in human hepatocellular carcinoma cells.

Purpose: This study evaluates the proapoptotic function of integrin β3 in human hepatocellular carcinoma (HCC). Experimental Design: The expression of integrin β3 in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin β3 in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin β3) was determined through colony formation, serum starvation, and anoikis assay. Results: Compared with neighboring pathologically normal liver tissue, ∼60% of the HCC specimens showed a significant down-regulated level of integrin β3 expression. Transient expression of integrin β3 in SMMC-7721 resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721 with stable expression of integrin β3 in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin αvβ3 in liver, was inhibited, which was correlated with the decreased integrin β3 expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin β3. Conclusions: Down-regulation of integrin β3 and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin β3 in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.

An inhibitory role of NEK6 in TGFβ/Smad signaling pathway.

The NEK6 (NIMA-related kinases 6) is reported to play po-tential roles in tumorigenesis. Although it is suggested to function in several cellular pathways, the underlying mechanism in tumorigenesis is still largely unknown. In the present study, we discovered interaction of NEK6 with Smad4, a key member of transforming growth factor beta (TGFβ) pathway. Over-expression of NEK6 in hepatocellular carcinoma (HCC) cell lines suppresses TGFβ-mediated transcription activity in a kinase activity-dependent manner. In addition, NEK6 suppresses the cell growth arrest induced by TGFβ. Mechanically, NEK6 blocks nuclear translocation of Smad4, which is essential for TGFβ function. Moreover, we identified that NEK6 could be regulated by TGFβ and hypoxia. Our study sheds new light on the roles of NEK6 in canonical TGFβ/Smad pathway and tum-origenesis. [BMB Reports 2015; 48(8): 473-478]

Membrane palmitoylated protein 3 promotes hepatocellular carcinoma cell migration and invasion via up-regulating matrix metalloproteinase 1

Membrane associated guanylate kinase (MAGUK) family, has been extensively studied in cellular adhesion and signal transduction at sites of cell–cell contact. Recently, growing attention has been paid to its role in the initiation and progression of various cancers. However, its role in hepatocellular carcinoma (HCC) has been rarely investigated. In this study, we found that membrane palmitoylated protein 3 (MPP3), a member of MAGUK family, was significantly up-regulated in both high metastatic potential cell lines and clinical tissue samples of HCC, and the most significant increase was observed in the tumors invading the portal veins. Higher level of MPP3 correlated with poorer survival of patients with HCC. Forced expression of MPP3 significantly enhanced HCC cell migration and invasion, whereas knockdown of this gene inhibited this oncogenic effect. Mechanismly, we found that MPP3 promoted HCC cell migration and invasion via up-regulating matrix metalloproteinase 1 (MMP1). These findings indicate that MPP3 play an important role in HCC metastasis by promoting cell migration and invasion, suggesting that it may serve as a novel prognostic marker and molecular target for therapy of HCC.

Link of Dlk/ZIP kinase to cell apoptosis and tumor suppression

Death-associated protein kinase (DAPk) family has emerged as a novel subfamily of pro-apoptotic serine/threonine kinase in the last 10 years. Although the functions of DAPk have been well documented, those of other family members remain uncertain. In this work, we characterized the expression pattern of human DAPk like kinase/Zipper interacting protein kinase (Dlk/ZIP kinase) in cancer specimens and cell lines. Dlk expression level was significantly down-regulated in cervical carcinoma cells compared to the surrounding non-tumorous tissues. Overexpression of Dlk led to cell morphological changes, suppressed colony formation and elevated cell apoptosis in cancer cell lines. Both the kinase activity and the cytoplasmic localization were required for its pro-apoptotic tendency. Mechanism exploration revealed that upon serum deprivation, Dlk overexpression could sensitize cells to apoptosis while overexpression of the kinase inactive mutant (Dlk-K42A) was able to rescue apoptotic cell death. Our data thus implicates that Dlk plays a positive role in modulating death-related signaling pathways. Reconstitution of Dlk expression might bring a potential therapeutic approach to cervical carcinoma treatments.

TCP10L acts as a tumor suppressor by inhibiting cell proliferation in hepatocellular carcinoma.

TCP10L (T-complex 10 (mouse)-like) has been identified as a liver and testis-specific gene. Although a potential transcriptional suppression function of TCP10L has been reported previously, biological function of this gene still remains largely elusive. In this study, we reported for the first time that TCP10L was significantly down-regulated in clinical hepatocellular carcinoma (HCC) samples when compared to the corresponding non-tumorous liver tissues. Furthermore, TCP10L expression was highly correlated with advanced cases exceeding the Milan criteria. Overexpression of TCP10L in HCC cells suppressed colony formation, inhibited cell cycle progression through G0/G1 phase, and attenuated cell growth in vivo. Consistently, silencing of TCP10L promoted cell cycle progression and cell growth. Therefore, our study has revealed a novel suppressor role of TCP10L in HCC, by inhibiting proliferation of HCC cells, which may facilitate the diagnosis and molecular therapy in HCC.

Proapoptotic Function of Integrin 3 in Human Hepatocellular Carcinoma Cells

Purpose: This study evaluates the proapoptotic function of integrin β3 in human hepatocellular carcinoma (HCC). Experimental Design: The expression of integrin β3 in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin β3 in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin β3) was determined through colony formation, serum starvation, and anoikis assay. Results: Compared with neighboring pathologically normal liver tissue, ∼60% of the HCC specimens showed a significant down-regulated level of integrin β3 expression. Transient expression of integrin β3 in SMMC-7721 resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721 with stable expression of integrin β3 in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin αvβ3 in liver, was inhibited, which was correlated with the decreased integrin β3 expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin β3. Conclusions: Down-regulation of integrin β3 and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin β3 in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.

Proapoptotic function of integrin β 3 in human hepatocellular carcinoma cells

Purpose: This study evaluates the proapoptotic function of integrin β3 in human hepatocellular carcinoma (HCC). Experimental Design: The expression of integrin β3 in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin β3 in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin β3) was determined through colony formation, serum starvation, and anoikis assay. Results: Compared with neighboring pathologically normal liver tissue, ∼60% of the HCC specimens showed a significant down-regulated level of integrin β3 expression. Transient expression of integrin β3 in SMMC-7721 resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721 with stable expression of integrin β3 in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin αvβ3 in liver, was inhibited, which was correlated with the decreased integrin β3 expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin β3. Conclusions: Down-regulation of integrin β3 and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin β3 in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.

Nogo‐B promotes tumor angiogenesis and provides a potential therapeutic target in hepatocellular carcinoma

Tumor angiogenesis is one of the hallmarks of cancer as well as an attractive target for cancer therapy. Characterization of novel pathways that act in parallel with the VEGF/VEGFR axis to promote tumor angiogenesis may provide insights into novel anti‐angiogenic therapeutic targets. We found that the expression level of Nogo‐B is positively correlated with tumor vessel density in hepatocellular carcinoma (HCC). While Nogo‐B depletion inhibited tumor angiogenesis, Nogo‐B overexpression promoted tumor angiogenesis in a tumor xenograft subcutaneous model of the human HCC cell line. Mechanically, Nogo‐B regulates tumor angiogenesis based on its association with integrin αvβ3 and activation of focal adhesion kinase. Moreover, Nogo‐B antibody successfully abolished the function of Nogo‐B in tumor angiogenesis in vitro and in vivo. Collectively, our results strongly suggest that Nogo‐B is an important tumor angiogenic factor and blocking Nogo‐B selectively inhibits tumor angiogenesis.