Nobuyuki Takaoka

Twist expression promotes migration and invasion in hepatocellular carcinoma.

BackgroundTwist, a transcription factor of the basic helix-loop-helix class, is reported to regulate cancer metastasis. It is known to induce epithelial-mesenchymal transition (EMT). In this study, we evaluated the expression of twist and its effect on cell migration in hepatocellular carcinoma (HCC).MethodsWe examined twist expression using immunohistochemistry in 20 tissue samples of hepatocellular carcinoma, and assessed twist expression in HCC cell lines by RT-PCR and Western blot analysis. Ectopic twist expression was created by introducing a twist construct in the twist-negative HCC cell lines. Endogenous twist expression was blocked by twist siRNA in the twist-positive HCC cell lines. We studied EMT related markers, E-cadherin, Vimentin, and N-cadherin by Western blot analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. We used immunofluorescent vinculin staining to visualize focal adhesion.ResultsWe detected strong and intermediate twist expression in 7 of 20 tumor samples, and no significant twist expression was found in the tumor-free resection margins. In addition, we detected twist expression in HLE, HLF, and SK-Hep1 cells, but not in PLC/RPF/5, HepG2, and Huh7 cells. Ectopic twist-expressing cells demonstrated enhanced cell motility, but twist expression did not affect cell proliferation. Twist expression induced epithelial-mesenchymal transition together with related morphologic changes. Focal adhesion contact was reduced significantly in ectopic twist-expressing cells. Twist-siRNA-treated HLE, HLF, and SK-Hep1 cells demonstrated a reduction in cell migration by 50, 40 and 18%, respectively.ConclusionTwist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition.

Des-γ-carboxy Prothrombin Is a Potential Autologous Growth Factor for Hepatocellular Carcinoma

Des-γ-carboxyl prothrombin (DCP) is a well recognized tumor marker for hepatocellular carcinoma (HCC). In the present study, we demonstrate that DCP has a mitogenic effect on HCC cell lines. Purified DCP stimulated DNA synthesis of Hep3B and SK-Hep-1 cells in a dose-dependent manner. DCP was found to bind with cell surface receptor Met causing Met autophosphorylation and also to activate STAT3 signaling pathway through Janus kinase 1. Luciferase gene reporter analysis showed that DCP induced STAT3-related transcription. Small interfering RNAs against both STAT3 and Met abrogated DCP-induced cell proliferation. DCP did not affect the mitogen-activated protein kinase pathway, Myc signaling pathway, or phosphoinositide 3-kinase/Akt pathway. Based on these results, we believe that DCP acts as an autologous mitogen for HCC cell lines. The Met-Janus kinase 1-STAT3 signaling pathway may be a major signaling pathway for DCP-induced cell proliferation.

Des-γ-carboxyl Prothrombin-promoted Vascular Endothelial Cell Proliferation and Migration

Des-γ-carboxyl prothrombin (DCP) is a well recognized tumor marker for hepatocellular carcinoma. Previously, we have demonstrated that DCP stimulates cell proliferation in hepatocellular carcinoma cell lines through Met-Janus kinase 1 signal transducer and activator of transcription 3 signaling pathway. In the present study, we demonstrated that DCP induces both cell proliferation and migration in human umbilical vein endothelial cells. DCP was found to bind with the kinase insert domain receptor (KDR), alternatively referred to as vascular endothelial growth factor receptor-2. Furthermore, DCP induced autophosphorylation of KDR and its downstream effector phospholipase C-γ and mitogen-activated protein kinase (MAPK). To support these results, we showed that DCP-induced cell proliferation and cell migration were inhibited by KDR short interfering RNA, KDR kinase inhibitor, or MAPK inhibitor. In conclusion, these results indicate that DCP is a novel type of vascular endothelial growth factor that possesses potent mitogenic and migrative activities.

Restored expression of the tumor suppressor gene RUNX3 reduces cancer stem cells in hepatocellular carcinoma by suppressing Jagged1-Notch signaling

Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene that is downregulated in various cancers. In the present study, we analyzed the regulatory function of RUNX3 on Jagged-1 (JAG1) expression and cancer stem cell (CSC) signaling in hepatocellular carcinoma (HCC). Eleven HCC cell lines and 30 human HCC tissues were used. RUNX3 and JAG1 expression levels were analyzed by immunoblotting and immunohistochemistry. Ectopic RUNX3 expression was induced by introducing RUNX3 cDNA into the RUNX3-negative HCC cell line Hep3B and Huh7 cells. Furthermore endogenous RUNX3 expression was knocked down by RUNX3 siRNA in SK-Hep-1 cells. In order to analyze JAG1 transcriptional regulation, we conducted reporter assays, chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSAs). Tumorigenicity was analyzed using a SCID mouse liver injection model. An inverse correlation was observed between RUNX3 expression and JAG1 expression in most HCC cell lines and tissues. Restoring RUNX3 expression decreased the expression of JAG1 in Hep3B and Huh7 cells, whereas JAG1 expression was upregulated in RUNX3 siRNA-treated SK-Hep-1 cells. Reporter assays, ChIP assays and EMSAs revealed that RUNX3 directly bound to the transcriptional regulatory region of JAG1 and suppressed JAG1 transcription. Moreover, RUNX3 restoration downregulated CSCs by suppressing JAG1-mediated Notch signaling. The tumorigenic capacity of RUNX3-expressing Hep3B cells was lower compared to that of control Hep3B cells. RUNX3 expression suppressed JAG1 expression and resulted in downregulation of tumorigenesis by suppression of JAG1-mediated CSCs.

Runt-related transcription factor 3 reverses epithelial–mesenchymal transition in hepatocellular carcinoma

Loss or decreased expression of runt‐related transcription factor 3 (RUNX3), a tumor suppressor gene involved in gastric and other cancers, has been frequently observed in hepatocellular carcinoma (HCC). The objective of this study was to identify the regulatory mechanism of the epithelial–mesenchymal transition (EMT) by RUNX3 in HCC. Human HCC cell lines, Hep3B, Huh7, HLF and SK‐Hep1, were divided into low‐ and high‐EMT lines, based on their expression of TWIST1 and SNAI2, and were used in this in vitro study. Ectopic RUNX3 expression had an anti‐EMT effect in low‐EMT HCC cell lines characterized by increased E‐cadherin expression and decreased N‐cadherin and vimentin expression. RUNX3 expression has previously been reported to reduce jagged‐1 (JAG1) expression; therefore, JAG1 ligand peptide was used to reinduce EMT in RUNX3‐expressing low‐EMT HCC cells. Immunohistochemical analyses were performed for RUNX3, E‐cadherin, N‐cadherin and TWIST1 in 33 human HCC tissues, also divided into low‐ and high‐EMT HCC, based on TWIST1 expression. E‐cadherin expression was correlated positively and N‐cadherin expression was correlated negatively with RUNX3 expression in low‐EMT HCC tissues. Correlations between EMT markers and RUNX3 mRNA expression were analyzed using Oncomine datasets. Similarly, mRNA expression of E‐cadherin was also significantly correlated with that of RUNX3 in low‐EMT HCC, while mRNA expression of JAG1 was negatively correlated with that of RUNX3. These results suggest a novel mechanism by which loss or decreased expression of RUNX3 induces EMT via induction of JAG1 expression in low‐EMT HCC.

Des-γ-carboxyl prothrombin is associated with tumor angiogenesis in hepatocellular carcinoma

Background and Aim:  Hepatocellular carcinoma (HCC) is a hypervascular tumor, and angiogenesis plays an important role in its development. Previously, we demonstrated that des‐γ‐carboxyl prothrombin (DCP) promotes both cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) by inducing the autophosphorylation of kinase insert domain receptor (KDR). In the present study, DCP‐associated tumor angiogenesis was assessed by comparing hypovascular and common hypervascular HCC.

Loss of runt-related transcription factor 3 expression leads hepatocellular carcinoma cells to escape apoptosis

BackgroundRunt-related transcription factor 3 (RUNX3) is known as a tumor suppressor gene for gastric cancer and other cancers, this gene may be involved in the development of hepatocellular carcinoma (HCC).MethodsRUNX3 expression was analyzed by immunoblot and immunohistochemistry in HCC cells and tissues, respectively. Hep3B cells, lacking endogenous RUNX3, were introduced with RUNX3 constructs. Cell proliferation was measured using the MTT assay and apoptosis was evaluated using DAPI staining. Apoptosis signaling was assessed by immunoblot analysis.ResultsRUNX3 protein expression was frequently inactivated in the HCC cell lines (91%) and tissues (90%). RUNX3 expression inhibited 90 ± 8% of cell growth at 72 h in serum starved Hep3B cells. Forty-eight hour serum starvation-induced apoptosis and the percentage of apoptotic cells reached 31 ± 4% and 4 ± 1% in RUNX3-expressing Hep3B and control cells, respectively. Apoptotic activity was increased by Bim expression and caspase-3 and caspase-9 activation.ConclusionRUNX3 expression enhanced serum starvation-induced apoptosis in HCC cell lines. RUNX3 is deleted or weakly expressed in HCC, which leads to tumorigenesis by escaping apoptosis.

Exon 2 deletion splice variant of γ‐glutamyl carboxylase causes des‐γ‐carboxy prothrombin production in hepatocellular carcinoma cell lines

Using GGCX gene‐specific real‐time PCR, exon 2 deletion splice variant of vitamin K‐dependent γ‐glutamyl carboxylase (GGCX) mRNA was identified in HCC cell lines. Expressions of wild type and exon 2 deletion variant of GGCX were analyzed with relevance to DCP production in HCC cell lines. Hep3B, HepG2, HuH1, HuH7, and PLC/PRF/5 produced DCP, while SK‐Hep‐1, HLE, HLF, and JHH1 produced no detectable level of DCP. DCP‐producing cells expressed exon 2 deletion variant of GGCX mRNA and protein, while DCP‐negative cells expressed no detectable level of exon 2 deletion variant of GGCX. These results suggest that exon 2 deletion splice variant of GGCX causes dysfunction of GGCX enzyme activity resulting in DCP production in HCC cell lines.

Cimetidine inhibits epidermal growth factor-induced cell signaling.

Background:  Cimetidine, a histamine‐2 (H2) receptor antagonist, has been demonstrated to have anticancer effects on colorectal cancer, melanoma and renal cell carcinoma. In the current study, we clarified that cimetidine inhibits both epidermal growth factor (EGF)‐induced cell proliferation and migration in hepatocellular carcinoma (HCC) cell lines.