Yasuo Hirono

The relation between the growth patterns of gastric carcinoma and the expression of hepatocyte growth factor receptor (c‐met), autocrine motility factor receptor, and urokinase‐type plasminogen activator receptor

Hepatocyte growth factor receptor (c‐met), autocrine motility factor receptor (AMFR), and urokinase‐type plasminogen activator receptor (uPAR) are known to play important roles in tumor cell migration, invasion, and metastasis. The authors studied the relation between the expression patterns of these genes and the growth patterns of human gastric carcinoma.

Amplification of the c-met, c-erbB-2 and Epidermal Growth Factor Receptor Gene in Human Gastric Cancers: Correlation to Clinical Features

We examined amplification of the c-met, c-erbB-2, and epidermal growth factor receptor (EGFR) gene in the patients with primary gastric cancer, and compared the data with clinical features in order to clarify the relationship between oncogenic abnormality and clinical features. Oncogene amplifications were examined by slot blot hybridization using DNAs extracted from formalin-fixed and paraffin-embedded tissues of primary gastric cancers. Seven of the seventy cancers (10.0%) had c-met gene amplification, nine (12.9%) had c-erbB-2 gene amplification, and six (8.6%) had EGFR gene amplification, respectively. Eighteen cases (25.7%) exhibited one or multiple oncogene amplification, and two cases (2.9%) exhibited simultaneous amplification of the three genes. The cases with c-met gene amplification tend to show invasive character and were related to peritoneal dissemination. The cases with c-erbB-2 gene amplification were related to lymph node metastasis. The cases with EGFR gene amplification had large tumors and were in highly advanced stage. The survival rate in patients with oncogene amplification was significantly lower than that in patients without amplification. Our data indicated that these genes were related to growth and metastasis of gastric cancer. Furthermore, this study about the three genes suggested that the type of activated gene might decide on the type of metastasis and clinical features.

Angiogenesis and Tumor Proliferation/Metastasis of Human Colorectal Cancer Cell Line SW620 Transfected with Endocrine Glands-Derived-Vascular Endothelial Growth Factor, As a New Angiogenic Factor

Endocrine glands-derived-vascular endothelial growth factor (EG-VEGF) was recently cloned as a new angiogenic factor that selectively acts on the endothelium of endocrine gland cells. We evaluated the involvement of EG-VEGF in colorectal cancer. The expression of EG-VEGF was confirmed in all of the colorectal cancer cell lines. (On the other hand, the expression of EG-VEGF mRNA was not detected in colorectal normal mucosae.) Stable EG-VEGF infectors of colorectal cancer cell line SW620 were produced, EG-VEGF transfectants were implanted into cecum and s.c., and cell proliferation was evaluated. Angiogenesis was evaluated by dorsal air sac method. Liver metastasis was evaluated after the implantation of EG-VEGF transfectants into the mouse spleen. Tumor proliferation (cecum, s.c.) was significantly higher in the EG-VEGF transfectants than in the control cells. The small vessels were significantly increased in EG-VEGF transfectants as compared with those in control cells. Also, liver metastatic ratio was higher in the EG-VEGF transfectants than in the control cells. In this study, EG-VEGF, a new angiogenic factor, may lead to angiogenesis, promoting cell proliferation and liver metastasis in colorectal cancers. When the EG-VEGF gene-overexpressing colorectal cancer cell line that had been treated with phosphorothioate antisense EG-VEGF oligonucleotides was injected s.c. into mice, angiogenesis and tumor growth were inhibited. Although the novel angiogenesis factor EG-VEGF was not expressed in the normal colorectal mucosa, it was expressed in colorectal cancer cells, which indicates that it is a cancer-specific and possibly tissue-specific angiogenesis factor in the large intestine, and which suggests that it can be targeted by a novel antiangiogenesis therapy.

Expression of autocrine motility factor receptor correlates with disease progression in human gastric cancer.

Up-regulation of autocrine motility factor receptor (AMF-R) expression has been shown to be associated with invasion and metastasis of experimental tumour systems and human neoplasms. Monoclonal antibodies against AMF-R (gp78) were used to stain 221 primary gastric cancer specimens, and level of expression was examined in relation to pathological stage and prognostic values. In 125 out of 221 (56.6%) patients, gp78 was detected. Expression of gp78 was associated with macroscopic type, lymphatic and venous invasions, and lymph node and peritoneal metastasis. The level of gp78 expression in the cancer specimens was associated with histopathological stage and grade of tumour penetration. Positive gp78 expression was significantly associated with poor prognosis (P < 0.001). This significant relationship remained among patients in stage II and III. The results suggest that gp78 expression could be used as a prognostic marker in gastric cancer patients.

Amplification of Epidermal Growth Factor Receptor Gene and Its Relationship to Survival in Human Gastric Cancer

The correlation between the clinical features in 103 patients with primary gastric carcinoma and amplification of epidermal growth factor receptor (EGFR) gene was analyzed retrospectively. EGFR gene amplification was examined by slot-blot hybridization using DNA extracted from formalin-fixed, paraffin-embedded tissues. EGFR expression was also examined immunohistochemically using the same tissues with a monoclonal antibody that is monospecific for EGFR. In 5 of 103 cases (4.9%), a 2- to 11-fold amplification of EGFR gene was detected. Four of these 5 cases were poorly differentiated adenocarcinomas. All of them had overexpressions of EGFR. The cumulative survival rate of patients with EGFR gene amplification was significantly lower than that of the patients without amplification (p < 0.05) and all of them died within 3 years. Except for tumor size (p < 0.03), there were no significant clinicopathologic differences between the two groups. On the other hand, 41 of 103 cases (39.8%) exhibited expression of EGFR. However, there was no significant correlation between EGFR expression and clinicopathologic factors or prognosis. These results indicate that EGFR gene amplification may occur in advanced stages during the progression and be an important indicator of poor short-term prognosis in gastric carcinoma.

The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

BackgroundIn addition to regulating platelet function, the G protein-coupled sub-family member Proteinase-activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer.MethodsWe established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (α-thrombin and TFLLR-NH2). We also quantified NF-κB activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of α-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR).ResultPAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF-κB and EGFR phosphorylation initially were triggered by the activation of PAR1 with α-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-κB target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C-neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed.ConclusionOur data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF-κB, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer.

Survivin-3B Gene Decreases the Invasion-Inhibitory Effect of Colon Cancer Cells With 5-Fluorouracil

The expression of survivin molecules has been confirmed in many types of cancer cells, including colon cancer cells, and they are considered important antiapoptotic molecules. Recent studies have revealed the existence of different splicing forms of survivin molecules; however, no studies have examined their expression in gastrointestinal cancers. In 2004, we reported the existence of the survivin-3B gene, a novel splice variant of survivin. In this study, we investigated the relationship between human colon cancer and our recently cloned survivin-3B gene with a coding region of 594 bp. In the first examination, survivin-3B expression was analyzed by RT-PCR in human colon cancer and adjacent normal mucosal tissues. The associations of its expression status with clinicopathological parameters and the prognosis were also examined. Survivin-3B mRNA expression was observed in 37 (46.3%) of 80 primary colon cancers, but not in the adjacent normal colonic mucosal tissue. The rate of survivin-3B gene expression was significantly higher in colon cancer with serosal invasion. The 5-year survival rate of patients with survivin-3B gene-positive primary colon cancer was significantly poorer, at 48.7%, than that (75.4%) of survivin-3B gene-negative patients. In the second examination, after the introduction of the survivin-3B gene into cells of the colon cancer cell line DLD-1, 5-fluorouracil-induced changes in their invasive capacity was examined. The invasion-inhibitory effect of 5-fluorouracil on survivin-3B gene-transfected DLD-1 cells was significantly lower than their empty vector gene-transfected counterparts. We speculate that survivin-3B expression in colon cancer is an important factor involved in the invasive capacity of cancer cells in the presence of anticancer drug.

PAR1 participates in the ability of multidrug resistance and tumorigenesis by controlling Hippo-YAP pathway

The Hippo pathway significantly correlates with organ size control and tumorigenesis. The activity of YAP/TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in cancer stem cells (CSCs). But, upstream signals that control the mammalian Hippo pathway have not been well understood. Here, we reveal a connection between the Protease-activated receptor 1 (PAR1) signaling pathway and the Hippo-YAP pathway in gastric cancer stem-like cells. The selective PAR1 agonist TFLLR-NH2 induces an increase in the fraction of side population cells which is enriched in CSCs, and promotes tumorigenesis, multi cancer drug resistance, cell morphological change, and cell invasion which are characteristics of CSCs. In addition, PAR1 activation inhibits the Hippo-YAP pathway kinase Lats via Rho GTPase. Lats kinase inhibition in turn results in increased nuclear localization of dephosphorylated YAP. Furthermore, PAR1 activation confers CSCs related traits via the Hippo-YAP pathway, and the Hippo-YAP pathway correlates with epithelial mesenchymal transition which is induced by PAR1 activation. Our research suggests that the PAR1 signaling deeply participates in the ability of multi drug resistance and tumorigenesis through interactions with the Hippo-YAP pathway signaling in gastric cancer stem-like cells. We presume that inhibited YAP is a new therapeutic target in the treatment human gastric cancer invasion and metastasis by dysregulated PAR1 or its agonists. The Hippo pathway significantly correlates with organ size control and tumorigenesis. The activity of YAP/TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in cancer stem cells (CSCs). But, upstream signals that control the mammalian Hippo pathway have not been well understood. Here, we reveal a connection between the Protease-activated receptor 1 (PAR1) signaling pathway and the Hippo-YAP pathway in gastric cancer stem-like cells. The selective PAR1 agonist TFLLR-NH2 induces an increase in the fraction of side population cells which is enriched in CSCs, and promotes tumorigenesis, multi cancer drug resistance, cell morphological change, and cell invasion which are characteristics of CSCs. In addition, PAR1 activation inhibits the Hippo-YAP pathway kinase Lats via Rho GTPase. Lats kinase inhibition in turn results in increased nuclear localization of Dephosphorylated YAP. Furthermore, PAR1 activation confers CSCs related traits via the Hippo-YAP pathway, and the Hippo-YAP pathway correlates with epithelial mesenchymal transition which is induced by PAR1 activation. Our research suggests that the PAR1 signaling deeply participates in the ability of multi drug resistance and tumorigenesis through interactions with the Hippo-YAP pathway signaling in gastric cancer stem-like cells. We presume that inhibited YAP is a new therapeutic target in the treatment human gastric cancer invasion and metastasis by dysregulated PAR1 or its agonists.

The activation of proteinase-activated receptor-1 (PAR1) promotes gastric cancer cell alteration of cellular morphology related to cell motility and invasion

Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. Tumor invasion and metastasis is directly related to cell motility. We showed that stimulation of proteinase-activated receptor-1 (PAR1) can trigger an array of responses that would promote tumor cell growth and invasion. Thus, we examined aspects of PAR1 activation related to cell morphological change that might contribute to cell motility. We established a PAR1 stably transfected MKN45 gastric cancer cell line (MKN45/PAR1). We examined morphological changes, Rho family activation and overexpression of cytoskeletal protein in cells exposed to PAR1 agonists (α-thrombin and TFLLR-NH2). MKN45/PAR1 grows with an elongated and polarized morphology, extending pseudopodia at the leading edge. However, in the presence of PAR1 antagonist, MKN45/PAR1 did not show any changes in cell shape upon addition of either α-thrombin or TFLLR-NH2. Activated PAR1 induced RhoA and Rac1 phosphorylation, and subsequent overexpression of myosin IIA and filamin B which are stress fiber components that were identified by PMF analysis of peptide mass data obtained by MALDI-TOF/MS measurement. Upon stimulation of MKN45/PAR1 for 24 h with either α-thrombin or TFLLR-NH2, the distribution of both myosin IIA and filamin B proteins shifted to being distributed throughout the cytoplasm to the membrane, with more intense luminescence signals than in the absence of stimulation. These results demonstrate that PAR1 activation induces cell morphological change associated with cell motility via Rho family activation and cytoskeletal protein overexpression, and has a critical role in gastric cancer cell invasion and metastasis.

Thrombin conducts epithelial‑mesenchymal transition via protease‑activated receptor‑1 in human gastric cancer

Epithelial-mesenchymal transition (EMT) is thought to be a key step for cancer metastasis. Using an immunohistochemical approach with gastric carcinoma tissue, we found the expression of protease-activated receptor-1 (PAR1), along with a metalloproteinase known to activate PAR1, were associated with poorer prognosis, compared with expression-negative tumors, and activated PAR1 promotes gastric cancer cell invasion and proliferation in vivo. In this study we observed EMT induction by the PAR1 agonist α-thrombin, in human gastric cell lines stably expressing PAR1. We investigated α-thrombin-induced changes in the cell forms of pcDNA3.1-MKN45 (MKN45/Mock), pcDNA3.1‑PAR1 transfected MKN45 (MKN45/PAR1), and MKN74. Expression levels of epithelial and mesenchymal markers as well as the distribution of transcriptional factors of E-cadherin in the cytoplasm and nucleus were also noted in these cell lines. We observed α-thrombin-induced morphological changes in MKN45/PAR1 and MKN74 cells. Western blotting and immunohistochemistry of these cells indicated a fall in the expression level of E-cadherin and an increase in fibronectin expression after 48 h. PAR1 activation also induced significant increases in nuclear levels of the Snail which is a repressor of E-cadherin gene expression. We found EMT in gastric cancer cell lines that underwent α-thrombin-induced PAR1 activation.