Candidate Oncogenes, ARHGAP4, NOS3, and OR51B5, for the Development of Scirrhous-type Gastric Cancer.

BACKGROUND/AIM: Scirrhous-type gastric cancer (SGC), one of the most intractable cancer subtypes, is characterized by rapid cancer cell proliferation and infiltration accompanied by extensive stromal fibrosis. One of the reasons for its poor prognosis may be the lack of molecular target drugs for SGC, because of the unknown driver genes. Exploration of somatic mutations in the human samples of SGC using next-generation sequencing (NGS) has been hampered by abundant fibrous tissues in these samples. Therefore, this study aimed to determine a novel oncogene by RNA-sequencing using SGC cell lines, avoiding contamination with fibrosis. MATERIALS AND METHODS: In silico analysis of RNA-sequencing public data of the gastric cancer cell line, and RNA- sequencing using five of our unique SGC cell lines, OCUM1, OCUM2MLN, OCUM8, OCUM12, and OCUM14 were performed. RESULTS: We found three differentially expressed genes, ARHGAP4, NOS3, and OR51B5 that are significantly over-expressed in SGC cells. Immunohistochemical analysis indicated that the protein expression levels of these three genes were significantly higher in SGC than in other types of gastric cancer. The prognosis of patients with positive expression of these three genes was significantly poorer than those with negative expression. In particular, ARHGAP4 expression was an independent predictor of poor prognosis and recurrence. CONCLUSION: ARHGAP4, NOS3, and OR51B5 may be candidate driver genes for SGC. ARHGAP4 may be a promising molecular target for SGC.

Serine threonine kinase 11/liver kinase B1 mutation in sporadic scirrhous-type gastric cancer cells.

Scirrhous-type gastric carcinoma (SGC), which is characterized by the rapid proliferation of cancer cells accompanied by extensive fibrosis, shows extremely poor survival. A reason for the poor prognosis of SGC is that the driver gene responsible for SGC has not been identified. To identify the characteristic driver gene of SGC, we examined the genomic landscape of six human SGC cell lines of OCUM-1, OCUM-2M, OCUM-8, OCUM-9, OCUM-12 and OCUM-14, using multiplex gene panel testing by next-generation sequencing. In this study, the non-synonymous mutations of serine threonine kinase 11/liver kinase B1 (STK11/LKB1) gene were detected in OCUM-12, OCUM-2M and OCUM-14 among the six SGC cell lines. Capillary sequencing analysis confirmed the non-sense or missense mutation of STK11/LKB1 in the three cell lines. Western blot analysis showed that LKB1 expression was decreased in OCUM-12 cells and OCUM-14 cells harboring STK11/LKB1 mutation. The mammalian target of rapamycin (mTOR) inhibitor significantly inhibited the proliferation of OCUM-12 and OCUM-14 cells. The correlations between STK11/LKB1 expression and clinicopathologic features of gastric cancer were examined using 708 primary gastric carcinomas by immunochemical study. The low STK11/LKB1 expression group was significantly associated with SGC, high invasion depth and frequent nodal involvement, in compared with the high STK11/LKB1 expression group. Collectively, our study demonstrated that STK11/LKB1 mutation might be responsible for the progression of SGC, and suggested that mTOR signaling by STK11/LKB1 mutation might be one of therapeutic targets for patients with SGC.

Establishment of a New Scirrhous Gastric Cancer Cell Line with FGFR2 Overexpression, OCUM-14.

BACKGROUND: The prognosis of scirrhous gastric carcinoma (SGC), which is characterized by rapid infiltration and proliferation of cancer cells accompanied by extensive stromal fibrosis, is extremely poor. In this study, we report the establishment of a unique SGC cell line from a gastric cancer patient in whom an autopsy was performed. METHODS: A new SGC cell line, OCUM-14, was established from malignant ascites of a male patient with SGC. A postmortem autopsy was performed on the patient. Characterization of OCUM-14 cells was analyzed by microscopic examination, reverse transcription polymerase chain reaction, fluorescence in situ hybridization analysis, immunohistochemical examination, CCK-8 assay, and in vivo assay. RESULTS: OCUM-14 cells grew singly or in clusters, and were floating and round-shaped. Most OCUM-14 cells had many microvilli on their surfaces. The doubling time was 43.1 h, and the subcutaneous inoculation of 1.0 × 107 OCUM-14 cells into mice resulted in 50% tumor formation. mRNA expressions of fibroblast growth factor receptor 2 (FGFR2) and human epidermal growth factor receptor 2 (HER2) were observed in OCUM-14 cells. FGFR2, but not HER2, overexpression was found in OCUM-14 cells. The heterogeneous overexpression of FGFR2 was also found in both the primary tumor and metastatic lesions of the peritoneum, lymph node, bone marrow, and lung of the patient. The FGFR2 inhibitors AZD4547 and BGJ398 significantly decreased the growth of OCUM-14 cells, while paclitaxel and 5-fluorouracil significantly decreased the proliferation of OCUM-14 cells, but cisplatin did not. CONCLUSION: A new gastric cancer cell line, OCUM-14, was established from SGC and showed FGFR2 overexpression. OCUM-14 might be useful for elucidating the characteristic mechanisms of SGC and clarifying the effect of FGFR2 inhibitors on SGC.

CD9-positive exosomes from cancer-associated fibroblasts stimulate the migration ability of scirrhous-type gastric cancer cells.

This corrects the article DOI: 10.1038/bjc.2017.85.

Expression level of microRNA-200c is associated with cell morphology in vitro and histological differentiation through regulation of ZEB1/2 and E-cadherin in gastric carcinoma.

Scirrhous type gastric cancer is characterized by diffuse infiltration of poorly differentiated adenocarcinoma cells and poor prognosis. Although association of poorly differentiated histology with reduction in E-cadherin expression, as well as association of microRNA (miR)-200c with E-cadherin through regulation of ZEB1/2, has been reported, participation of miR-200c in gastric carcinogenesis is not fully understood. We used 6 cell lines originating from gastric cancers, and investigated levels of miR-200c along with its target mRNAs ZEB1/2 and E-cadherin by qRT-PCR. ZEB1 and E-cadherin protein expression was also assessed via western blotting. Furthermore, we investigated the expression levels of miR­200c by in situ hybridization, along with the expression of ZEB1 and E-cadherin by immunohistochemistry, in 97 gastric adenocarcinoma tissues. Inverse correlation between miR­200c and ZEB1 levels were obtained by qRT-PCR in cell lines (P<0.05). Cell lines with low miR-200c and high ZEB1 exhibited low E-cadherin expression in both qRT-PCR and western blotting, and exhibited spindle-shaped morphology, in contrast to round cell morphology in those cell lines with high miR-200c levels. Inverse correlations were also obtained between miR-200c and ZEB1 as well as between ZEB1 and E-cadherin levels in tissue samples (P<0.001). Cancer tissues with low miR-200c, high ZEB1, and low E-cadherin expression were associated with poorly differentiated histology, in contrast to tubular form in cancers with high miR-200c expression levels (P<0.001). Our data revealed that downregulation of miR-200c primarily regulated cell morphology by downregulation of E-cadherin through upregulation of ZEB1, leading to poorly differentiated histology in gastric cancer.

Effects of 5-fluorouracil, adriamycin and irinotecan on HSC-39, a human scirrhous gastric cancer cell line.

Therapies for patients with scirrhous gastric cancer remain ineffective. Current treatments for gastric cancer based on systemic therapy, such as the combination of S-1 with cisplatin or docetaxel, show good clinical response rates. S-1 plus cisplatin is the standard treatment for HER2-negative advanced scirrhous gastric cancer in Japan. In spite of recent advances in the treatment of gastric cancer, a standard chemotherapy regimen is yet to be established for scirrhous gastric cancer. To develop new therapeutic approaches based on characteristic biological features of cancer cells, we examined the mechanisms underlying the cytotoxicity of anticancer drugs and reactive oxygen species (ROS) toward a human scirrhous cancer cell line, HSC-39, in vitro. Anticancer drugs such as 5-fluorouracil (5-FU), adriamycin (ADR) and irinotecan (CPT-11), as well as ROS, were previously shown to have important cytotoxic effects on these tumor cells. We demonstrated that 5-FU effectively induced apoptosis in HSC-39 cells in a dose­dependent manner, while ADR and CPT-11 induced necrosis and/or aponecrosis. 5-FU effectively inhibited WST-1 decrease in the MTT viability assay, even at low doses where little LDH release was observed, while ADR and CPT-11 only inhibited WST-1 decrease at high doses where LDH release was induced. Moreover, HSC-39 cells showed high sensitivity to H2O2 and NOC-18, but less sensitivity to other ROS, suggesting a link between cell damage and membrane permeability changes induced by H2O2 and NOC-18 or related oxygen radical species such as OH· or ·O2. These results suggest that combination treatment of chemotherapeutics with a fluoropyrimidine such as 5-FU is effective chemotherapy for scirrhous gastric cancer.

Fibrolamellar carcinoma versus scirrhous hepatocellular carcinoma : diagnostic usefulness of CD68.

BACKGROUND: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of hepatocellular carcinoma that commonly affects young individuals without a prior history of liver disease. The principal differential diagnosis is conventional hepatocellular carcinoma especially the scirrhous variant. Despite their distinctive appearance, recent studies have demonstrated a lack of consistency in how FL-HCC are diagnosed by pathologists. AIM: To investigate the diagnostic utility of CD68 in differentiating between FL-HCC and scirrhous hepatocellular carcinoma. PATIENTS AND METHODS: In our retrospective study, we reviewed four cases of FL-HCC that were diagnosed at the pathology department of Mongi Slim hospital over a thirteen-year period (2002-2014). Relevant clinical information and microscopic slides were available in all cases and were retrospectively reviewed. Immunohistochemical analysis was performed using the avidin-biotin complex technique with antibodies against CD68 and CK7. RESULTS: Our study group included one man and three women (sex ratio M/F=0.33) aged between 23 and 34 years (mean=28 years). All cases arose in non-cirrhotic liver. Immunohistochemically, all cases were positive for CK7 and for CD68 (n=4). CONCLUSIONS: CD68 immunostaining is a sensitive marker for FL-HCC that may be of use in routine diagnostic surgical pathology. Lack of CD68 staining should suggest caution in making a diagnosis of FL-HCC.

Epigenetic modulation and repression of miR-200b by cancer-associated fibroblasts contribute to cancer invasion and peritoneal dissemination in gastric cancer.

Cancer-associated fibroblasts (CAFs) have recently been linked to the invasion and metastasis of gastric cancer. In addition, the microRNA (miR)-200 family plays a central role in the regulation of the epithelial-mesenchymal transition process during cancer metastasis, and aberrant DNA methylation is one of the key mechanisms underlying regulation of the miR-200 family. In this study, we clarified whether epigenetic changes of miR-200b by CAFs stimulate cancer invasion and peritoneal dissemination in gastric cancer. We evaluated the relationship between miR-200b and CAFs using a coculture model. In addition, we established a peritoneal metastasis mouse model and investigated the expression and methylation status of miR-200b. We also investigated the expression and methylation status of miR-200b and CAFs expression in primary gastric cancer samples. CAFs (CAF-37 and CAF-50) contributed to epigenetic changes of miR-200b, reduced miR-200b expression and promoted tumor invasion and migration in NUGC3 and OCUM-2M cells in coculture. In the model mice, epigenetic changes of miR-200b were observed in the inoculated high-frequency peritoneal dissemination cells. In the 173 gastric cancer samples, the low miR-200b expression group demonstrated a significantly poorer prognosis compared with the high miR-200b expression group and was associated with peritoneal metastasis. In addition, downregulation of miR-200b in cancer cells was significantly correlated with alpha-smooth muscle actin expression. Our data provide evidence that CAFs reduce miR-200b expression and promote tumor invasion through epigenetic changes of miR-200b in gastric cancer. Thus, CAFs might be a therapeutic target for inhibition of gastric cancer.

Stromal fibroblasts mediate extracellular matrix remodeling and invasion of scirrhous gastric carcinoma cells.

Scirrhous gastric carcinoma (SGC) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.

Cancer-associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor-ß signaling.

Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells (CSCs) have been proposed to have an important role in cancer progression. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM-12 and OCUM-2MD3, and non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74, were used. OCUM-12/side population (SP) cells and OCUM-2MD3/SP cells were sorted by flow cytometry as CSC-rich cells from the parent cells. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12/SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM-12/SP and OCUM-2MD3/SP cells, but not that of MKN-45/SP and MKN-74/SP cells. Taken together, CM from CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM-12/SP and OCUM-2MD3/SP cells. These stimulating-activities by CM were significantly decreased by TGFß inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM-12/SP cells with CAFs was significantly high in comparison with that by OCUM-12/SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFß signaling.

The relevance of the intrinsic subtype to the clinicopathological features and biomarkers in Japanese breast cancer patients.

BACKGROUND: Breast cancer is a disease rich in diversity, and it can be categorized into the immunohistochemical intrinsic subtypes : ER/PR + and HER2-, ER/PR + and HER2+, HER2 type, basal-like and unclassified. METHODS: In this study, in addition to the clinicopathological features potentially associated with the intrinsic subtypes, protein expression and genetic mutations of key molecules associated with breast cancer prognosis and treatment sensitivity were analyzed. The distribution of subtypes in the patient population and the differences in marker distribution across the subtypes were investigated. RESULTS: The immunohistochemical features of 471 consecutive surgical cases of women with primary breast cancer, treated in a single institution, were examined. There were 306 patients who were ER/PR + HER2- (65%); 41 who were ER/PR + HER2+ (8.7%); 59 with HER2 type (12.5%); 37 with basal-like (7.9%); and 28 patients whose breast cancer was unclassified (5.9%). There were no significant differences between the subtypes regarding age, menopausal status, disease stage, lymphatic invasion, blood vessel invasion and lymph node metastasis. Statistically significant differences were found for histological type and grade. Regarding protein expression and genetic mutation, significant differences were found in the distribution within each subtype for six out of 12 molecules investigated. CONCLUSIONS: This study revealed that subtypes differ not only in their clinical pathological profiles, such as histological types and histological grades, but also in molecular expression. The molecular expression patterns observed for each intrinsic subtype may help the selection of an optimal treatment strategy.

Paracrine activation of MET promotes peritoneal carcinomatosis in scirrhous gastric cancer.

Scirrhous gastric cancer is associated with abundant stroma and frequently develops into peritoneal carcinomatosis with malignant ascites. Although malignant ascites is among the most deadly diseases worldwide, its molecular pathogenesis is poorly understood. We investigated the role of hepatocyte growth factor (HGF) in the production of peritoneal carcinomatosis with malignant ascites. We examined three scirrhous and three non-scirrhous human gastric cancer cell lines for the production of peritoneal carcinomatosis in vivo and responses to HGF in vitro. Furthermore, clinical scirrhous gastric cancer specimens were examined for HGF production. Among the six cell lines examined, only two scirrhous cell lines (NUGC4 and GCIY) produced peritoneal carcinomatosis with massive ascites after intraperitoneal injection in nude mice. Their proliferation was stimulated by exogenous HGF in vitro. On the other hand, a non-scirrhous cell line, MKN45, with MET amplification generated peritoneal tumors but not ascites. MET tyrosine kinase inhibitors, crizotinib and TAS-115, inhibited HGF-stimulated proliferation of NUGC4 and GCIY as well as constitutive proliferation of MKN45. Furthermore, crizotinib and TAS-115 prolonged the survival of mice bearing established tumors by NUGC4 or MKN45. In clinical specimens, HGF was markedly produced by stromal fibroblasts. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high levels of HGF. Our results strongly suggest that paracrine HGF-induced activation of MET-mediated signaling pathways plays an important role in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, MET signaling pathway may be a potential therapeutic target for peritoneal carcinomatosis of gastric cancer, even without MET amplification.

HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer.

The molecular mechanisms underlying the peritoneal dissemination of gastric cancer remain unclear. Using in vivo metastatic models, this study attempted to clarify the role of hypoxia inducible factor (HIF)-1α in the development of peritoneal dissemination of gastric cancer. HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9. Using KD and control (SC) cells, the presence of peritoneal dissemination was assessed in orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. A series of in vitro analyses were also conducted. Finally, tumor angiogenesis was immunohistochemically analyzed. In the o.i. model, peritoneal dissemination was more frequently observed in the SC mice (93%) compared to the KD mice (13%) (P<0.001). In the i.p. model, peritoneal dissemination occurred at a high rate in both types of mice; however, a greater number of nodules was observed in the KD mice (P=0.017). The in vitro assays showed that HIF-1α exerts unfavorable effects on anoikis resistance and adhesion to extracellular matrix. Angiogenesis and vascular invasion were more aggressive in the SC gastric tumors. Vascular invasion was present in the intratumoral regions of the disseminated nodules in the SC o.i., but not the i.p., mice. HIF-1α was found to be crucial for the development of peritoneal dissemination in o.i. model, which mimics natural metastasis. In contrast, HIF-1α played an inhibitory role in suppressing peritoneal dissemination in the i.p. model. These results indicate that peritoneal dissemination in o.i. mice may not act through a seeding mechanism. An immunohistochemical analysis demonstrated HIF-1α-activated angiogenesis and vascular invasion in stomach tumors. Furthermore, the results showed that the disseminated nodules observed in SC o.i. mice were formed via extravasation of cancer cells. We provide a possible mechanism in which peritoneal dissemination of gastric cancer develops via a vascular network whereby HIF-1α activates tumor angiogenesis.

Primary culture of breast cancer: a model system for epithelial-mesenchymal transition and cancer stem cells.

BACKGROUND: A novel model system to study the cellular, molecular, and genetic characteristics of breast cancer stem cells is needed. Personalized prognostic models are indispensable for assessing the effect of so-called tailor-made adjuvant therapy. MATERIALS AND METHODS: Surgically-extirpated tissues were dispersed with Dispase and cultured in commonly used medium. The expression of tumor markers was detected with immunohistochemistry, and gene expression profiles of tissues and cells were analyzed using the Agilent Human Microarray. RESULTS: We established a primary culture, which exhibited mesenchymal morphology. The gene expression profile of the primary culture revealed that the cells underwent epithelial-mesenchymal transition and had cancer stem cell properties. CONCLUSION: A primary culture of breast cancer cells can be easily established and could be used for studying breast cancer stem cells and assessing treatment of a patient.

Mesothelial cells differentiate into fibroblast-like cells under the scirrhous gastric cancer microenvironment and promote peritoneal carcinomatosis in vitro and in vivo.

Peritoneal metastases are one reason for the poor prognosis of scirrhous gastric cancer (SGC), and myofibroblast provides a favorable environment for the peritoneal dissemination of gastric cancer. The aim of this study was to determine whether myofibroblast originates from peritoneal mesothelial cells under the influence of the tumor microenvironment. Immunohistochemical studies of peritoneal biopsy specimens from patients with peritoneal lavage cytological (+) status demonstrate the expression of the epithelial markers cytokeratin in fibroblast-like cells entrapped in the stroma, suggesting that these cells stemmed from local conversion of mesothelial cells. To confirm this hypothesis in vitro, we co-incubated mesothelial cells with SGC or non-SGC to investigate morphology and function changes. As we expected, mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype with loss of epithelial morphology and decrease in the expression of cytokeratin and E-cadherin when exposed to conditioned medium from HSC-39, and the induction of mesothelial cells can be abolished using a neutralizing antibody to transforming growth factor-beta1 (TGF-ß1) as well as by pre-treatment with SB431542. Moreover, we found that these mesothelial cells-derived cells exhibit functional properties of myofibroblasts, including the ability to increase adhesion and invasion of SGC. In summary, our current data demonstrated that mesothelial cells are a source of myofibroblasts under the SGC microenvironment which provide a favorable environment for the dissemination of gastric cancer; TGF-ß1 produced by autocrine/paracrine in peritoneal cavity may play a central role in this pathogenesis.

Hepatitis B virus reactivation in adjuvant chemotherapy for breast cancer.

Immunosuppressive therapy, such as chemotherapy or the use of corticosteroids, may stimulate reactivation of hepatitis B virus (HBV). Most of these episodes occur in patients whose hepatitis B surface antigens are positive (HBsAg+). We report a case of HBV reactivation in a patient with negative HBsAg during chemotherapy for breast cancer, in spite of avoiding corticosteroids. A 68-year-old woman received adjuvant chemotherapy for breast cancer. Her serological examinations showed that HBsAg, HBeAg, and HBV-DNA were all negative. Her chemotherapy consisted of CAF (cyclophosphamide 500 mg/m(2), doxorubicin 50 mg/m(2), fluorouracil 500 mg/m(2)) without administration of corticosteroids. She received six cycles of CAF. At day 27 after her sixth CAF, she was admitted to the hospital with acute hepatitis B virus (HBV) reactivation. She received glycyrrhizinic acid by intravenous injection (80 mg/day), ursodeoxycholic acid (300 mg/day), and entecavir (0.5 mg/day). Then she received interferon by intravenous injection (3 million units/day), prednisolon by intravenous injection (45 mg/day), and plasma exchange therapy. However, she died of liver failure. This is a rare case in which HBV reactivation occurred in an HBsAg negative patient during chemotherapy without using corticosteroids. This episode suggests that HBV reactivation may occur during chemotherapy in any patient with a history of HBV infection. Therefore, we recommend checking HBsAg, HBsAb, and HBcAb before starting chemotherapy. Moreover, with positive HBsAb or/and HBcAb patients, HBV-DNA should be checked before starting chemotherapy and monitored during chemotherapy by a sensitive PCR method.

N-myc downstream regulated gene 1 (NDRG1) promotes metastasis of human scirrhous gastric cancer cells through epithelial mesenchymal transition.

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.

PI3K/Akt signalling is required for the attachment and spreading, and growth in vivo of metastatic scirrhous gastric carcinoma.

BACKGROUND: PI3K/Akt (PKB) pathway has been shown in several cell types to be activated by ligands to cell surface integrins, leading to the metastasis of tumour cells. The signalling pathways involved in the metastatic spread of human scirrhous gastric carcinoma cells have not been defined. METHODS: The role of the PI3K/Akt pathway in an extensive peritoneal-seeding cell line, OCUM-2MD3 and a parental cell line, OCUM-2M, was investigated by assessing in vitro adhesion and spreading assay, and in vivo peritoneal metastatic model. We also examined the correlation of PI3K/Akt pathway with integrin signals by immunoprecipitations, using cells by transfection with mutant p85 (Δp85). RESULTS: Adhesiveness and spreading of OCUM-2MD3 cells on collagen type IV was significantly decreased by PI3K inhibitors and expression of mutant p85, but not by inhibitors of protein kinase C (PKC) or extracellular signal-regulated kinase (ERK). Immunoprecipitation studies indicated that the PI3K/Akt pathway was associated with integrin signalling through Src and vinculin. In an in vivo experimental metastasis model, p85 inhibition reduced peritoneal metastasis of OCUM-2MD3 cells. CONCLUSION: PI3K/Akt signalling may be required for integrin-dependent attachment and spreading of scirrhous gastric carcinoma cells, and would be translated into generating better strategies to optimise their use in cancer clinical trials.

Upregulation of cancer-associated myofibroblasts by TGF-ß from scirrhous gastric carcinoma cells.

BACKGROUND: Myofibroblasts in the cancer microenvironment have recently been implicated in tumour growth and metastasis of gastric cancer. However, the mechanisms responsible for the regulation of myofibroblasts in cancer-associated fibroblasts (CAFs) remain unclear. This study was performed to clarify the mechanisms for regulation of myofibroblasts in gastric cancer microenvironment. METHODS: Two CAFs (CaF-29 and CaF-33) from the tumoural gastric wall and a normal fibroblast (NF-29) from the nontumoural gastric wall, 4 human gastric cancer cell lines from scirrhous gastric cancer (OCUM-2MD3 and OCUM-12), and non-scirrhous gastric cancer (MKN-45 and MKN-74) were used. Immunofluorescence microscopy by triple-immunofluorescence labelling (α-SMA, vimentin, and DAPI) was performed to determine the presence of α-SMA-positive myofibroblasts. Real-time RT-PCR was performed to examine α-SMA mRNA expression. RESULTS: Immunofluorescence microscopy showed that the frequency of myofibroblasts in CaF-29 was greater than that in NF-29. The number of myofibroblasts in gastric fibroblasts gradually decreased with serial passages. Transforming growth factor-ß (TGF-ß) significantly increased the α-SMA expression level of CAFs. Conditioned medium from OCUM-2MD3 or OCUM-12 cells upregulated the α-SMA expression level of CAFs, but that from MKN-45 or MKN-74 cells did not. The α-SMA upregulation effect of conditioned medium from OCUM-2MD3 or OCUM-12 cells was significantly decreased by an anti-TGF-ß antibody or Smad2 siRNA. CONCLUSION: Transforming growth factor-ß from scirrhous gastric carcinoma cells upregulates the number of myofibroblasts in CAFs.