Gene expression analysis of TGF-beta signaling-disrupting gastric cancer cell line 2MLN
ABSTRACT: Diffuse-type gastric carcinoma is a poor-prognostic cancer with high expression of transforming growth factor (TGF)-β and thick stromal fibrosis. However, detailed investigations on the roles of TGF-β signaling in diffuse-type gastric carcinoma have not been performed. We generated two diffuse-type gastric carcinoma cell lines, dominant-negative TGF-β type II receptor expressing cells (2MLN-dnTβRII) and GFP-expressing cells (2MLN-GFP). Cells were subcutaneously or orthotopically injected into nude mice. Although dnTβRII did not affect the growth of OCUM-2MLN in vitro, it accelerated the growth of subcutaneously or orthotopically transplanted tumors in vivo. By microarray analysis, we found gene expression of TSP-1, an angiogenic inhibitor, was down-regulated in dnTβRII tumors. This results suggested disruption of TGF-β signaling in diffuse-type gastric carcinoma cells leads to alteration of tumor microenvironment and acceleration of tumor growth. Overall design: We generated two OCUM-2MLN-derived gastric carcinoma cell lines; 2MLN-dnTβRII, which expresses dominant negative form of TβRII, and control 2MLN-GFP, which expresses GFP. The transplanted tumors of these cell lines were subjected to gene expression analysis with Affymetrix U133 plus2 arrays.
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:Diffuse-type gastric carcinoma is a poor-prognostic cancer with high expression of transforming growth factor (TGF)-β and thick stromal fibrosis. However, detailed investigations on the roles of TGF-β signaling in diffuse-type gastric carcinoma have not been performed. We generated two diffuse-type gastric carcinoma cell lines, dominant-negative TGF-β type II receptor expressing cells (2MLN-dnTβRII) and GFP-expressing cells (2MLN-GFP). Cells were subcutaneously or orthotopically injected into nude mice. Although dnTβRII did not affect the growth of OCUM-2MLN in vitro, it accelerated the growth of subcutaneously or orthotopically transplanted tumors in vivo. By microarray analysis, we found gene expression of TSP-1, an angiogenic inhibitor, was down-regulated in dnTβRII tumors. This results suggested disruption of TGF-β signaling in diffuse-type gastric carcinoma cells leads to alteration of tumor microenvironment and acceleration of tumor growth. We generated two OCUM-2MLN-derived gastric carcinoma cell lines; 2MLN-dnTβRII, which expresses dominant negative form of TβRII, and control 2MLN-GFP, which expresses GFP. The transplanted tumors of these cell lines were subjected to gene expression analysis with Affymetrix U133 plus2 arrays.
Project description:BACKGROUND: Diffuse-type gastric carcinoma is a cancer with poor prognosis that has high levels of transforming growth factor beta (TGF-beta) expression and thick stromal fibrosis. However, the association of TGF-beta signaling with diffuse-type gastric carcinoma has not been investigated in detail. METHODS: We used a lentiviral infection system to express a dominant-negative TGF-beta type II receptor (dnTbetaRII) or green fluorescent protein (GFP) as a control in the diffuse-type gastric carcinoma cell lines, OCUM-2MLN and OCUM-12. These infected cells and the corresponding parental control cells were subcutaneously or orthotopically injected into nude mice. Angiogenesis was inhibited by infecting cells with a lentivirus carrying the gene for angiogenic inhibitor thrombospondin-1 or by injecting mice intraperitoneally with the small-molecule angiogenic inhibitor sorafenib or with anti-vascular endothelial growth factor (VEGF) neutralizing antibody (six or eight mice per group). Expression of phospho-Smad2 and thrombospondin-1 was investigated immunologically in human gastric carcinoma tissues from 102 patients. All statistical tests were two-sided. RESULTS: Expression of dnTbetaRII into OCUM-2MLN cells did not affect their proliferation in vitro, but it accelerated the growth of subcutaneously or orthotopically transplanted tumors in vivo (eg, for mean volume of subcutaneous tumors on day 10 relative to that on day 0: dnTbetaRII tumors = 3.49 and GFP tumors = 2.46, difference = 1.02, 95% confidence interval [CI] = 0.21 to 1.84; P = .003). The tumors expressing dnTbetaRII had higher levels of angiogenesis than those expressing GFP because of decreased thrombospondin-1 production. Similar results were obtained with OCUM-12 cells. Expression of thrombospondin-1 in the dnTbetaRII tumor or treatment with sorafenib or anti-VEGF antibody reduced tumor growth, whereas knockdown of thrombospondin-1 expression resulted in more accelerated growth of OCUM-2MLN tumors than of GFP tumors (eg, mean tumor volumes on day 14 relative to those on day 0: thrombospondin-1-knockdown tumors = 4.91 and GFP tumors = 3.79, difference = 1.12, 95% CI = 0.80 to 1.44; P < .001). Positive association between phosphorylated Smad2 and thrombospondin-1 immunostaining was observed in human gastric carcinoma tissues. CONCLUSIONS: Disruption of TGF-beta signaling in diffuse-type gastric carcinoma models appeared to accelerate tumor growth, apparently through increased tumor angiogenesis that was induced by decreased expression of thrombospondin-1.
Project description:Gastric carcinoma is one of the most common human malignancies and remains the second leading cause of cancer-associated mortality worldwide. Gastric carcinoma is characterized by early-stage metastasis and is typically diagnosed in the advanced stage. Previous results have indicated that bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) overexpression has been demonstrated to inhibit growth and metastasis of gastric cancer cells. However, the molecular mechanisms of the BAMBI-mediated signaling pathway in the progression of gastric cancer are poorly understood. In the present study, to assess whether BAMBI overexpression inhibited the growth and aggressiveness of gastric carcinoma cells through regulation of transforming growth factor (TGF)-β/epithelial-mesenchymal transition (EMT) signaling pathway, the growth and metastasis of gastric carcinoma cells were analyzed following BAMBI overexpression and knockdown in vitro and in vivo. Molecular changes in the TGF-β/EMT signaling pathway were studied in gastric carcinoma cells following BAMBI overexpression and knockdown. DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway was investigated in gastric carcinoma cells. Tumor growth in tumor-bearing mice was analyzed after mice were subjected to endogenous overexpression of BAMBI. Results indicated that BAMBI overexpression significantly inhibited gastric carcinoma cell growth and aggressiveness, whereas knockdown of BAMBI significantly promoted its growth and metastasis compared with the control (P<0.01). The TGF-β/EMT signaling pathway was downregulated in BAMBI-overexpressed gastric carcinoma cells; however, signaling was promoted following BAMBI knockdown. In addition, it was observed that BAMBI overexpression significantly downregulated the DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway (P<0.01). Furthermore, RNA interference-mediated BAMBI overexpression also promoted apoptosis in gastric cancer cells and significantly inhibited growth of gastric tumors in murine xenografts (P<0.01). In conclusion, the present findings suggest that BAMBI overexpression inhibited the TGF-β/EMT signaling pathway and suppressed the invasiveness of gastric tumors, suggesting BAMBI may be a potential target for the treatment of gastric carcinoma via regulation of the TGF-β/EMT signaling pathway.
Project description:Gastric cancer is classified into two subtypes, diffuse and intestinal. The diffuse-type gastric cancer (DGC) has poorer prognosis, and the molecular pathology is not yet fully understood. The purpose of this study was to identify functional secreted molecules involved in DGC progression. We integrated the secretomics of six gastric cancer cell lines and gene expression analysis of gastric cancer tissues with publicly available microarray data. Hierarchical clustering revealed characteristic gene expression differences between diffuse- and intestinal-types. GDF15 was selected as a functional secreted molecule owing to high expression only in fetal tissues. Protein expression of GDF15 was higher in DGC cell lines and tissues. Serum levels of GDF15 were significant higher in DGC patients as compared with healthy individuals and chronic gastritis patients, and positively correlated with wall invasion and lymph node metastasis. In addition, the stimulation of GDF15 on NIH3T3 fibroblast enhanced proliferation and up-regulated expression of extracellular matrix genes, which were similar to TGF-β stimulation. These results indicate that GDF15 contributes to fibroblast activation. In conclusion, this study revealed that GDF15 may be a novel functional secreted molecule for DGC progression, possibly having important roles for cancer progression via the affecting fibroblast function, as well as TGF-β.
Project description:BACKGROUND: Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-beta (TGF-beta) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-beta receptor (TbetaR) phosphorylation inhibitor on the invasiveness of gastric cancer cells. METHODS: Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TbetaR type I (TbetaR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TbetaR-I. We investigated the expression levels of TbetaR and phospho-Smad2, and the effects of TGF-beta in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells. RESULTS: TbetaR-I, TbetaR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-beta1 in scirrhous gastric cancer cells. Transforming growth factor-beta1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-beta1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-beta1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-beta1 or Ki26894 treatment. CONCLUSION: A TbetaR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.
Project description:A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma.
Project description:Transforming growth factor (TGF)-beta plays a pivotal role in regulation of progression of cancer through effects on tumor microenvironment as well as on cancer cells. TGF-beta inhibitors have recently been shown to prevent the growth and metastasis of certain cancers. However, there may be adverse effects caused by TGF-beta signaling inhibition, including the induction of cancers by the repression of TGF-beta-mediated growth inhibition. Here, we present an application of a short-acting, small-molecule TGF-beta type I receptor (TbetaR-I) inhibitor at a low dose in treating several experimental intractable solid tumors, including pancreatic adenocarcinoma and diffuse-type gastric cancer, characterized by hypovascularity and thick fibrosis in tumor microenvironments. Low-dose TbetaR-I inhibitor altered neither TGF-beta signaling in cancer cells nor the amount of fibrotic components. However, it decreased pericyte coverage of the endothelium without reducing endothelial area specifically in tumor neovasculature and promoted accumulation of macromolecules, including anticancer nanocarriers, in the tumors. Compared with the absence of TbetaR-I inhibitor, anticancer nanocarriers exhibited potent growth-inhibitory effects on these cancers in the presence of TbetaR-I inhibitor. The use of TbetaR-I inhibitor combined with nanocarriers may thus be of significant clinical and practical importance in treating intractable solid cancers.
Project description:Genes involved in the transforming growth factor beta (TGF-beta) signaling pathway are frequently altered in several types of cancers, and a gastric tumor suppressor RUNX3 appears to be an integral component of this pathway. We reported previously that apoptosis is notably reduced in Runx3-/- gastric epithelial cells. In the present study, we show that a proapoptotic gene Bim was transcriptionally activated by RUNX3 in the gastric cancer cell lines SNU16 and SNU719 treated with TGF-beta. The human Bim promoter contains RUNX sites, which are required for its activation. Furthermore, a dominant negative form of RUNX3 comprised of amino acids 1 to 187 increased tumorigenicity of SNU16 by inhibiting Bim expression. In Runx3-/- mouse gastric epithelium, Bim was down-regulated, and apoptosis was reduced to the same extent as that in Bim-/- gastric epithelium. We confirmed comparable expression of TGF-beta1 and TGF-beta receptors between wild-type and Runx3-/- gastric epithelia and reduction of Bim in TGF-beta1-/- stomach. These results demonstrate that RUNX3 is responsible for transcriptional up-regulation of Bim in TGF-beta-induced apoptosis.
Project description:To investigate the pathobiological behaviors of gastric mixed-type (MT) carcinomas and gastric carcinogenesis, the clinicopathological characteristics of MT carcinomas were analyzed and compared with intestinal-type (IT) and diffuse-type (DT) carcinomas. The expression of Ki-67, caspase-3, p53, fragile histine triad (FHIT), maspin, extracellular matrix metalloproteinase inducer (EMMPRIN), vascular growth factor (VEGF), MUC-2, 4, 5AC and 6, CD44, E-cadherin, beta-catenin, and phosphorylated glycogen synthase kinase 3beta-ser9 (P-GSK3beta-ser9) was examined on tissue microarrays using immunohistochemistry. It was found that MT carcinomas exhibited large size, deep invasion, frequent local invasion, and lymph node metastasis in comparison with IT and DT carcinomas (p < 0.05). All the markers except MUC-5AC showed higher expression in IT than DT carcinomas (p < 0.05). The expression of maspin, EMMPRIN, VEGF, MUC-4, and membrane E-cadherin was stronger in MT intestinal than diffuse component (p < 0.05). Immunoreactivities to Ki-67, EMMPRIN, and VEGF were weaker in IT carcinoma than in the MT intestinal portion (p < 0.05), while the opposite was true for CD44, MUC-2, and MUC-6 (p < 0.05). The MT diffuse component displayed a higher expression of FHIT, VEGF, and P-GSK3beta-ser9 than DT carcinoma (p < 0.05). The accumulative survival rate of the IT carcinoma patients was higher than the other types (p < 0.05). The invasive depth, venous invasion, lymph node, peritoneal or liver metastasis, and Lauren's classification were independent prognostic factors for gastric carcinomas (p < 0.05). These findings suggested that MT carcinomas were also indicated to be more aggressive than IT and DT carcinomas. Significant differences were observed in the proliferation, apoptosis, angiogenesis, mucin secretion, and cell adhesion between IT and DT carcinomas, whereas only a few of these characteristics showed differences between the MT intestinal and diffuse parts, thus suggesting that both the MT components might originate from the stem cells with similar genetic traits, but follow different histogenic pathways.
Project description:Adenocarcinomas exhibiting gastric differentiation represent a recently described and uncommon subtype of non-human papillomavirus (HPV)-related cervical adenocarcinoma. They comprise a spectrum from a well-differentiated variant (adenoma malignum/mucinous variant of minimal deviation adenocarcinoma) to a more poorly differentiated overtly malignant form, generally referred to as gastric-type adenocarcinoma. Rarely, such tumors have also been described as primary vaginal neoplasms. Gastric-type adenocarcinomas exhibit considerable morphologic overlap with adenocarcinomas originating outside the female genital tract, especially mucinous adenocarcinomas arising in the pancreas and biliary tract. Moreover, they often metastasize to unusual sites, such as the ovary and peritoneum/omentum, where they can be mistaken for metastatic adenocarcinomas from other, nongynecologic sites. There is little information regarding the immunophenotype of gastric-type adenocarcinomas, and knowledge of this is important to aid in the distinction from other adenocarcinomas. In this study, we undertook a detailed immunohistochemical analysis of a large series of cervical (n=45) and vaginal (n=2) gastric-type adenocarcinomas. Markers included were cytokeratin (CK)7, CK20, CDX2, carcinoembryonic antigen, CA125, CA19.9, p16, estrogen receptor, progesterone receptor, MUC6, PAX8, PAX2, p53, hepatocyte nuclear factor 1 beta, carbonic anhydrase IX, human epidermal receptor 2 (HER2), and mismatch repair (MMR) proteins. All markers were classified as negative, focal (<50% of tumor cells positive), or diffuse (≥50% tumor cells positive) except for p53 (classified as "wild-type" or "mutation-type"), HER2 (scored using the College of American Pathologists guidelines for gastric carcinomas), and MMR proteins (categorized as retained or lost). There was positive staining with CK7 (47/47-45 diffuse, 2 focal), MUC6 (17/21-6 diffuse, 11 focal), carcinoembryonic antigen (25/31-12 diffuse, 13 focal), carbonic anhydrase IX (20/24-8 diffuse, 12 focal), PAX8 (32/47-20 diffuse, 12 focal), CA125 (36/45-5 diffuse, 31 focal), CA19.9 (11/11-8 diffuse, 3 focal), hepatocyte nuclear factor 1 beta (13/14-12 diffuse, 1 focal), CDX2 (24/47-4 diffuse, 20 focal), CK20 (23/47-6 diffuse, 17 focal), and p16 (18/47-4 diffuse, 14 focal). Most cases were negative with estrogen receptor (29/31), progesterone receptor (10/11), PAX2 (18/19), and HER2 (25/26). p53 showed "wild-type" and "mutation-type" staining in 27 of 46 and 19 of 46 cases, respectively. MMR protein expression was retained in 19 of 20 cases with loss of MSH6 staining in 1 patient with Lynch syndrome. Molecular studies for HPV were undertaken in 2 tumors, which exhibited diffuse "block-type" immunoreactivity with p16, and both were negative. This is the first detailed immunohistochemical study of a large series of gastric-type adenocarcinomas of the lower female genital tract. Our results indicate immunophenotypic overlap with pancreaticobiliary adenocarcinomas but suggest that PAX8 immunoreactivity may be especially useful in distinguishing gastric-type adenocarcinomas from pancreaticobiliary and other nongynecologic adenocarcinomas, which are usually negative. Diffuse "block-type" p16 immunoreactivity in a cervical adenocarcinoma is not necessarily indicative of a high-risk HPV-associated tumor.