Role of trefoil factor 1 in gastric cancer and relationship between trefoil factor 1 and gastrokine 1

Trefoil factor 1 (TFF1) is a small cysteine-rich secreted protein which is principally expressed in the superficial cells of gastric mucosa. In gastric cancer, TFF1 is downregulated and plays an important role. Gastrokine 1 (GKN1) is a secreted protein with similar expression and biological functions to TFF1. This study aimed to determine the expression and biological functions of TFF1 and the relationships between TFF1 and GKN1 in gastric cancer. RT-PCR and immunohistochemistry were performed to detect TFF1 expression in gastric cancer cell lines and tissues. The transfected and co-transfected AGS cells which stably expressed TFF1 or both TFF1 and GKN1 were generated. Phenotypic changes such as cell viability, apoptosis and cell cycle modulation were assayed in the transfected cells. We found that TFF1 expression was significantly downregulated or lost in gastric cancer cell lines, gastric dysplasia and cancer. Restoration of TFF1 expression in AGS cells suppressed tumor cell viability and arrested AGS cells in the G1-S transition phase after olomoucine treatment. However, TFF1 was unable to induce cell apoptosis. In co-transfected cells, we found that TFF1 and GKN1 did not directly interact at the protein level. GKN1 was unable to cooperate with TFF1 on cell viability suppression, cell apoptosis and differentiation. Together, these results indicate that TFF1 expression is significantly downregulated in gastric cancer. TFF1 inhibited cell proliferation by delaying G1-S phase transition but not by inducing apoptosis. TFF1 may not interact or cooperate with GKN1 at the protein and functional level.     

Inhibition of cyclooxygenase-2 causes regression of gastric adenomas in trefoil factor 1 deficient mice

Cyclooxygenase-2 (Cox-2) expression is a marker of reduced survival in gastric cancer patients, and inhibition of Cox-2 suppresses gastrointestinal carcinogenesis in experimental animal models. To investigate the role of Cox-2 in gastric carcinogenesis in vivo, we utilized trefoil factor 1 (Tff1) deficient mice, which model the neoplastic process of the stomach by developing gastric adenomas with full penetrance. These tumors express Cox-2 protein and mRNA, and we have now investigated the effects of genetic deletion of the mouse Cox-2 gene [also known as prostaglandin-endoperoxide synthase 2 (Ptgs2)] and a Cox-2 selective drug celecoxib. Our results show that genetic deletion of Cox-2 in the Tff1 deleted background resulted in reduced adenoma size and ulceration with a chronic inflammatory reaction at the site of the adenoma. To characterize the effect of Cox-2 inhibition in more detail, mice that had already developed an adenoma were fed with celecoxib for 8-14 weeks, which resulted in disruption of the adenoma that ranged from superficial erosion to deep ulcerated destruction accompanied with chronic inflammation. Importantly, mice fed with celecoxib for 16 weeks, followed by control food for 9 weeks, redeveloped a complete adenoma with no detectable inflammatory process. Finally, we determined the identity of the Cox-2 expressing cells and found them to be fibroblasts. Our results show that inhibition of Cox-2 is sufficient to reversibly disrupt gastric adenomas in mice.     

Clinicopathologic significance of hypoxia-inducible factor 1alpha overexpression in gastric carcinomas

BACKGROUND: Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a key role in responses to hypoxia and expression of HIF-1alpha downstream genes leads to both an adapted metabolism and increased oxygen supply. We investigated the clinical significance of HIF-1alpha expression in gastric carcinoma. METHODS: We examined HIF-1alpha, vascular endothelial growth factor (VEGF), and insulin-like growth factor-2 (IGF-2) expression patterns immunohistochemically in 126 specimens of gastric carcinoma. CD34 antigen levels were also examined by immunohistochemistry to determine microvessel density (MVD) within tumors and correlations between HIF-1alpha expression, clinicopathological features, and survival were examined. RESULTS: HIF-1alpha expression correlated with tumor size (P<0.005), depth of invasion (P=0.018), VEGF expression (P=0.03), and intra-tumor MVD (P<0.005). IGF-2 expression was more prevalent in HIF-1alpha positive than in HIF-1alpha negative tumors and the 5-year survival rate was 58.4% for HIF-1alpha positive patients and 81.5% for HIF-1alpha negative patients (P=0.009). HIF-1alpha expression is an independent prognostic factor in gastric carcinoma (P=0.032). CONCLUSIONS: Overexpression of HIF-1alpha in gastric carcinomas may upregulate its downstream gene products leading to VEGF-mediated angiogenesis, and resulting in a poor prognosis for patients.     

Cyclooxygenase-2 expression and effect of celecoxib in gastric adenomas of trefoil factor 1-deficient mice

Expression of cyclooxygenase-2 (Cox-2) is elevated in gastric adenocarcinomas and precursor lesions leading to this disease. Mice deficient for trefoil factor 1 (TFF1) develop a pyloric adenoma with full penetrance. Because inhibition of Cox-2 suppresses tumor growth in several animal models, we studied expression of Cox-2 and effect of a selective Cox-2 inhibitor celecoxib in gastrointestinal tissues of the TFF1-deficient mice. Cox-2 mRNA and protein were strongly expressed in the pyloric adenomas of the TFF1(-/-) mice as detected by in situ hybridization and immunohistochemistry. Nonneoplastic gastrointestinal tissues of wild-type or TFF1(-/-) mice expressed low or nondetectable levels of Cox-2. Celecoxib (1600 ppm p.o. for 3 months) caused ulceration and inflammation of the adenoma in all treated TFF1(-/-) mice (n = 7). This effect of the drug was adenoma specific, because no histological alterations were observed in the non-neoplastic gastric or intestinal tissues in the TFF1(-/-) or wild-type mice receiving the drug treatment. All untreated TFF1(-/-) mice had an adenoma (n = 7), but none demonstrated the combination of ulceration and inflammation. Our data show that Cox-2 is expressed in gastric adenomas of the TFF1(-/-) mice and suggest that inhibition of Cox-2 disturbs the integrity of the adenoma by promoting ulceration and inflammation. These findings support the effort to initiate clinical studies to investigate the effect of Cox-2 inhibitors as a chemotherapeutic modality for dysplasias of the stomach.     

三叶因子3在胃癌组织中的表达及与血管形成的关系

目的　研究三叶肽因子 3(trefoilfactor3,TFF3)在人胃癌组织中的表达及其与胃癌血管形成的关系。方法　应用免疫组织化学S-P法检测 40例原发性胃癌及 10例正常胃粘膜组织中TFF3的表达,同时检测微血管密度(microvesseldensity,MVD),以抗CD34标记。结果　10例正常胃黏膜组织TFF3全部阴性表达, 40例胃癌组织TFF3表达阳性率为 60% (24 /40)。其阳性表达与胃癌的淋巴结转移有关 (P<0. 05),而与性别、肿瘤的浸润程度、细胞的分化程度无关.胃癌组织MVD明显高于正常胃粘膜组织,TFF3阳性及阴性表达组MVD均值分别为 32. 71±8. 04、26. 94±9. 08,两组间MVD均值差异有统计学意义 (P<0. 05)。结论　胃癌组织中TFF3表达增高,其高表达与淋巴结转移,肿瘤微血管形成有关。     

The pro-apoptotic FAS-associated factor 1 is specifically reduced in human gastric carcinomas

The Fas-associated factor 1, FAF1, is a protein, which was first identified as an interaction partner of the death receptor Fas. Not much is known about the function of FAF1, but it has been found that it is able to potentiate Fas-induced apoptosis in cell lines. To clarify the role of FAF1 in human cancer, a number of tumors from different organs were screened for expression of the protein, and it was only found reduced in gastric carcinoma tissue. Thus, 58 human gastric carcinomas were collected, and the expression of FAF1 was analyzed by Western blotting and in a few cases also by immunohistochemistry. The hypothesis was that since FAF1 is able to potentiate apoptosis, it would likely be reduced in the gastric carcinomas in order for them to escape apoptosis. We found that FAF1 was reduced in 50% (29/58) of the gastric carcinomas analyzed as compared to non-neoplastic gastric mucosa from the same patients. 26 of the investigated carcinomas contained signet ring cells, and FAF1 was significantly reduced in 69% of these (p=0.017), whereas it was only reduced in 34% of the carcinomas without signet ring cells. The observed reduction of FAF1 was predominantly caused by proteolytic cleavage of the protein. Additionally, 31 colorectal carcinomas were analyzed for expression of FAF1. Here, FAF1 was only reduced in 16% of the carcinomas when compared to non-neoplastic colorectal mucosa. Our findings support the hypothesis that FAF1 is reduced in gastric carcinomas compared to non-neoplastic tissue, and there was a significant relation between FAF1 reduction and content of signet ring cells in the gastric carcinomas. Also, the reduction of FAF1 is likely to be specific for gastric cancer, which might be due to the fact that signet ring cells are most frequently found in gastric cancers.     

Beclin 1 expression is an independent prognostic factor for gastric carcinomas

Beclin 1, an important autophagy-related protein in human cells, is involved in autophagy, differentiation, anti-apoptosis, and cancer progression, which is increased during periods of cell stress and extinguished during the cell cycle. In order to clarify the role of Beclin 1 in gastric carcinogenesis and subsequent progression, its expression was examined by immunohistochemistry and in situ hybridization (ISH) on tissue microarrays containing gastric carcinomas, adjacent non-neoplastic mucosa, and metastatic lymph node. Gastric carcinoma tissue and cell lines were studied for Beclin 1 expression by Western blot or RT-PCR, respectively. The results demonstrated that Beclin 1 was distinctively expressed in GES-1, AGS, BGC-823, GT-3 TKB, HGC-27, KATO-III, MGC-803, MKN28, MKN45, SCH, SGC-7901, or STKM-2 at both mRNA and protein levels. However, Beclin 1 mRNA was highly expressed in gastric carcinoma than matched mucosa by real-time PCR and ISH (P?<?0.05). Beclin 1 expression was negatively related to distant metastasis and poor prognosis of gastric carcinoma (P?<?0.05). Beclin 1 was highly expressed in male than female patients with gastric carcinoma (P?<?0.05). The 65-year-elder patients with gastric carcinoma had higher Beclin 1 expression than the younger ones (P?<?0.05). The diffuse-type carcinomas showed less Beclin 1 expression than intestinal- and mixed-type ones (P?<?0.05). In intestinal-type gastric carcinoma, Beclin 1 expression was inversely associated with venous invasion, lymph node metastasis, and tumor–node–metastasis (TNM) staging (P?<?0.05). Kaplan–Meier analysis indicated that Beclin 1 expression was positively linked to favorable prognosis of the patients with overall and intestinal-type carcinoma (P?<?0.05). Cox’s proportional hazard model indicated that venous invasion, lymph node metastasis, distant metastasis, TNM staging, and Beclin 1 expression were independent prognostic factors for gastric carcinomas (P?<?0.05). It was suggested that aberrant Beclin 1 expression is closely linked to pathogenesis, metastasis, and differentiation of gastric carcinoma. Beclin 1 expression might be employed to indicate the favorable prognosis of gastric carcinomas as an independent factor.     

Intestinal trefoil factor: a marker of poor prognosis in gastric carcinoma.

PURPOSE: Intestinal trefoil factor (ITF) is a marker of intestinal differentiation that may also play a role in cancer cell biology by inhibiting cell adhesion, promoting cell invasion, and blocking apoptosis. Gastric adenocarcinomas can arise through a process of intestinalization, but no study has yet comprehensively examined the expression of ITF in gastric cancer or correlated ITF expression with clinical outcome in any cancer type. EXPERIMENTAL DESIGN: Patients (209) with primary gastric adenocarcinoma were evaluated for ITF expression by immunohistochemistry. Results of immunostaining were correlated with clinicopathological variables and overall survival. RESULTS: In normal gastric mucosa, ITF expression was absent, whereas areas of intestinal metaplasia revealed strong ITF expression by goblet cells. A portion of gastric cancers (55%) demonstrated ITF expression. Women were more likely than men to express ITF in gastric cancers. However, in men, the expression of ITF correlated with aggressive phenotype of tumors (advanced stage, infiltrative growth pattern, and positive lymph nodes). Multivariate analysis revealed that expression of ITF was associated with a poor prognosis, independent of tumor stage. CONCLUSIONS: This is the first study to correlate ITF expression with clinicopathological features or outcome in any cancer type. ITF expression in gastric cancer exhibited a curious gender-associated relationship, being more frequently expressed in tumors of women, but associated with more aggressive pathological features in men. The poor prognosis of patients with ITF-positive gastric cancers further implicates ITF in cancer cell biology.     

miRNA423-5p regulates cell proliferation and invasion by targeting trefoil factor 1 in gastric cancer cells

TFF1 is a small, secreted protein in the TFF family that has a pivotal role as a motogenic factor in epithelial restitution and cell motility, and as a tumor suppressor gene in the stomach. In this study, we identified TFF1 as a novel target gene of miRNA-423-5p. miRNA-423-5p negatively regulated the expression of TFF1 by binding to its 3′UTR and participated in proliferation/invasion-related processes via a TFF1-dependent manner in gastric cancer cells. Our findings suggested that miR-423-5p may be a novel target for the future development of specific therapeutic interventions for gastric cancer.     

Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcinomas

Expression profiling analysis revealed ectopic high expression of mouse TFF3 in non-tumor liver tissues from the hepatocellular carcinoma (HCC) susceptible PWK/Rbrc strain. TFF3 is a member of the trefoil factor family peptides, which are small secreted proteins regulating mucosal regeneration and repair, and which are overexpressed during inflammatory processes and cancer progression. We, therefore, analyzed the TFF3 expression extensively in mouse and human HCCs. Expression of the mouse TFF3 gene was significantly increased in 6 out of 7 HCCs from a PWK spontaneous tumor model and in all 7 HCCs from an SV40T antigen-induced transgenic MT-D2C57BL/6 model. In humans, 8 of 20 HCCs (40%) had overexpression of TFF3 in both mRNA level and protein level. We then analyzed DNA methylation patterns of the TFF3 promoter region to evaluate expression regulation of promoter methylation. In mouse HCCs, we demonstrated that two CpGs, at positions -992 and +109, were hypomethylated in 13 of 14 mouse HCCs. In human HCCs, hypomethylation at CpG -260 was associated with TFF3 overexpression (p=0.04). These results indicate that TFF3 overexpression may be a critical process in mouse and human hepatocellular carcinogenesis, and the specific promoter CpG hypomethylation may be one of the regulation mechanisms of TFF3 overexpression in HCCs.     

Expression of epidermal growth factor receptor in human gastric and colonic carcinomas

The expression of epidermal growth factor (EGF) receptor was examined immunohistochemically in a total of 122 gastric and 61 colonic carcinomas, out of which 16 gastric and 8 colonic carcinomas were also examined by 125I-labeled EGF binding analysis and Western blotting. The values of EGF binding were 12.68 +/- 1.98 (SE; n = 16) fmol/mg protein in gastric carcinomas and 5.72 +/- 2.15 (n = 8) fmol/mg protein in nonneoplastic gastric mucosa, the difference being significant (P less than 0.01). In the colonic tissue, the binding capacities in carcinomas and nonneoplastic mucosa were 13.29 +/- 4.17 (n = 8) and 10.68 +/- 0.41 (n = 3) fmol/mg protein, respectively. Scatchard analysis of 125I-labeled EGF binding indicated a single class of receptors in gastric and colonic carcinomas with an apparent Kd value of from 111 to 277 (n = 4) and from 87.4 to 341 fM (n = 5), respectively, except for one gastric carcinoma having two classes of receptors (Kd = 15.9 and 896 fM). In Western blotting using monoclonal anti-EGF receptor antibody, various levels of EGF receptor expression were detected in 12 (85.7%) of the 14 gastric carcinomas and in 7 (87.5%) of the 8 colonic carcinomas. Immunohistochemically, EGF receptor immunoreactivity was detected in one (3.8%) of the 26 early gastric carcinomas, while it was observed in 33 (34.4%) of the 96 advanced gastric carcinomas, the incidence between the two being significantly different (P less than 0.01). In the colonic carcinomas, 47 (77.1%) of the 61 cases showed positive immunoreactivity to EGF receptor, which did not differ by histological type.     

Expression of epidermal growth factor receptors on normal human gastric epithelia and gastric carcinomas

Tissues of normal human gastric mucosae and 15 advanced gastric carcinomas were studied immunohistologically for the presence of receptors for epidermal growth factor (EGF) by use of a murine monoclonal antibody (528IgG), which reacts with the binding domain of human EGF receptor. On normal gastric mucosae, only parietal cells showed positive staining. On cancer tissues, definite staining was observed in 9 of 15 cases. Their staining intensities were variable and weaker in general compared to those of either gastric parietal cells or normal tonsilar squamous epithelium. No apparent correlation of EGF receptor staining with the grade of histologic differentiation or lymph node metastases of these gastric carcinomas was noted.     

Decreased expression of biliary glycoprotein in hepatocellular carcinomas

Biliary glycoprotein (BGP) is an adhesion and anti-cell-growth molecule of the carcinoembryonic antigen family. We have earlier demonstrated that BGP mRNA is expressed in hepatocellular carcinomas (HCCs) and the adjacent non-cancerous regions, neither of which express CEA and NCA mRNA. To define an expression level and pattern of BGP at the protein level in HCCs, TS135, a monoclonal antibody (MAb) against BGP, was prepared. This MAb clearly reacted with BGP with a molecular weight of 110 kDa and 85 kDa (BGP-110/85). It cross-reacted weakly with NCA-90 from NCA transfectants, but not at all with CEA-200 from the serum of a colon-cancer patient. The BGP transfectants of cultured hepatocellular carcinoma cHc-4 cells showed Ca²⁻-dependent cell aggregation, which was partially inhibited by modulating BGP on the cell surface with MAb TS135. Immunostaining of non-cancerous liver tissues with MAb TS135 indicated that BGP could be expressed in the bile canalicular domain of hepatocytes. In HCCs, the expression of BGP was predominantly found in the well-differentiated type, where the bile canaliculi and the apical portion of pseudoglands were positively stained, although their staining intensity and stained area were lower and more limited, respectively, than those of non-cancerous regions. The percentage of faintly positive and negative cases (n = 22) from the total (n = 30) was 73%. This suggests that the expression level of BGP decreased in HCCs as compared with adjacent non-cancerous regions. Int. J. Cancer 74:15–19. © 1997 Wiley-Liss, Inc.     

Expression of telomeric repeat binding factor 1 and 2 and TRF1-interacting nuclear protein 2 in human gastric carcinomas

According to studies on a variety of malignant tumors from different organs MUC1 mucin antigen presents as a valuable marker of cancer progression and prognosis. During recent years, a great number of monoclonal antibodies (mabs) directed to MUC1 was generated. Their epitopes can be classified according to their position within the tandem repeat domain of the mucin and with respect to effects exerted by site-specific glycosylation. In this study, eight mabs from different clusters were selected to correlate their epitope specificity with their binding pattern in human cancer specimens. By applying an immunohistochemical ABC-peroxidase method, ten carcinomas derived from breast, pancreas, stomach and colon were characterized. A positive reaction of all mabs could be observed in the majority of the carcinomas, however, the extent of the stained tumor area varied significantly. In general, mabs M38, VA1 and BC3 exhibited the strongest staining reaction. Mab BW835 showed a similar binding intensity, especially in pancreatic and gastric carcinomas. It is tempting to speculate that the different binding patterns may reflect differences in epitope specificity. In conclusion, future immunohistochemical, immunoserological and therapeutic studies involving MUC1 antigen should prefer well-characterized and highly reactive mabs detecting defined peptide epitopes.     

Expression of the MDR1 gene in human gastric and colorectal carcinomas

We measured expression of the MDR1 gene (also known as the PGY1 gene) in the human gastrointestinal tract. MDR1 messenger RNA (mRNA) levels were elevated in 13 of 15 colorectal carcinoma specimens and in six of 13 gastric carcinoma specimens. Well-differentiated colorectal carcinomas contained significantly higher concentrations of MDR1 mRNA than moderately differentiated colorectal carcinomas. Similarly, moderately differentiated gastric carcinomas contained higher concentrations of MDR1 mRNA than poorly differentiated gastric carcinomas. MDR1 gene expression in normal colorectal and gastric tissues adjacent to carcinomas was similar to that in the carcinomas. MDR1 gene expression in xenografts of colorectal and gastric carcinomas in nude mice was also investigated. Elevated expression of the MDR1 gene was seen in only four of 18 xenografts of colorectal carcinoma and was not seen in any xenografts of gastric carcinoma. P-glycoprotein was distributed over the luminal surface of the colorectal carcinoma. These results imply that the higher levels of MDR1 mRNA found in well-differentiated carcinomas derived from colorectal tissues are the results of increased expression of the MDR1 gene in the luminal surface cells. The level of expression of the MDR1 gene in colorectal and gastric carcinomas appears to correlate with the degree of differentiation and also appears to be affected by transplantation into nude mice.     

KAI1及Caveolin-1在胃癌组织中的表达及其意义

目的探讨正常胃黏膜、不典型增生胃黏膜及胃癌组织中KAI1及Caveolin-1蛋白的表达。方法应用免疫组化SP法检测22例正常胃黏膜、65例不典型增生胃黏膜及74例胃癌组织中KAI1蛋白、Caveolin-1蛋白的表达状况。结果正常胃黏膜、不典型增生胃黏膜及胃癌组织中,KAI1和Caveolin-1阳性率呈递减趋势,且组间差异有显著性（χ2=24.022,P〈0.05;χ2=44.315,P〈0.05）。经χ2和Fisher精确概率法检验,KAI1蛋白的表达在不同浸润深度、有无淋巴结转移组、有无脉管侵犯组内表达率的差异有显著性（P〈0.05）,在年龄及性别组内表达率的差异性无显著性（P〉0.05）。Caveolin-1蛋白阳性表达率与有无淋巴结转移、浸润深度有关（P〈0.05）,而与有无脉管转移、年龄及性别（P〉0.05）无关。Spearman等级相关分析显示,KAI1蛋白与Caveolin-1蛋白表达呈正相关（rs=0.827,P〈0.05）。结论 KAI1、Caveolin-1在胃癌组织中表达的下调甚至缺失可能是胃癌发生、发展以及浸润转移的重要原因之一。