MT2-MMP expression associates with tumor progression and angiogenesis in human lung cancer

Matrix metalloproteinases (MMPs) are a family of important proteolytic enzymes that play an important role in the remodeling of the tumor microenvironment and associate with tumorigenesis and metastasis. We previously reported that membrane type-2 MMP (MT2-MMP) is highly expressed in human esophageal cancer tissues, and its expression level is positively correlated to tumor size and intratumoral angiogenesis. In order to reveal whether MT2-MMP expression is operative in human lung cancer and its underlying physio-pathological role, in the present study, we examined both mRNA and protein expression levels of MT2-MMP in non-small cell lung caner (NSCLC) tissues and in adjacent normal tissues by using real-time RT-PCR and immunohistochemistry respectively, which showed that both MT2-MMP mRNA (P=0.0359) and protein (P<0.0001) expression levels were significantly increased in cancer tissues in contrast to adjacent normal tissues. Moreover, we also found that the MT2-MMP protein level in cancer tissues positively correlated to lymph node metastasis (P=0.0483), tumor stage (P=0.0483), intra-tumoral microvessel density (MVD) (P=0.0445). We have not found statistically significant correlation between MT2-MMP expression and patients’ prognoses, but we found that the patients with both higher MT2-MMP protein expression and higher intra-tumoral microvessel density showed better prognoses than that of the patients with either higher MT2-MMP protein expression or higher intra-tumoral microvessel density (P=0.0311). Thus, our data suggest that MT2-MMP expression positively involves in NSCLC, and might play an important role in promoting the tumor progression and intra-tumoral angiogenesis in NSCLC.     

Clinical significance of expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase-2 in human head and neck squamous cell carcinoma

Three different membrane-type matrix metalloproteinases (MT-MMPs) activate in vitro the latent form of matrix metalloproteinase-2 (MMP-2), which is one of the key proteinases in invasion and metastasis of various cancers. We examined the mRNA expression of MT1, 2, and 3-MMPs and MMP-2 in cell lines of head and neck squamous cell carcinoma (HNSCC) and quantitated the relative expression levels in human HNSCC tissues by Northern blotting. The tissue localization of MT1-MMP and MMP-2 was determined by immunohistochemistry and in situ hybridization. Their implications in clinicopathologic factors were statistically evaluated. All cell lines examined consistently expressed MT1-MMP and MMP-2, but not MT2, 3-MMP. In the clinical specimens, there was a significant correlation in coexpression of messenger of RNA (P = .0005) and colocalization by immunohistochemistry (P < .0001) for MT1-MMP and MMP-2. Relative mRNA expression levels of MT1-MMP and MMP-2 in the carcinoma tissues were significantly higher than those of the control tissues (P = .0045 and P = .0122, respectively). Both mRNA expression level and immunopositivity of MT1-MMP significantly correlated with lymph node metastasis (P = .0081 and P = .0193, respectively), which was confirmed by multivariate logistic regression analysis. Immunoreaction of MT1-MMP and its mRNA expression were observed in both carcinoma cells and stromal cells. The localization of MMP-2 closely corresponded to that of MT1-MMP. These observations suggest that MT1-MMP possesses a role as a determinant of lymph node metastasis in HNSCC, and that concurrent expression of MT1-MMP and MMP-2 are involved in progression of HNSCC.     

Interaction of MT1-MMP and laminin-5γ2 chain correlates with metastasis and invasiveness in human esophageal squamous cell carcinoma

To gain insights into metastatic mechanisms in esophageal squamous cell carcinoma (ESCC), we established sublines (MLuB1 and MLuC1) with different capacity of spontaneous lung metastasis by subcutaneous injection of a human ESCC cell line (EC 9706) into nude mices. The incidence of the mice with lung metastasis produced by MLuC1 (87%) was significantly higher than that of MLuB1 (22%). The gene expression profiles of the two sublines were compared with cDNA arrays containing 5,000 known genes, and 47 genes were differentially expressed ≥2.0 fold. Laminin-5γ2 chain (Ln-5γ2) was one of the up-regulated genes in MLuC1 cells. Proteolytically processed forms of γ2 are known to promote migration of a multitude of epithelial cells in vitro. Western-blotting analysis revealed that degraded fragments of Ln-5γ2 and active form of membrane-type matrix metalloproteinase-1 (MT1-MMP) in MLuC1 was significantly higher than those in MLuB1. Expression of MT1-MMP was observed in 60 of 75 Ln-5γ2-positive carcinoma tissues (80%). Co-expression of the two proteins was significantly associated with depth of invasion (P = 0.012). Moreover, proteolytic fragments of Ln-5γ2 and active forms of MT1-MMP were frequently found in tumor tissues, whereas in the corresponding normal esophageal tissues there were only intact forms of γ2 and MT1-MMP. siRNA-mediated silencing of MT1-MMP significantly reduced production of γ2′ and γ2x in MLuC1 cells and inhibited cell migration. The results suggest that MT1-MMP is an enzyme responsible for Ln-5γ2 cleavage in ESCC, and interaction between them may play a critical role in promoting invasion and metastasis of human ESCC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

15-PGDH expression as a predictive factor response to neoadjuvant chemotherapy in advanced gastric cancer

Given the various clinical and pathologic responses to neoadjuvant chemotherapy (NACT) in gastric cancer (GC), potential biomarkers that reflecting the efficacy of NACT on GC should be investigated. The aim of this study was to investigate the 15-PGDH expression response to NACT in GC patients and its relationship with prognosis of GC. Immunohistochemical method was used to assess the level of 15-PGDH expression in 56 GC patients who received NACT before surgery and 46 patients who underwent surgical treatment without NACT as well as their corresponding adjacent non-neoplastic tissues. We found that there was no correlation of 15-PGDH expression between non-cancerous gastric tissues and GC tissues (P=0.519), while 15-PGDH expression level in NACT group was higher than that in nNACT group (P=0.015). In patients with NACT, the higher level of 15-PGDH expression was significantly associated with well-moderately differentiated grade (P=0.023), I/II stage (P=0.014) and with no lymph node metastasis (P=0.016). Moreover, statistically significant differences in overall survival (OS) were found among 15-PGDH expression (log-rank test, P<0.001) and TNM stage (log-rank test, P=0.032). Most importantly, expression of 15-PGDH was found to be an independent predictive factor by multivariate analysis (Hazard ratio (HR) 0.315 [0.120-0.827], P=0.019). These findings indicated that NACT could increase 15-PGDH expression in advanced GC patients, and 15-PGDH may serve as a candidate prognostic biomarker of advanced GC response to NACT.     

Impact of chemokine receptor CXCR3 on tumor-infiltrating lymphocyte recruitment associated with favorable prognosis in advanced gastric cancer

Chemokine receptor CXCR3 has been proved to play an important role in tumorigenesis and tumor progression in many malignancies, but its precise efficacy on gastric cancer (GC) has not been evaluated yet. The present study was aimed to explore the correlation of chemokine receptor CXCR3 with tumor-infiltrating lymphocytes (TILs) and prognosis in advanced gastric cancer (GC). Expression of CXCR3 and CD4+, CD8+ TILs was conducted in 192 advanced GC specimens and 48 corresponding paracancerous tissues by immunohistochemical (IHC) analysis. CXCR3 expression in GC tissues was significantly higher than that in paracancerous tissues (P<0.001) and CD8+, CD4+ TILs infiltration increased with high CXCR3 expression (P=0.032 and P<0.001, respectively). Our study showed significantly lower CXCR3 expression in patients with greater tumor invasion depth and lymph node metastasis compared with patients with lesser tumor invasion depth and without lymph node metastasis (P=0.002 and P=0.001, respectively). Univariate analysis indicated that patients with high CXCR3 expression and high CD8+ TILs infiltration had longer overall survival (OS) (log-rank test, P<0.001 and P=0.002, respectively). Univariate and multivariate analyses indicated that CXCR3 expression was an independent prognostic factor for OS (P=0.002). The present study suggested that CXCR3 expression was upregulated in advanced GC and was associated with increased CD4+, CD8+ TILs infiltration and improved OS. Therefore, CXCR3 overexpression is implicated as a favorable prognostic biomarker in human advanced GC.     

Expression of MT-1 MMP, MMP2, MMP9 and TIMP2 mRNAs in ductal carcinoma in situ and invasive ductal carcinoma of the breast

We investigated the expression of membrane type-1 (MT1)-MMP, MMP2, MMP9 and TIMP2 mRNAs and their roles in ductal carcinoma in situ (DCIS) and T1 and T2 invasive ductal carcinoma of the breast. We further compared these two types of carcinomas for differences in microvessel density, and expression of angiogenic factors and CD44std. MT1-MMP, MMP2, MMP9 and TIMP2 mRNA were expressed in both DCIS and invasive ductal carcinomas. Expression rates of MT1-MMP, MMP2, MMP9 and TIMP2 mRNAs were not statistically different between DCIS and invasive ductal carcinomas, nor did they differ statistically when grouped by tumor size, histologic grade or nuclear grade of invasive ductal carcinoma. Microvessel density and expression of VEGF and TGF-beta1 were not statistically different between DCIS and invasive ductal carcinoma. CD44std expression was significantly increased in DCIS compared to invasive ductal carcinoma (p < 0.05) and it was also significantly increased in lower clinical stage, histologic grade and nuclear grade of invasive ductal carcinoma (p < 0.05). Axillary node metastasis was significantly correlated with MT1-MMP mRNA, VEGF and TGF-beta1 expression (p < 0.05) and MT1-MMP mRNA was positively correlated with VEGF expression and TIMP2 mRNA (p < 0.05). In summary, patterns of MMP mRNA expression in DCIS and invasive ductal carcinoma suggest that the invasive potential of breast carcinoma is already achieved before morphologically overt invasive growth is observed. As MT1-MMP mRNA expression is significantly correlated with axillary nodal metastasis, it may be useful as a prognostic indicator of invasive ductal carcinoma. Considering the positive correlation of MT1-MMP mRNA and TIMP2mRNA expression, our finding supports a role for TIMP2 in tumor growth, as well as the utility of CD44std as a prognostic indicator of breast cancer.     

Differential Expression and Origin of Membrane-Type 1 and 2 Matrix Metalloproteinases (MT-MMPs) in Association with MMP2 Activation in Injured Human Livers

Matrix metalloproteinase-2 (MMP2) activation is associated with basement membrane remodeling that occurs in injured tissues and during tumor invasion. The newly described membrane-type MMPs (MT-MMPs) form a family of potential MMP2 activators. We investigated the localization and steady-state levels of MT1-MMP and MT2-MMP mRNA, compared with those of MMP2 and tissue inhibitor of MMP-2 in 22 hepatocellular carcinomas, 12 liver metastases from colonic adenocarcinomas, 13 nontumoral samples from livers with metastases, 10 benign tumors, and 6 normal livers. MMP2 activation was analyzed by zymography in the same series. The expression of MT1-MMP mRNA and the activation of MMP-2 were increased in hepatocellular carcinomas, metastases, and cholestatic nontumoral samples. MT2-MMP mRNA was rather stable in the different groups. MT1-MMP mRNA levels, but not MT2-MMP mRNA, correlated with MMP-2 and tissue inhibitor of MMP-2 mRNA levels and with MMP2 activation. In situ hybridization showed that MT1-MMP mRNA was expressed in stromal cells, and MT2-MMP mRNA was principally located in both hepatocytes and biliary epithelial cells. Consistently, freshly isolated hepatocytes expressed only MT2-MMP mRNA, and culture-activated hepatic stellate cells showed high levels of MT1-MMP mRNA. These results indicate that in injured livers, MMP2 activation is related to a coordinated high expression of MMP2, tissue inhibitor of MMP-2, and MT1-MMP. Furthermore, the finding of a preferential expression of MT2-MMP in hepatocytes, together with our previous demonstration that the activation of stellate cell-derived MMP2 in co-culture requires interactions with hepatocytes (Am J Pathol 1997, 150:51–58), suggests that parenchymal cells might play a pivotal role in the MMP2 activation process.     

Correlation analysis of riboflavin,RFT2 and Helicobater pylori in gastric carcinoma

Objective: To investigate the relationship between tissue riboflavin level and riboflavin transporter 2 (RFT2) protein expression, and the relationship between Helicobacter pylori (H.pylori) infection and the plasma riboflavin level in gastric carcinoma (GC). Methods: Enzyme-linked immunosorbent assay (ELISA) was used to detect tissue riboflavin level in patients with GC. Western blotting was applied to analyze the expression of RFT2 protein in 60 tissue samples from gastric carcinoma together with their normal tissues. The Warthin-starry method, rapid urease test and ¹⁴C-UBT were administered to detect the infection of H.pylori. High performance liquid chromatography (H.PYLORILC) was performed to detect plasma riboflavin level in the GC. Results: A significant decrease in the tissue riboflavin level was detected in GC samples compared to those in the normal mucous membrane (17.02±3.91 vs. 21.0±4.73; P = 0.043), and a significant decrease in the RFT2 protein was found in GC samples compared to those in the normal mucous membrane (0.92±0.39 vs. 1.23±0.51; P = 0.042). A positive correlation of tissue riboflavin level with defective expression of RFT2 protein was observed in GC patients (χ² = 1.969; P = 0.039). Plasma riboflavin level in gastric cancer without H.pylori infection group (1.6674 ng/mL ±0.37009 ng/mL) was higher than H.pylori infection group (1.2207 ng/mL ±0.17727 ng/mL, P = 0.043). Conclusion: The results indicate that RFT2 plays an important role in gastric carcinogenesis by modulating riboflavin absorption. H.pylori infection affects plasma riboflavin level and the prognosis of patients with gastric cancer.     

Clinicopathological and prognostic significance of C/EBP homologous protein (CHOP) in advanced gastric cancer

Background

Few studies have reported the clinical and prognostic significance of C/EBP homologous protein (CHOP) in advanced gastric cancer (GC). Therefore, the present study investigated the expression of CHOP in advanced GC patients to determine its potential prognostic role.

Methylation status of TRAF2 is associated with the diagnosis and prognosis of gastric cancer

The purpose was to investigate whether the expression level of TRAF2 gene was regulated by DNA methylation and explore the role of TRAF2 methylation in the diagnosis and prognosis of gastric cancer (GC). Firstly, we detected the expression of TRAF2 both at mRNA level and protein level. And the up-regulated of TRAF2 expression at two different levels were both found (P<0.001). Then we measured the methylated status of TRAF2 by MSP and got a result of that TRAF2 was hypomethylated in GC patients compared with healthy controls (P<0.001). Meanwhile, the relationship between TRAF2 methylation and clinicopathologic characteristics was estimated through chi-square. The outcome proved that TRAF2 methylation was impacted by age (P=0.024), lymph node metastasis (P=0.046), TNM stage (P=0.021), distant metastasis (P=0.002) and depth of invasion (P=0.002). The AUC of 0.795 accompanying a sensitivity of 66.7% and a specificity of 94.7% were obtained from Receiver Operating Characteristic (ROC) curve which indicated the diagnostic value of TRAF2 methylation was high. At last, we researched the prognostic value of TRAF2 methylation. Kaplan-Meier showed that patients with TRAF2 hypomethylation had lived much shorter than those with TRAF2 hypermethylation (log rank test, P<0.001). Cox regression analysis revealed TRAF2 hypomethylation (HR=18.827, 95% CI=3.103-114.222, P=0.001), lymph node metastasis (HR=0.154, 95% CI=0.047-0.512, P=0.002), distant metastasis (HR=3.032, 95% CI=1.116-8.237, P=0.030), as well as differentiation (HR=0.287, 95% CI=0.113-0.731, P=0.009) were all vital prognostic factors in GC. Taken together, TRAF2 expression was increased in GC patients by DNA hypomethylation and this methylation could be an independent diagnostic and prognostic indicator in GC.     

High levels of MMP-2, MMP-9, MT1-MMP and TIMP-2 mRNA correlate with poor survival in ovarian carcinoma

The object of this study was to analyze the potential association between the expression of MMP-2, MMP-9, MT1-MMP and TIMP-2, and disease outcome in advanced-stage ovarian carcinomas. Sections from 70 paraffin-embedded blocks (36 primary ovarian carcinomas and 34 metastatic lesions) from 45 patients diagnosed with advanced stage ovarian carcinomas (FIGO stages III–IV) were studied using mRNA in situ hybridization (ISH) technique. Patients were divided retrospectively in two groups based on disease outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period for patients that were diagnosed with advanced-stage carcinoma was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Intense mRNA signals were detected more frequently in tumor cells of short-term survivors with use of all four probes. Comparable findings were observed in peritumoral stromal cells with ISH for MMP-2, MMP-9 and TIMP-2 mRNA. Notably, primary tumors with intense mRNA signal for TIMP-2 (No = 14) were uniformly associated with a fatal outcome. In univariate analysis of primary tumors, mRNA levels of TIMP-2 in stromal cells (P = 0.0002), as well as for MMP-9 (P = 0.012) and TIMP-2 (P = 0.02) in tumor cells, correlated with poor outcome. In univariate analysis of metastatic lesions, mRNA levels of TIMP-2 in stromal cells (P = 0.031), as well as for MMP-2 (P = 0.027) and MT1-MMP (P = 0.008) in tumor cells, correlated with poor outcome. Interestingly, the presence of MT1-MMP in stromal cells correlated with longer survival (P = 0.025). In a multivariate analysis of ISH results for primary tumors, TIMP-2 levels in stromal cells (P = 0.006) and MMP-9 levels in tumor cells (P = 0.011) retained their predictive value. We conclude that MMP-2, MMP-9, MT1-MMP and TIMP-2 are valid markers of poor survival in advanced-stage ovarian carcinoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Membrane-type 1 matrix metalloproteinase enhances lymph node metastasis of gastric cancer

The mechanisms of the lymph node metastasis remain unclear. We demonstrate the role of MT1-MMP on the lymph node metastasis using in vivo experimental model of lymph node metastasis by orthotopic implantation of MT1-MMP transfected gastric cancer cell line in the stomach of nude rats. TMK-1 cell line without expression of MT1-MMP was transfected with the pcDNA3 plasmid containing a 3.4-kb MT1-MMP cDNA fragment by calcium phosphate method, and the transfected cell line was designated as TMK-MT. Western blot and RT-PCR analyses showed the specific bands corresponding to MT1-MMP in the TMK-MT cells. By gelatin zymography, the activated form (62-kDa) of MMP-2 was identified in the medium of TMK-MT cell line, but was not detected in TMK-1 cells. Six weeks after orthotopic implantation of TMK-1 and TMK-MT xenografts of nude mouse-subcutaneus tumor into the stomach of nude rats, gastric tumors were found in all the animals. Histologically, the lymphatic invasion was found in the submucosa of the TMK-MT gastric tumors. Lymph node metastasis was not detected in nude rats bearing TMK-1 gastric tumor (0/8). In contrast, lymph node metastasis was detected in five out of 8 rats, bearing TMK-MT gastric tumor. MT1-MMP immunoreactivity was found on the cell membrane and cytoplasm of TMK-MT cells not only in the lymph node metastasis but also in the stomach tumor. These results suggest that MT1-MMP overexpression induced by transfection of its gene may promote lymph node metastasis of transformed cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of long noncoding RNA TC0101441 as a novel biomarker for diagnosis and prognosis of gastric cancer

The present work aimed to explore the prognostic values of lncRNA TC0101441 (TC0101441) in patients with gastric cancer (GC). The expression of TC0101441 in a total of 159 GC specimens and matched normal specimens was detected by quantitative RT-PCR. ROC assays were conducted to determine the diagnostic value of TC0101441 expression in GC patients. The association of TC0101441 expression with clinical characteristics of 159 patients was analyzed using chi-square test. Kaplan-Meier methods were employed to determine the prognostic value of TC0101441 expression in the survival rate of GC patients. Multivariate Cox regression assays were used to identify whether TC0101441 could be a prognostic biomarker for GC patients. We found that TC0101441 expression was significantly increased in GC specimens compared to that in the normal specimens (P < 0.01). High TC0101441 expression was correlated with lymphatic metastasis (P = 0.027) and TNM stage (P = 0.015). TC0101441 could distinguish GC specimens from adjacent normal gastric specimens with an area under the receiver operating characteristic curve (AUC) of 0.8082. Survival data revealed that patients with high TC0101441 expression had worse overall survival (P = 0.0009) and disease-free survival (P < 0.0001) rates than those with low TC0101441 expression. Multivariate assays showed that TC0101441 expression was an independent biomarker for GC patients. The present study suggested that TC0101441 expression was increased in GC and may be aprognostic and diagnostic biomarker for GC.