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.     

胸腺上皮肿瘤组织中基质金属蛋白酶-2活性与Ⅰ型膜型基质金属蛋白酶 mRNA表达的相关性

目的探讨胸腺瘤和胸腺癌中基质金属蛋白酶（MMP）-2、Ⅰ型膜型（MT1）-MMP、金属蛋白酶组织抑制剂（TIMP）-2mRNA的表达和MMP-2蛋白活性的关系。方法分别用real-time逆转录．聚合酶链反应（RT-PCR,Taqman法）、明胶酶谱法和Filmin situ gelatin-Zymography（FIZ）对正常的胸腺组织（2例）、胸腺瘤（12例）和胸腺癌（2例）患者的新鲜肿瘤组织中MMP-2、MT1-MMP、TIMP-2mRNA的表达,pro-MMP-2的活性率及活性蛋白的定位进行测定。结果MMP-2、MT1-MMP及TIMP-2mRNA在Ⅰ期与Ⅱ期和Ⅲ期与Ⅳ期中的表达差异均无统计学意义（P〉0．05）,在Ⅰ～Ⅱ期与Ⅲ～Ⅳ期和胸腺癌3组中差异均有统计学意义（P〈0．01）。在AB、B1型（混合型和淋巴细胞为主型）与B2、B3型（皮质型和多角细胞为主型）以及胸腺癌3组中差异均有统计学意义（P〈0．05）。MMP-2的蛋白活性率（MMP-2／pro—MMP-2＋MMP-2）在Ⅰ～Ⅱ期、Ⅲ～Ⅳ期和胸腺癌各组中差异有统计学意义（P〈0．05）,在AB、B1型与B2、B3型以及胸腺癌各组中的差异均具有统计学意义（P〈0．05）。胸腺瘤各期及各型中MT1-MMP、TIMP-2mRNA与MMP-2蛋白活性表达均呈正相关,且相关程度相似（r=0．7235、r=0．7647、P〈0．005）。MMP-9的蛋白表达在各组间差异均无统计学意义。结论MMP-2、MT1-MMP及TIMP-2的mRNA表达与胸腺瘤临床分期、病理分型相关,MMP-2的活性与MT1-MMP和TIMP-2的表达升高正相关。推测MT1-MMP通过TIMP-2对MMP-2的激活起促进作用。     

胃癌MT1-MMP、RECK蛋白的表达及其意义

目的探讨胃癌组织中膜型基质金属蛋白酶一1（MT1一MMP）和RECK蛋白表达状况和两者之间的关系,及其表达与胃癌临床诊断之间的关系。方法采用免疫组织化学法（两步法）对胃癌切除标本进行研究。结果在44例胃癌标本中,有37（84．1％）例MT1-MMP表达呈阳性反应,31（70．5％）例RECK免疫组化为阳性。MT1-MMP表达与胃癌分化具有一定的关系,低分化组织标本中,MT1一MMP表达较多,而中、高分化组织标本中,MT1-MMP表达相对较弱,具有统计学意义（P=0．015）。胃癌中MT1-MMP表达与肿瘤细胞浸润深度相关（P=0．007）。但MT1-MMP表达与性别以及有无淋巴结转移之间未见统计学差异。与此相反,在胃癌标本中,高分化标本中RECK蛋白表达相对较多,中分化标本其次,在低分化标本中表达较少。RECK表达与肿瘤分化具有统计学意义（P：0．006）。RECK表达与性别、肿瘤细胞浸润深度以及有无淋巴结转移无关,且与MT1-MMP表达之间亦无统计学意义。结论MT1-MMP过表达在胃癌分化、转移中发挥着重要的作用,而RECK的表达有益于胃癌向高分化发展。     

Activation of pro-MMP-2 mediated by MT1-MMP in human salivary gland carcinomas: possible regulation of pro-MMP-2 activation by TIMP-2

Matrix metalloproteinases (MMPs), a family of extracellular matrix-degrading enzymes, are considered to play important roles in cancer invasion and metastasis. The present study examined the production levels of eight different MMPs (MMP-1, 2, 3, 7, 8, 9 and 13, and MT1-MMP) and two tissue inhibitors of metalloproteinases (TIMP-1 and 2) in homogenates of human salivary gland carcinomas [mucoepidermoid carcinomas (MECs), adenoid cystic carcinomas (ACCs), and adenocarcinomas (ADEs)] and non-neoplastic control salivary glands using sandwich enzyme immunoassay systems. The levels of MMP-1, MMP-2, MMP-13, MT1-MMP, and TIMP-1 were significantly higher in the carcinoma samples than in the controls (p < 0.05). Gelatin zymography demonstrated that the activation ratio of the MMP-2 zymogen (pro-MMP-2) was significantly higher in the carcinomas than in the controls (p < 0.05). In addition, the activation ratio in MECs was significantly higher than that in ACCs or ADEs (p < 0.01) and also correlated with histological grade and lymph node metastasis in MECs (p < 0.05), whereas the ratio showed no such correlation in ACCs or ADEs. Although the production levels of pro-MMP-2 and MT1-MMP were similar among these carcinoma groups, TIMP-2 levels were significantly higher in ACCs and ADEs than in MECs (p < 0.01). In carcinoma samples, the pro-MMP-2 activation ratio correlated directly with the MT1-MMP/TIMP-2 ratio (r = 0.736, n = 23; p < 0.01). Immunohistochemistry and in situ zymography demonstrated localization of MMP-2, MT1-MMP, and TIMP-2 to carcinoma cells, but only in MECs did carcinoma cell nests exhibit gelatinolytic activity, which was inhibited by 1,10-phenanthroline. These results suggest that enhanced activation of pro-MMP-2 mediated by MT1-MMP is implicated in the invasion and metastasis of MECs and that TIMP-2 may regulate pro-MMP-2 activation in salivary gland carcinomas.     

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM– counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7ADR), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM–, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such ÔfibroblastoidÕ carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.     

Differential expression patterns of MMPs and their role in the invasion of epithelial premalignant tumors and invasive cutaneous squamous cell carcinoma

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumors. MMP-2, MMP-9, TIMP-2, and MT1-MMP are thought to be involved in the process of destruction of basement membranes and stromal invasion by neoplastic epithelial cells. In this study, we investigated the expression and role of MMPs in cutaneous oncogenesis. Tissue microarray consisting of 62 squamous cell carcinomas (SCC), 32 Bowen's disease (BD) samples, 25 normal epidermis samples were obtained for the study. MMP-2,-9, MT1-MMP and TIMP-2 proteins were examined by immunohistochemical staining and mRNA level was detected by quantitative RT-PCR in fresh tissues consisting of 5 cutaneous SCCs and paired normal epidermis samples. Gelatinase activity of MMP-2 and MMP-9 was investigated by gelatin zymography and protein levels of MT1-MMP and TIMP-2 were measured by western blot in 2 human SCC cell lines. The invasive property was evaluated with invasion assays using Transwell filters. SCC exhibited significantly increased MMP-2, MT1-MMP and decreased TIMP-2 mRNA and protein expression compared to that of the normal epithelium. Immunohistochemical staining revealed that MT1-MMP was strongly expressed on the invasive front of SCCs, whereas BD exhibited higher expression around the dyskeratotic cells in the epithelium. In comparison with the expression observed in BD, SCC exhibited significantly increased MMP-2 expression. In addition, high MMP-2 and MT1-MMP expression and low TIMP-2 expression had a significant positive correlation with the invasiveness of SCC cell lines in vitro. Our results revealed significantly increased MT1-MMP and MMP-2 expression and decreased TIMP-2 expression in cutaneous SCC, and the expression correlated with the invasiveness of SCC cell lines. Therefore, the expression of these factors in cutaneous tumors may serve as an indicator of tumor aggressiveness and invasion.     

基质金属蛋白酶及其组织抑制因子在人前列腺组织及各种类型前列腺细胞中的表达

目的：研究基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）在人前列腺组织及各种类型细胞中的表达。方法： 用半定量RT－PCR的方法，对癌变和非癌变部分的前列腺组织、原代培养的平滑肌细胞、成纤维细胞、上皮细胞以及4种前列腺上皮细胞系（BPH－1、LNCaP、DU－145和PC－3）中MMP2、MMP7和MMP9、膜型基质金属蛋白酶1和3（MT1－MMP和MT3－MMP）及其组织抑制因子1和2（TIMP－1和TIMP－2）的mRNA 水平进行了测定。结果：MMP-2主要在前列腺基质细胞中表达；MMP-7和MMP-9则在前列腺上皮细胞中有较高的表达；MT1-MMP、MT3-MMP、TIMP-1和TIMP-2在前列腺基质细胞和上皮细胞中均有表达，但MT1-MMP和MT3-MMP在成纤维细胞中的表达量较高；另外，各种基质金属蛋白酶及其组织抑制因子在各种前列腺细胞系中也存在差异表达。结论： MMPs和TIMPs在前列腺组织及其各种类型细胞中的差异表达提示：它们可能在前列腺癌的转移中起着不同的作用。     

浸润性乳腺癌中基质金属蛋白酶-13蛋白的表达及其与其他相关因子和预后的关系

目的 探讨浸润性乳腺癌中基质金属蛋白酶(MMP)-13蛋白在肿瘤细胞和间质细胞中的表达及其与肿瘤临床病理、生物学指标以及预后的相关性.方法 采用组织芯片免疫组织化学染色sP法检测263例浸润性乳腺癌组织中MMP-13、ER、PR、HER2、MMP-2、MMP-9、基质金属蛋白酶组织抑制因子(TIMP)-1、TIMP-2蛋白的表达及表达间的相关性.结果 MMP-13在肿瘤细胞及间质纤维母细胞中均表达,肿瘤细胞中MMP-13过表达与淋巴结受累和高组织学分级相关(均P<0.01),且与HER2表达(P=0.015)和肿瘤细胞TIMP-1蛋白表达相关(P<0.01).MMP-13过表达与患者总生存期缩短相关,分层分析显示MMP-13的过表达分别与淋巴结阳性患者总生存期和HER2阳性和阴性表达状态下的患者总生存期有关.间质纤维母细胞表达MMP-13尽管与肿瘤细胞表达者有相关性,且与淋巴结状态和HER2表达也相关,但评估预后的价值较弱.经单因素分析,肿瘤直径、组织学分级、淋巴结和PR、HER2表达状态及肿瘤表达的TIMP-1和MMP-13均是有意义的预后指标;但多因素分析显示仅肿瘤直径、组织学分级、淋巴结状态、HER2表达、肿瘤中TIMP-1和MMP-13表达是独立预后因素.结论 MMP-13蛋白与浸润性乳腺癌的侵袭和转移相关,有可能作为预后不良的指标.     

The clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-9 according to their localization in invasive breast carcinoma

AIMS: To investigate the clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-9 proteins expression in invasive breast carcinoma and their relationship to tumour proliferation and expression of c-erbB2 and peroxisome proliferator-activated receptor (PPAR) gamma. METHODS: Immunohistochemistry was carried out on 175 paraffin-embedded breast tissue specimens to detect MT1-MMP, MMP-9, oestrogen receptor (ER), progesterone receptor, c-erbB-2, Ki67, topoisomerase IIalpha (topo IIalpha) and PPARgamma protein expression. RESULTS: Both MT1-MMP and MMP-9 were expressed in the cytoplasm of the malignant cells and the peritumoral stroma. Cytoplasmic MT1-MMP was more often observed in ER+ tumours (P = 0.022), of a lower nuclear grade (P = 0.020) and with reduced expression of Ki67 and topo IIalpha (P = 0.027 and P = 0.006, respectively). Moreover, cytoplasmic MT1-MMP was positively associated with MMP-9 (P = 0.010) and PPARgamma (P < 0.0001). Cytoplasmic MMP-9 was inversely associated with Ki67 (P = 0.034) and topo IIalpha (P = 0.004), whereas its relationship with MT1-MMP (P = 0.034) and PPARgamma (P = 0.024) was found to be positive. Stromal MMP-9 was more often observed in c-erbB2+ tumours (P = 0.043) and had an unfavourable impact on overall and relapse-free survival in both univariate (P = 0.0157 and P = 0.0274, respectively) and multivariate analyses (P = 0.007 and P = 0.024, respectively). CONCLUSIONS: Cytoplasmic MT1-MMP and MMP-9 seem to be related to well-differentiated tumours, with a low proliferation potential, while stromal MMP-9 is associated with an aggressive tumour phenotype and is recognized as an independent poor prognostic indicator.     

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.     

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.     

Expression of matrix metalloproteinase-2 (MMP-2) and of membrane-type-1-matrix metalloproteinase (MT1-MMP) in transitional cell carcinoma of the upper urinary tract

Tumor cell invasion and metastasis are biologically dependent on the proteolytic destruction of surrounding matrix components. Matrix metalloproteinase-2 (MMP-2) is able to cleave type IV collagen, and membrane-type-1-matrix metalloproteinase (MT1-MMP) induces activation of proMMP-2. We investigated the expression of MMP-2 and MT1-MMP using in situ hybridization and immunohistochemistry in 102 cases of transitional cell carcinoma of the upper urinary tract (TCC-UUT). A positive expression of each metalloproteinase was recognized in all samples and was apparent within the cytoplasm of tumor epithelial cells and/or stromal cells situated at the interface between tumor and stroma. Our analysis of clinicopathologic findings showed a relationship between MMP-2 and MT1-MMP expression and stage. The correlation between the MMP-2 protein staining score for tumor epithelial cells and overall survival rate reached significance in the univariate analysis. However, only stage was associated with disease-free and overall survivals in the multivariate analysis. In conclusion, the detection of MMP-2 and MT1-MMP would appear to be of limited value in informing the prognosis of TCC-UUT.