Endometrial carcinoma is the most common malignant tumor of the female genital tract. It typically affects postmenopausal women and is increasingly frequent in many advanced countries, composing 6% of all malignancies in women in the U.S. and 4% in Australia.1, 2 The invasion of endometrial cancer cells through the myometrium and to nearby lymph nodes is a key factor related to poor prognosis and involves the escape of tumor cells from their site of origin, their penetration of blood or lymph vessel walls, and their subsequent growth at a new site. Metastatic spread requires the interaction of tumor cells with components of the extracellular matrix (ECM) and with other cells. Such interactions depend on factors from the cellular surface, such as surface bound proteolytic enzymes, growth factors, and growth factor receptors and cell adhesion molecules. The ECM is the main physical barrier to the movement of cells in each step of tumor progression. This comprises both the interstitial ECM, whose fibrillar framework is composed mainly of Types I, II and III collagens, and the basement membrane, comprising predominantly Type IV collagen along with laminin and nidogen.
The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases which participate in the degradation of collagens and other extracellular matrix macromolecules3 and are therefore implicated in tissue destruction in various pathologic conditions, including inflammation, tumor invasion, and metastasis. In addition, MMPs play a role in releasing and activating certain bioactive molecules, which in turn alter cell function.4, 5 Matrix metalloproteinases can be subgrouped based on substrate specificities and sequence characteristics into the stromelysins, collagenases, gelatinases, and the membrane type MMPs. Most MMPs are secreted as inactive zymogens (proMMPs) and are activated extracellularly; membrane-type MMPs are an exception, as they are anchored within the cellular membrane. The activation process is a key step for the action of the proteinases in vivo and generally occurs extracellularly. The tissue inhibitors of metalloproteinases (TIMPs) partly regulate MMP activity,6–8 which form high affinity, non-covalent 1:1 complexes with active forms of all MMPs.9 In addition, complexes are formed between specific TIMPs and the C-termini of progelatinases: TIMP-1 with proMMP-9 and TIMP-2 with proMMP-2.6, 10 It is now clear that, along with membrane type 1 (MT1)-MMP, TIMP-2 plays an important role in the activation of pro-MMP-2 in cell membranes.11 As it is associated with the insoluble ECM, TIMP-3 differs from the other family members.12
The enhanced expression of many MMPs has been correlated with the malignant phenotype of human cancer cells both in vivo and in vitro.13 In particular, the gelatinases are implicated with tumor invasion, as they degrade constituents of both the interstitial stroma and the subendothelial basement membrane matrix. Clinicopathologic studies have shown a correlation between the expression of MMP-2 and MMP-9 mRNA and the invasion and metastasis of human carcinomas, such as gastric,14 thyroid,15 breast,16 and ovarian carcinomas.17 Studies have shown MT1-MMP to play a significant role in the activation of proMMP-2 in breast18 and thyroid carcinomas.19 The immunoreactive enzyme proteins likewise have been shown in a wide variety of human carcinomas and to stroma or to tumor cells within these carcinomas,14, 16, 18, 20 the pattern and intensity of immunostaining varying with each carcinoma type. In many instances, immunostaining may reflect MMP binding to various ECM-bound molecules, such as integrins, fibronectin, and laminin.
Several studies have reported mRNA synthesis or the immunohistochemic localization of the gelatinases in the malignant endometrium. Such studies have localized MMP-2 and MMP-9 mRNA to stromal cells, including endothelial cells, macrophages, and fibroblasts, particularly in areas within or surrounding tumor aggregates,21, 22 and also, to varying degrees, to epithelial cells of tumor origin.21 Furthermore, the frequency of MMP-2 and MMP-9 expression increases with advancing histologic grade and with increasing depth of myometrial invasion.22 Studies on MMP immunolocalization in endometrial carcinoma are limited: matrilysin (MMP-7) was localized predominantly to carcinoma cells,19 MMP-2, MMP-1, and TIMP-1 proteins were correlated with histologic stage, depth of infiltration, histologic type and age,23 while MMP-9 proteins were mainly localized to tumor cells.24 Studies have localized TIMP-1 mRNA to the stromal compartment of the malignant endometrium, while TIMP-2, TIMP-3, and MT1-MMP mRNA have been localized to both the stromal and epithelial compartments.20 As in the case of the gelatinases, increased mRNA expression was observed in peripheral regions of the tumors and in relation to the lower differentiated tumors.20 However, to our knowledge, no studies have examined both the cellular site of synthesis (mRNA) and the location of the proteins for these enzymes in the same tissues. Neither is it known whether the gelatinases are active in endometrial carcinoma tissues.
In the current study we examined the cellular localization of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, and TIMP-3 proteins in human endometrial carcinomas of varying histologic grades by immunohistochemistry, along with MMP-2 and MMP-9 mRNA by in situ hybridization, and have shown correlations with increasing tumor grade and invasion. To our knowledge, we have shown for the first time the presence of active forms of gelatinases in endometrial carcinoma tissues.
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- MATERIALS AND METHODS
The current study has shown, firstly, that increasing MMP-9 and MMP-2 protein production in endometrial tumor cells is associated with advanced histologic grade and, secondly, that MMP-9 and MT1-MMP, but not MMP-2, are associated with the presence of myometrial invasion and with vascular/lymphatic invasion. A key determinant of invasive behavior is MMP activation. In situ zymography has shown, to our knowledge for the first time, that a proportion of MMPs detected in endometrial carcinoma tissues are in their active forms and that this activity is associated with tumor cells.
These data are overall in accord with other studies investigating MMPs in endometrial carcinoma. Simultaneous angiogenesis and over-expression of MMP-2 and MMP-9 mRNA have been shown during endometrial carcinoma progression. In particular, MMP-2 and MMP-9 detected by in situ hybridization were significantly over-expressed from histologic Grade 1 to Grade 3 carcinomas and with increasing depth of myometrial invasion.22 Inoue et al.23 showed that MMP-9 protein was associated with histologic Grade 3, vessel invasion, and lymph node metastasis. No previous studies have examined MT1-MMP protein in endometrial carcinomas.
Using immunohistochemistry, we have shown that the gelatinases are predominantly localized to tumor epithelial cells. However, some stromal staining was also observed at both the mRNA and protein levels, and this was identified in stromal fibroblasts, endothelial cells, and leukocytes. Matrix metalloproteinase-9 protein was particularly associated with tissue macrophages, in agreement with the study by Iurlaro et al.,22 who localized MMP-2 and MMP-9 mRNA to these stromal compartments. Therefore the regulation of tumor progression in endometrial carcinoma may involve several cell types, including both malignant and non-malignant cells in the tumor stroma.
In the current study, MT1-MMP protein was mainly localized to neoplastic epithelial cells, as also reported in lung and gastric carincomas.24, 25 The current data support previous studies showing that endometrial tumor cells are the main producers of MT1-MMP mRNA in endometrial carcinoma26 and that high MT1-MMP gene expression in primary endometrial carcinoma tissue correlates with MT1-MMP expression in metastatic lesions.27 We observed a correlation of MT1-MMP protein expression with both increasing depth of myometrial invasion and the presence of vascular/lymphatic invasion. Collectively, these studies suggest important roles for MT1-MMP in endometrial carcinoma progression. One likely role is the activation of proMMP-2, supported by the observation of MT1-MMP, MMP-2, and TIMP-2 co-localization. The presence of gelatinase activity at the cellular surface, as shown by in situ zymography, further supports this model. However, MT1-MMP also functions in degradation of ECM components.28, 29
The pattern of MMP expression is usually similar in different cases of the same type of cancer, while it varies between different types of cancer. For example, in neuroblastomas, MMP-9 is expressed by stromal cells,30 in tumors of the brain, it is expressed by malignant epithelium.31 In prostate carcinoma, MMP-2 is expressed by malignant epithelial cells only,32 while in breast carcinoma, MMP-2 is generally expressed ubiquitously by both epithelial and stromal cells.33 Tissue-specific differences in MMP expression may reflect local microenvironments, possibly in response to matrix components or from signals derived from an inflammatory reaction which often accompanies the establishment of a malignant tumor. In the case of endometrial carcinoma, the predominance of MMP-2, MMP-9, and MT1-MMP expression by epithelial cells may reflect the fact that these proteinases are expressed by this cell type at various stages of the normal menstrual cycle.34, 35
In general, MMPs are secreted in pro-enzyme form, which are then activated following limited proteolysis. However, over-production of pro-enzyme and generation of proteolytic activity are not equivalent. Therefore, the detection of active MMPs is critical. Active gelatinases have recently been detected in carcinoma cell nests of oral squamous cell carcinoma36 and in ovarian neoplasms37 using in situ zymography. Using this technique, we report the presence of active gelatinases in endometrial carcinoma. The current results show significant gelatinase activity even at the early stages of tumor progression (i.e., histologic Grades 1 and 2). It is likely that such early gelatinase activity results in local ECM breakdown in the endometrium, which is necessary for local tumor invasion. Significant gelatinase activity was also observed in malignant endometrium from tumors which had penetrated the myometrium and invaded the lymph nodes.
The regulation of MMP expression can be linked to a number of inducing agents, including growth factors, cytokines, oncogenes, and ECM-derived signals.38 As well as ECM degradation, MMPs process and release a variety of molecules, which are regulators of vascular growth or function, including fibroblast growth factor receptor type I,39 tumor necrosis factor-α,40 and heparin-binding-epidermal growth factor.41 Thus the metalloproteinases are not limited to simply degrading structural proteins that surround the cell, but have a more generalized role in the interactions of cells with their matrix environment, affecting basic cellular processes such as differentiation, proliferation and apoptosis.
Matrix metalloproteinases are not only regulated at the level of gene transcription and activation but are also regulated locally by the TIMPs. An imbalance between MMPs and TIMPs is linked to the degradation of the extracellular matrix in tumor progression. Using immunohistochemistry we localized TIMP-1 to the stromal compartment in all grades of endometrial carcinoma, with variable tumor epithelial cell localization in Grades 2 and 3 carcinomas only. Tumor cells from all histologic grades stained intensely for TIMP-2 and –3, with varying degrees of stromal staining also observed. These data partially support studies of Määtta et al.,26 who found TIMPs-2 and –3 mRNA in tumor epithelial cells, with the latter also being expressed by stromal and endothelial cells. They also found TIMP-1 mRNA localized to stromal and endothelial cells with no clear epithelial cell expression.25 The TIMPs are expressed in most tissues, including the non-malignant endometrium, where high levels of TIMPs 1-3 presumably maintain tissue integrity and ECM homeostasis.42 However, TIMPs have additional functions in cellular proliferation43, 44 and cell survival pathways,45 with TIMPs-1 and –2 having specific roles in controlling the activations of proMMP-9 and proMMP-2 respectively.46–48
In conclusion, MMPs, along with TIMPs, are likely to play a complex role in the progression of endometrial carcinoma. Semiquantitative analysis has shown correlations between MMP-2, MMP-9, and MT1-MMP immunoreactive protein and clinicopathologic features of endometrial carcinoma, such as the presence of myometrial invasion, vascular/lymphatic invasion, and increasing histologic grade. Most importantly, active gelatinases are present, which would facilitate tumor invasion and subsequent metastasis. In order to fully understand the complex roles that MMPs play in endometrial carcinoma, further questions need to be addressed, including the precise mechanisms of MMP regulation/activation and the interactions of MMPs/TIMPs with other molecules.