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Abstract
Epithelial-mesenchymal transition (EMT) is critical for carcinoma invasiveness and metastasis. To investigate the role of membrane-type-2 matrix metalloproteinase (MT2-MMP) in EMT, we generated lentiviral constructs of wild-type (WT) and an inactive Glu260Ala (E260A) mutant MT2-MMP and derived stably transfected HCT116 and A549 cell lines. WT-transfected cells appeared mesenchymal-like, whereas cells transfected with the E260A mutant were epithelial-like, as were cells treated with an MMP inhibitor (GM6001). Expression of E-cadherin, β-catenin, and zonula occludens-1 was lower in cells transfected with WT MT2-MMP compared to vector controls, cells treated with GM6001, or cells transfected with the E260A mutant. An 80-kD N-terminal fragment of E-cadherin was immunoprecipitated in conditioned medium from WT MT2-MMP cells, but not in the medium from vector controls, cells treated with GM6001, or E260A mutant cells. When endogenous expression of MT2-MMP in A2780 human ovarian cancer cells was inhibited using GM6001 or MT2-MMP-specific siRNA, levels of the 80-kD E-cadherin fragment in conditioned medium were decreased. Chick embryo chorioallantoic membrane invasion assays demonstrated that cells transfected with WT MT2-MMP were more invasive than cells transfected with control vector, treated with GM6001, or transfected with the E260A mutant. These results suggest that MT2-MMP degrades adherens and tight junction proteins and results in EMT, making it a potential mediator of EMT in carcinomas.
Highlights
Epithelial-mesenchymal transition (EMT) is the transition of immotile, polarized epithelial cells into highly motile, fibroblastoid-like cells
Expression of E-cadherin, β-catenin, and zonula occludens-1 was lower in cells transfected with WT membrane-type-2 matrix metalloproteinase (MT2-matrix metalloprotease (MMP)) compared to vector controls, cells treated with GM6001, or cells transfected with the E260A mutant
The human MT2-MMP open reading frame (ORF) was cloned into lentiviral vector GV287 at the AgeI/AgeI site, in order to encode a FLAG epitope-tagged form of human MT2MMP (Figure 1A)
Summary
Epithelial-mesenchymal transition (EMT) is the transition of immotile, polarized epithelial cells into highly motile, fibroblastoid-like cells. EMT is a critical step for the acquisition of an invasive and metastatic morphology in cancer cells and their ultimate dissemination and colonization in distant locations [1,2,3,4]. Epithelial cells are intimately associated with each other through specialized cell-cell contact structures, such as adherens junctions and tight junctions, which are absent in mesenchymal cells [5]. These contacts are disrupted during the process of cancer EMT. The cytoplasmic domain of E-cadherin associates directly with β-catenin and γ-catenin, which link to the actin cytoskeleton through α-catenin [1, 6]. In addition to transcriptional regulation, E-cadherin is reportedly cleaved by proteases, such as A disintegrin and metalloprotease (ADAM)-10 and -15 [9, 10], plasmin www.impactjournals.com/oncotarget [11], cathepsins B, L, and S [12], kallikreins-6 and -7 [13, 14], and matrix metalloprotease (MMP)-3 and -7 [15, 16]
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