The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. Here we investigate the functional role of EZH2 in cancer cell invasion and breast cancer progression. EZH2 transcript and protein were consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrated that EZH2 protein levels were strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorageindependent growth and cell invasion. EZH2-mediated cell invasion required an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation.
The physical limits of cell migration in dense porous environments are dependent upon the available space and the deformability of the nucleus and are modulated by matrix metalloproteinases, integrins and actomyosin function.
Cancer cells are able to proliferate at accelerated rates within the confines of a three-dimensional (3D) extracellular matrix (ECM) that is rich in type I collagen. The mechanisms used by tumor cells to circumvent endogenous antigrowth signals have yet to be clearly defined. We find that the matrix metalloproteinase, MT1-MMP, confers tumor cells with a distinct 3D growth advantage in vitro and in vivo. The replicative advantage conferred by MT1-MMP requires pericellular proteolysis of the ECM, as proliferation is fully suppressed when tumor cells are suspended in 3D gels of protease-resistant collagen. In the absence of proteolysis, tumor cells embedded in physiologically relevant ECM matrices are trapped in a compact, spherical configuration and unable to undergo changes in cell shape or cytoskeletal reorganization required for 3D growth. These observations identify MT1-MMP as a tumor-derived growth factor that regulates proliferation by controlling cell geometry within the confines of the 3D ECM.
Tissue invasion during metastasis requires cancer cells to negotiate a stromal environment dominated by cross-linked networks of type I collagen. Although cancer cells are known to use proteinases to sever collagen networks and thus ease their passage through these barriers, migration across extracellular matrices has also been reported to occur by protease-independent mechanisms, whereby cells squeeze through collagen-lined pores by adopting an ameboid phenotype. We investigate these alternate models of motility here and demonstrate that cancer cells have an absolute requirement for the membrane-anchored metalloproteinase MT1-MMP for invasion, and that protease-independent mechanisms of cell migration are only plausible when the collagen network is devoid of the covalent cross-links that characterize normal tissues.
As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity. Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM. Using fibroblasts isolated from gene-targeted mice, a matrix metalloproteinase (MMP)–dependent activity is identified that drives invasion independently of plasminogen, the gelatinase A/TIMP-2 axis, gelatinase B, collagenase-3, collagenase-2, or stromelysin-1. In contrast, deleting or suppressing expression of the membrane-tethered MMP, MT1-MMP, in fibroblasts or tumor cells results in a loss of collagenolytic and invasive activity in vitro or in vivo. Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity.
Accumulating evidence indicates that hyperactive Wnt signalling occurs in association with the development and progression of human breast cancer. As a consequence of engaging the canonical Wnt pathway, a beta-catenin-T-cell factor (TCF) transcriptional complex is generated, which has been postulated to trigger the epithelial-mesenchymal transition (EMT) that characterizes the tissue-invasive phenotype. However, the molecular mechanisms by which the beta-catenin-TCF complex induces EMT-like programmes remain undefined. Here, we demonstrate that canonical Wnt signalling engages tumour cell dedifferentiation and tissue-invasive activity through an Axin2-dependent pathway that stabilizes the Snail1 zinc-transcription factor, a key regulator of normal and neoplastic EMT programmes. Axin2 regulates EMT by acting as a nucleocytoplasmic chaperone for GSK3beta, the dominant kinase responsible for controlling Snail1 protein turnover and activity. As dysregulated Wnt signalling marks a diverse array of cancerous tissue types, the identification of a beta-catenin-TCF-regulated Axin2-GSK3beta-Snail1 axis provides new mechanistic insights into cancer-associated EMT programmes.
During angiogenesis, endothelial cells penetrate fibrin barriers via undefined proteolytic mechanisms. We demonstrate that the fibrinolytic plasminogen activator (PA)-plasminogen system is not required for this process, since tissues isolated from PA- or plasminogen-deficient mice successfully neovascularize fibrin gels. By contrast, neovessel formation, in vitro and in vivo, is dependent on fibrinolytic, endothelial cell-derived matrix metalloproteinases (MMP). MMPs directly regulate this process as invasion-incompetent cells penetrate fibrin barriers when transfected with the most potent fibrinolytic metalloproteinase identified in endothelium, membrane type-1 MMP (MT1-MMP). Membrane display of MT1-MMP is required, as invasion-incompetent cells expressing a fibrinolytically active, transmembrane-deleted form of MT1-MMP remain noninvasive. These observations identify a PA-independent fibrinolytic pathway wherein tethered MMPs function as pericellular fibrinolysins during the neovascularization process.
RESULTS Snail1 depletion ameliorates UUO-induced fibrosis Supplementary Fig. 1), impedes Snail1 expression in Cadherin-16 positive cells. Snail1 expression is silent in the adult kidney but it is reactivated after UUO in the cortex and in the medulla (Supplementary Fig. 1c). During renal development, Snail1 is a strong Cadh16 repressor 5 , and it is only when Snai1 is downregulated that renal cells express Cadherin-16 and epithelialization occurs. As Snail1 is silenced in the adult, preventing Snail1 activation in Cadherin-16 positive cells in the kidney does not have any impact in healthy mice. As expected, while Snail1 was highly reactivated in the kidney upon UUO, this was not the case in recombined renal epithelial cells in SFKC mice (Fig. 1a-c). We analyzed overall morphology, collagen deposition and expression of alpha smooth muscle actin ( -SMA) and vimentin 7 or 15 days after UUO and found that kidneys from SFKC mice were protected from the development of overt fibrosis, although signs of tubular distension resulting from the obstruction 5 were readily evident (Fig. 1d,e and Supplementary Fig. 2a,b). We confirmed the attenuation of fibrosis by analysis of the expression of epithelial and mesenchymal markers and this was particularly evident 2 weeks after UUO (Fig. 1f). Quantification of Sirius Red staining (Fig. 1g) showed a 35% reduction in fibrosis in the cortex of obstructed kidneys from SFKC mice when compared to obstructed kidneys from WT or control (Ksp1.3-Cre-only) mice ( Fig. 1g and Supplementary Fig. 3a,b). In our SFKC model, Snail1 reactivation was also prevented in the collecting ducts, and we observed a much better preserved morphology and lower collagen deposition in the medulla compared to WT animals ( Supplementary Fig. 3c) indicating that the overall protection from fibrotic degeneration is higher than that reflected by the quantification of SiriusRed in the cortex.We next assessed the contribution of epithelial cells to the interstitium after UOO in our system. We generated a mouse model harboring Cre-loxP mediated expression of the Tomato fluorescent protein driven by the Ksp 1.3 promoter (Ksp1.3-Cre; Rosa-LSL-TdTomato). These mice allow the visualization and fate mapping of renal epithelial cells. Notably, we could not detect tdTomato+ cells in the interstitium 7 days after obstruction ( Supplementary Fig. 4a) and found less than 1% after 15 days (Fig. 2a). One of those rare cells leaving the tubule is shown in Supplementary Fig. 4b. Thus, renal epithelial cells do not delaminate from the tubules to contribute to myofibroblasts or other interstitial cells, also in keeping with the observation that the total number of tubular cross sections did not differ between kidneys from WT or SFKC mice 15 days after UUO (Fig. 2b), indicative of tubular integrity. 6Snail1 expression was reactivated after UUO in over 80% of tubular cells and also in activated interstitial cells as reported in cancer models 16 (Fig. 2c).Although tubular cells did not significantly contribute to myofibroblasts, UUO pr...
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