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.

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.

Down-regulation of E-cadherin marks the initiation of the epithelial-mesenchymal transition, a process exploited by invasive cancer cells. The zinc finger transcription factor, Snail, functions as a potent repressor of E-cadherin expression that can, acting alone or in concert with the Wnt/␤-catenin/T cell factor axis, induce an epithelial-mesenchymal transition. Although mechanisms that coordinate signaling events initiated by Snail and Wnt remain undefined, we demonstrate that Snail displays ␤-catenin-like canonical motifs that support its GSK3␤-dependent phosphorylation, ␤-TrCPdirected ubiquitination, and proteasomal degradation. Accordingly, Wnt signaling inhibits Snail phosphorylation and consequently increases Snail protein levels and activity while driving an in vivo epithelial-mesenchymal transition that is suppressed following Snail knockdown. These findings define a potential mechanism whereby Wnt signaling stabilizes Snail and ␤-catenin proteins in tandem fashion so as to cooperatively engage transcriptional programs that control an epithelial-mesenchymal transition.

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

If left untreated, a subset of high-grade squamous intraepithelial lesions (HSIL) of the cervix will progress to invasive squamous cell carcinomas (SCC). To identify genes whose differential expression is linked to cervical cancer progression, we compared gene expression in microdissected squamous epithelial samples from 10 normal cervices, 7 HSILs, and 21 SCCs using high-density oligonucleotide microarrays. We identified 171 distinct genes at least 1.5-fold up-regulated (and P < 0.001) in the SCCs relative to HSILs and normal cervix samples. Differential expression of a subset of these genes was confirmed by quantitative reverse transcription-PCR and immunohistochemical staining of cervical tissue samples. One of the genes up-regulated during progression, HOXC10, was selected for functional studies aimed at assessing its role in mediating invasive behavior of neoplastic squamous epithelial cells. Elevated HOXC10 expression was associated with increased invasiveness of human papillomavirus-immortalized keratinocytes and cervical cancer-derived cell lines in both in vitro and in vivo assays. Cervical cancer cells with high endogenous levels of HOXC10 were less invasive after short hairpin RNA-mediated knockdown of HOXC10 expression. Our findings support a key role for the HOXC10 homeobox protein in cervical cancer progression. Other genes with differential expression in invasive SCC versus HSIL may contribute to tumor progression or may be useful as markers for cancer diagnosis or progression risk.

EMT ͉ extracellular matrix ͉ Snail T he transition of carcinoma in situ to a frankly carcinomatous lesion requires the cancer cells to acquire an ability to perforate and transmigrate the underlying basement membrane (BM), a specialized form of extracellular matrix (ECM) that subtends all epithelial cells (1, 2). Current evidence suggests that the induction of a BM-invasive phenotype may be linked to the expression of zinc-finger transcriptional repressors capable of promoting an epithelial-mesenchymal cell transition (EMT) which trigger epithelial cell-derived cancer cells to adopt a tissue-invasive, mesenchymal cell-like phenotype (2-4). Snail1 is a prototypical member of a family of EMT-inducing transcription factors, playing a required role in developmental programs, such as gastrulation, and capable of undergoing pathologic re-activation postnatally in neoplastic states (5). While Snail1 has been linked to cancer cell invasion programs, cancer recurrence, and the adoption of cancer stem cell-like properties (2, 5), the mechanisms by which the transcription factor induces BM degradation and invasion programs remain undefined.Recent efforts to delineate normal or neoplastic cell interactions with the intact BM in an in vivo setting have been limited largely to model organisms where changes in BM structure during invasive processes can be evaluated directly by microscopic imaging (6, 7). In vertebrate systems, experimental models are unavailable where carcinoma cells can be situated atop linear, unbroken stretches of BM and invasion monitored in a fashion that lends itself to molecular characterization in vivo. Herein, we have adopted a live chick chorioallantoic membrane (CAM) model to analyze the cancer cell-BM interactions that underlie the earliest steps in the carcinoma invasion program (3, 4, 8, 9). These studies demonstrate that Snail1 induces cancer cells to transmigrate BM barriers by mobilizing the membrane-type matrix metalloproteinases (MTMMPs), MT1-MMP and MT2-MMP (1). Working in tandem, these MT-MMP family members not only confer carcinoma cells with the ability to perforate BM structures in vivo, but also to trigger angiogenesis, cancer cell proliferation and dissemination of the transformed cells to distant sites through the host vasculature. These findings suggest that Snail1, and perhaps all EMT-inducing transcription factors, mobilize MT1-MMP and/or MT2-MMP as necessary co-factors during tumor progression. ResultsSnail1-Induced BM Degradation and Transmigration by Breast Carcinoma Cells. To define cancer cell-BM interactions in an in vivo setting, neoplastic cell populations were cultured atop the CAM, an extra-embryonic tissue consisting of a chorionic epithelium of ectodermal origin, an intermediate mesenchyme and an endodermal allantoic epithelium (Fig. 1A) (10). The upper chorionic epithelium is heavily vascularized by BM-encased capillaries and subtended by a continuous epithelial-derived BM that demarcates the epithelium from the underlying mesenchyme (Fig.

Protein O-phosphorylation often occurs reciprocally with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by glycogen synthase kinase-3b on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O-phosphorylation-mediated degradation, O-GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E-cadherin mRNA expression. Hyperglycaemic condition enhances O-GlcNAc modification and initiates EMT by transcriptional suppression of E-cadherin through Snail1. Thus, dynamic reciprocal O-phosphorylation and OGlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.