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  • Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1 Ichiro Otaa,b, Xiao-Yan Lia, Yuexian Hua, and Stephen J. Weissa,1

    aDivision of Molecular Medicine and Genetics, Department of Internal Medicine, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; and bDepartment of Otolaryngology, Nara Medical University, Nara 634-8522, Japan

    Communicated by David Ginsburg, University of Michigan Medical School, Ann Arbor, MI, September 25, 2009 (received for review June 8, 2009)

    The ability of carcinoma cells arising at primary sites to cross their underlying basement membrane (BM), a specialized form of extra- cellular matrix that subtends all epithelial cells, and to access the host vasculature are central features of the malignant phenotype. The initiation of the invasive phenotype has been linked to the aberrant expression of zinc-finger transcriptional repressors, like Snail1, which act by triggering an epithelial-mesenchymal cell-like transformation (EMT-like) via the regulation of largely undefined, downstream ef- fectors. Herein, we find that Snail1 induces cancer cells to (i) degrade and perforate BM barriers, (ii) initiate angiogenesis, and (iii) and intravasate vascular networks in vivo via a matrix metalloproteinase (MMP)-dependent process. Unexpectedly, the complete Snail1 inva- sion program can be recapitulated by expressing directly either of the membrane-anchored metalloproteinases, MT1-MMP or MT2-MMP. The pro-invasive, angiogenic, and metastatic activities of MT1-MMP and MT2-MMP are unique relative to all other metalloproteinase family members and cannot be mimicked in vivo by the secreted MMPs, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, or MMP-13. Further, siRNA-specific silencing of MT1-MMP and MT2-MMP ablates com- pletely the ability of Snail1 to drive cancer cell BM invasion, induce angiogenesis, or trigger intravasation. Taken together, these data demonstrate that MT1-MMP and MT2-MMP cooperatively function as direct-acting, pro-invasive factors that confer Snail1-triggered cells with the key activities most frequently linked to morbidity and mortality in cancer.

    EMT � extracellular matrix � Snail

    The transition of carcinoma in situ to a frankly carcinomatouslesion 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 epithe- lial 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 transcrip- tion 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 (MT- MMPs), 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.

    Results Snail1-Induced BM Degradation and Transmigration by Breast Carci- noma 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 mesen- chyme (Fig. 1 A) (10). Immunohistochemical staining with chick type IV collagen-specific antibodies clearly identifies (i) capil- lary BMs interspersed within the chorionic epithelium, (ii) the upper chorionic and lower allantoic epithelial BMs, and (iii) the vascular BMs surrounding arterioles and venules that traverse the CAM mesenchyme (Fig. 1 A).

    When f luorescently labeled human MCF-7 breast carcinoma cells, a well-differentiated cancer cell line that displays mini- mal invasive activity in mouse model systems (3, 4), are cultured atop the live CAM surface, the underlying epithelial BM remains intact and no invasive activity can be discerned (Fig. 1). By contrast, when MCF-7 cells are induced to express Snail1 (i.e., MCF-7Snail1 cells), the epithelial BM is destroyed and the CAM mesenchyme is widely infiltrated by the tumor cells (Fig. 1). Interestingly, Snail1-dependent MCF-7 cell invasion occurs in tandem with a burst in chick angiogenic activity as well as cancer cell intravasation of the chick vasculature (see below). Consistent with a major role for one or more MMP family members, BM degradation and invasion by MCF-7Snail1 cells is abrogated completely in the presence of the pan-specific MMP inhibitors, BB-2516 or GM6001 (Fig. 1) (1). The ability of MCF-7Snail1 cells to degrade and transmi-

    Author contributions: I.O. and S.J.W. designed research; I.O., X.-Y.L., and Y.H. performed research; I.O. and X.-Y.L. analyzed data; and S.J.W. wrote the paper.

    The authors declare no conflict of interest.

    1To whom correspondence should be addressed. E-mail: sjweiss@umich.edu.

    This article contains supporting information online at www.pnas.org/cgi/content/full/ 0910962106/DCSupplemental.

    20318–20323 � PNAS � December 1, 2009 � vol. 106 � no. 48 www.pnas.org�cgi�doi�10.1073�pnas.0910962106

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    http://www.pnas.org/cgi/content/full/0910962106/DCSupplemental http://www.pnas.org/cgi/content/full/0910962106/DCSupplemental

  • grate the CAM BM is similar to that observed by MDA-MB- 231 carcinoma cells, an undifferentiated and highly invasive human breast cancer cells whose invasive activity is similarly blocked by MMP inhibitors (Fig. 1) (11, 12).

    MT-MMP Family Members Confer MCF-7 Cells with Pro-Invasive and Angiogenic Activities. The human MMP family is comprised of 23 members that include 15 secreted enzymes and eight membrane- anchored proteases (i.e., four type I transmembrane, one type II transmembrane, and two GPI-anchored enzymes) (1). While mul- tiple MMPs have been characterized as expressing BM-degradative activity in various model systems in vitro (1), only 3 membrane- anchored MMPs, MT1-MMP, MT2-MMP, and MT3-MMP, have been shown to confer recipient cells with the ability to degrade or invade native BM barriers ex vivo (13). To first determine the ability of MT1-MMP, MT2-MMP, or MT3-MMP to confer MCF-7 cells with BM-degradative activity in vivo, the breast carcinoma cells were engineered to express the respective MT-MMPs and cultured atop live CAMs. As shown in Fig. 2, MT1-MMP- as well as MT2-MMP- expressing MCF-7 cells are not only able to degrade the underlying BM, but also to infiltrate the underlying mesen- chyme while simultaneously triggering significant increases in an- giogenesis as well as tumor cell proliferation via an MMP inhibitor- sensitive process (Fig. 2 A–D). Furthermore, although control MCF-7 cells are unable to intravasate the chick vasculature as determined by monitoring the lower CAM for human-specific Alu sequences (14), MT1-MMP- as well as MT2-MMP-expressing cells successfully access the host vascular bed (Fig. S1 ). As expected, the ability of MT1-MMP or MT2-MMP to trigger MCF-7 intravasation is likewise inhibited completely by BB-2516 (Fig. S1). Although recent studies have reported that MT1-MMP can modify cell behavior independently of its catalytic activity by initiating signaling cascades dependent upon the MT1-MMP cytosolic tail (15–18), MCF-7 cells expressing a catalytically-inactive MT1-MMP mutant lose all BM-degradative and invasive activities and no longer support increased rates of angiogenesis, proliferation or intravasa- tion (Fig. 2 A–D and Fig. S1). Further, MCF-7 cells engineered to express a cytosolic tail-deleted form of MT1-MMP retain full BM-degradative, angiogenic, and proliferative activity in vivo while promoting intravasation as well (Fig. 2 A–D and Fig. S1). The ability of MT1-MMP or MT2-MMP to modulate MCF-7 cell be