6
Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1 Ichiro Ota a,b , Xiao-Yan Li a , Yuexian Hu a , and Stephen J. Weiss a,1 a Division of Molecular Medicine and Genetics, Department of Internal Medicine, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; and b Department 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 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 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 fluorescently 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-7 Snail1 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-7 Snail1 cells is abrogated completely in the presence of the pan-specific MMP inhibitors, BB-2516 or GM6001 (Fig. 1) (1). The ability of MCF-7 Snail1 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. 1 To whom correspondence should be addressed. E-mail: [email protected]. 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.orgcgidoi10.1073pnas.0910962106 Downloaded by guest on November 1, 2020

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Page 1: Induction of a MT1-MMP and MT2-MMP-dependent basement ...Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1 Ichiro Otaa,b,

Induction of a MT1-MMP and MT2-MMP-dependentbasement membrane transmigration programin cancer cells by Snail1Ichiro 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 theirunderlying basement membrane (BM), a specialized form of extra-cellular matrix that subtends all epithelial cells, and to access the hostvasculature are central features of the malignant phenotype. Theinitiation of the invasive phenotype has been linked to the aberrantexpression of zinc-finger transcriptional repressors, like Snail1, whichact 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) degradeand perforate BM barriers, (ii) initiate angiogenesis, and (iii) andintravasate 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 themembrane-anchored metalloproteinases, MT1-MMP or MT2-MMP.The pro-invasive, angiogenic, and metastatic activities of MT1-MMPand MT2-MMP are unique relative to all other metalloproteinasefamily members and cannot be mimicked in vivo by the secretedMMPs, 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, induceangiogenesis, or trigger intravasation. Taken together, these datademonstrate that MT1-MMP and MT2-MMP cooperatively function asdirect-acting, pro-invasive factors that confer Snail1-triggered cellswith the key activities most frequently linked to morbidity andmortality 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), aspecialized form of extracellular matrix (ECM) that subtends allepithelial cells (1, 2). Current evidence suggests that the inductionof a BM-invasive phenotype may be linked to the expression ofzinc-finger transcriptional repressors capable of promoting anepithelial-mesenchymal cell transition (EMT) which trigger epithe-lial cell-derived cancer cells to adopt a tissue-invasive, mesenchymalcell-like phenotype (2–4). Snail1 is a prototypical member of afamily of EMT-inducing transcription factors, playing a requiredrole in developmental programs, such as gastrulation, and capableof undergoing pathologic re-activation postnatally in neoplasticstates (5). While Snail1 has been linked to cancer cell invasionprograms, cancer recurrence, and the adoption of cancer stemcell-like properties (2, 5), the mechanisms by which the transcrip-tion factor induces BM degradation and invasion programs remainundefined.

Recent efforts to delineate normal or neoplastic cell interactionswith the intact BM in an in vivo setting have been limited largelyto model organisms where changes in BM structure during invasiveprocesses can be evaluated directly by microscopic imaging (6, 7).In vertebrate systems, experimental models are unavailable wherecarcinoma cells can be situated atop linear, unbroken stretches ofBM and invasion monitored in a fashion that lends itself tomolecular characterization in vivo. Herein, we have adopted a live

chick chorioallantoic membrane (CAM) model to analyze thecancer cell-BM interactions that underlie the earliest steps in thecarcinoma invasion program (3, 4, 8, 9). These studies demonstratethat Snail1 induces cancer cells to transmigrate BM barriers bymobilizing the membrane-type matrix metalloproteinases (MT-MMPs), MT1-MMP and MT2-MMP (1). Working in tandem, theseMT-MMP family members not only confer carcinoma cells with theability to perforate BM structures in vivo, but also to triggerangiogenesis, cancer cell proliferation and dissemination of thetransformed cells to distant sites through the host vasculature.These findings suggest that Snail1, and perhaps all EMT-inducingtranscription factors, mobilize MT1-MMP and/or MT2-MMP asnecessary co-factors during tumor progression.

ResultsSnail1-Induced BM Degradation and Transmigration by Breast Carci-noma Cells. To define cancer cell-BM interactions in an in vivosetting, neoplastic cell populations were cultured atop the CAM,an extra-embryonic tissue consisting of a chorionic epithelium ofectodermal origin, an intermediate mesenchyme and anendodermal allantoic epithelium (Fig. 1A) (10). The upperchorionic epithelium is heavily vascularized by BM-encasedcapillaries and subtended by a continuous epithelial-derived BMthat demarcates the epithelium from the underlying mesen-chyme (Fig. 1 A) (10). Immunohistochemical staining with chicktype IV collagen-specific antibodies clearly identifies (i) capil-lary BMs interspersed within the chorionic epithelium, (ii) theupper chorionic and lower allantoic epithelial BMs, and (iii) thevascular BMs surrounding arterioles and venules that traversethe CAM mesenchyme (Fig. 1 A).

When f luorescently labeled human MCF-7 breast carcinomacells, a well-differentiated cancer cell line that displays mini-mal invasive activity in mouse model systems (3, 4), arecultured atop the live CAM surface, the underlying epithelialBM remains intact and no invasive activity can be discerned(Fig. 1). By contrast, when MCF-7 cells are induced to expressSnail1 (i.e., MCF-7Snail1 cells), the epithelial BM is destroyedand the CAM mesenchyme is widely infiltrated by the tumorcells (Fig. 1). Interestingly, Snail1-dependent MCF-7 cellinvasion occurs in tandem with a burst in chick angiogenicactivity as well as cancer cell intravasation of the chickvasculature (see below). Consistent with a major role for oneor more MMP family members, BM degradation and invasionby MCF-7Snail1 cells is abrogated completely in the presence ofthe 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. performedresearch; 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: [email protected].

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

MT-MMP Family Members Confer MCF-7 Cells with Pro-Invasive andAngiogenic Activities. The human MMP family is comprised of 23members that include 15 secreted enzymes and eight membrane-anchored proteases (i.e., four type I transmembrane, one type IItransmembrane, and two GPI-anchored enzymes) (1). While mul-tiple MMPs have been characterized as expressing BM-degradativeactivity in various model systems in vitro (1), only 3 membrane-anchored MMPs, MT1-MMP, MT2-MMP, and MT3-MMP, havebeen shown to confer recipient cells with the ability to degrade orinvade native BM barriers ex vivo (13). To first determine the abilityof MT1-MMP, MT2-MMP, or MT3-MMP to confer MCF-7 cellswith BM-degradative activity in vivo, the breast carcinoma cellswere engineered to express the respective MT-MMPs and culturedatop live CAMs. As shown in Fig. 2, MT1-MMP- as well asMT2-MMP- expressing MCF-7 cells are not only able to degradethe 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 controlMCF-7 cells are unable to intravasate the chick vasculature asdetermined by monitoring the lower CAM for human-specific Alusequences (14), MT1-MMP- as well as MT2-MMP-expressing cellssuccessfully access the host vascular bed (Fig. S1 ). As expected, theability of MT1-MMP or MT2-MMP to trigger MCF-7 intravasationis likewise inhibited completely by BB-2516 (Fig. S1). Althoughrecent studies have reported that MT1-MMP can modify cellbehavior independently of its catalytic activity by initiating signalingcascades dependent upon the MT1-MMP cytosolic tail (15–18),MCF-7 cells expressing a catalytically-inactive MT1-MMP mutantlose all BM-degradative and invasive activities and no longersupport increased rates of angiogenesis, proliferation or intravasa-tion (Fig. 2 A–D and Fig. S1). Further, MCF-7 cells engineered toexpress a cytosolic tail-deleted form of MT1-MMP retain fullBM-degradative, angiogenic, and proliferative activity in vivo whilepromoting intravasation as well (Fig. 2 A–D and Fig. S1). The abilityof MT1-MMP or MT2-MMP to modulate MCF-7 cell behavior is,

however, completely lost when the protease is expressed in anactive, but secreted, form as a transmembrane-deleted mutant (Fig.2 A–D). Finally, while MT3-MMP has been demonstrated previ-ously to slowly remodel acellular BM sheets in vitro (13), theprotease is unable to confer MCF-7 cells with BM remodelingactivity in vivo, or any of the other ancillary activities associatedwith MT1-MMP or MT2-MMP activity despite similar levels ofexpression at the cell surface (Fig. 2 A–D).

Secreted MMPs Fail to Support MCF-7 Cell Invasion, Proliferation,Angiogenesis, or Intravasation in Vivo. A series of secreted MMPs,including MMP-1 (collagenase-1), MMP-2 (gelatinase A), MMP-3(stromelysin-1), MMP-7 (matrilysin), and MMP-9 (gelatinase B)have been postulated to alter the invasive properties of cancer cellsby virtue of their ability to either digest extracellular matrix com-ponents, cleave cell surface-associated targets, hydrolyze solublesubstrates or induce EMT programs (1, 19). However, in directcontrast with MT1-MMP or MT2-MMP, MMP-1, MMP-2,MMP-9, and MMP-7 do not affect MCF-7 cell invasion, angiogenicactivity, or proliferation in vivo (Fig. 3 A–C). Similarly, althoughMMP-3 has been reported to induce EMT-like changes in breastcarcinoma cells via a Snail1-dependent process (20, 21), the pro-tease is unable to induce BM degradative, invasive, proliferative orpro-angiogenic activity in MCF-7 cells (Fig. 3 A–C). Because eachof these MMPs are synthesized and secreted as zymogens, and theenzymatic activities of the proteases cannot be readily monitored inthe complex, in vivo setting, the functional status of the secretedproteases remains at issue. Nevertheless, when MMP-2, -9, -1, -3, or-7 are purposely expressed in MCF-7 cells as RXKR chimeras (R �Arg, X � any amino acid, K � Lys) that undergo efficientintracellular processing to their active forms within the trans-Golginetwork by proprotein convertase family members before theirextracellular secretion (13, 22), the engineered tumor cells continueto express the indolent activity characteristic of the parental cellpopulation (Fig. S2). Interestingly, MMP-13, expressed either as thewild-type or RXKR mutant, is able to trigger significant BMdegradation (Fig. S2). MMP-13-dependent BM remodeling did not,however, induce MCF-7 invasion, proliferation or angiogenesis(Fig. S2). Apparently, BM degradation alone is not sufficient totrigger the entire repertoire of activities associated with MT1-MMP

Fig. 1. Cancer cell-BM interactions in vivo. (A) CAMstructure and matrix composition of the developingchick egg at day 11 as assessed in hematoxylin andeosin (H&E)-stained cross-section (left panel). Immu-nofluorescent micrographs of the CAM reveal type IVcollagen staining of the ectodermal/endodermal BMs,intraepithelial capillaries (arrowheads) and interstitialblood vessels (right panel). (Scale bar, 10 �m.) (B) CAMinvasion by human cancer cells. Human breast cancerMCF-7MOCK, MCF-7Snail1 or MDA-MB-231 cells [labeledwith fluorescent nanospheres (green) or engineeredto express GFP] were inoculated atop the CAM of11-day-old chicks with or without doxycycline and cul-tured for 3 days in the absence or presence of the MMPinhibitors, BB-2516 or GM6001. Fluorescent micro-graphs of CAM cross-sections demonstrate the abilityof MCF-7Snail1 or MDA-MB-cells to perforate the CAMsurface through the chorionic epithelial BM (arrowhead). Red, type IV collagen; blue, DAPI staining forcell nuclei. Results are representative of four or moreexperiments performed. (Scale bar, 50 �m.)

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or MT2-MMP activity as the tumor cells must also traffic throughthe interstitial collagen-rich CAM mesenchyme (11, 23).

Tandem Expression of MT1-MMP and MT2-MMP Regulates the MDA-MB-231 Invasive Phenotype. MDA-MB-231 cells express tissue-invasive activity in vivo that is largely dependent on Snail1 (4, 24).Consistent with the potential existence of a Snail1-MT-MMP axis,both the BM-invasive and pro-angiogenic activities of MDA-MB-231 cells correlate with their ability to express MT1-MMP as wellas MT2-MMP (Fig. 3 A and B). To determine the degree to whichMDA-MB-231 phenotype is linked to endogenous MT1-MMP orMT2-MMP activity, each of the proteases was silenced alone or incombination. Following knockdown of either MT1-MMP or MT2-MMP alone, MDA-MB-231 cells continue to degrade the under-lying BM although invasive activity is attenuated significantly (Fig.3 A and B). Despite the decrease in MDA-MB-231 invasion into thechick mesenchyme, pro-angiogenic activity and carcinoma cellproliferation continue in an unabated fashion (Fig. 3 C and D). Bycontrast, when MT1-MMP and MT2-MMP are both silenced, theBM remains intact, and invasion is inhibited significantly in tandemwith comparable decreases in angiogenesis, proliferation and in-travasation (Fig. 3 A–E and Fig. S3). Consistent with the absenceof any siRNA-dependent off-target effects on cell function, theability of MT1-MMP/MT2-MMP-silenced MDA-MB-231 cells todegrade the underlying BM, invade the CAM mesenchyme, pro-liferate, trigger angiogenic activity and intravasate is rescued fol-lowing the expression of mouse orthologues of MT1-MMP orMT2-MMP that escape siRNA-mediated silencing (Fig. 3 A–E andFig. S3). While MDA-MB-231 cells also express MMP-1 andMMP-9, secreted MMPs whose expression has previously beenlinked indirectly with carcinoma cell invasion or pro-angiogenicactivities (25, 26), neither MMP-1 nor MMP-9 silencing affects BM

transmigration, intravasation, or angiogenic responses (Fig. 3 A–Eand Fig. S3).

Snail1 Triggers an MT1-MMP/MT2-MMP-Dependent Invasion Program.To characterize directly the role of MT1-MMP and/or MT2-MMPin regulating MCF-7Snail1 behavior, the expression of each of themembrane-anchored MMPs was examined in vivo following doxy-cycline induction of the invasive phenotype. Coincident with theability of MCF-7Snail1 cells to degrade and transmigrate the BM aswell as trigger angiogenesis and tumor cell intravasation, bothMT1-MMP and MT2-MMP expression are upregulated in responseto Snail1 induction (Fig. 4 A–E). Following silencing of eitherMT1-MMP or MT2-MMP in MCF-7Snail1 cells, invasion as well asangiogenesis is inhibited significantly, but not completely (Fig. 4A–C). However, following the tandem knockdown of MT1-MMPand MT2-MMP, the ability of MCF-7Snail1 cells to degrade the chickBM, mount an invasive response, stimulate angiogenesis or intra-vasate the host vasculature is blocked (Fig. 4 A–E). Under theseconditions, changes in apoptosis are not observed (Fig. S4). Asexpected, the inability of MT1/MT2-MMP-silenced MCF-7Snail1cells to mount a BM-invasive program is rescued following expres-sion of either mouse MT1-MMP or MT2-MMP (Fig. 4 A–E).Hence, the ability of Snail1 to induce an aggressive carcinomacell-like phenotype characterized by BM invasion, pro-angiogenicactivity, and intravasation is mediated by the combined expressionof MT1-MMP and MT2-MMP.

DiscussionThe first structural barrier encountered by invasive carcinoma cellsis the BM, a 100- to 300-nm thick ECM composite dominated byan interwoven network of type IV collagen fibrils (1, 27). DuringEMT, normal as well as neoplastic cells exhibit an ability toperforate the underlying BM by largely uncharacterized mecha-

Fig. 2. MT1-MMP and MT2-MMP confer MCF-7 cellswith tissue-invasive and pro-angiogenic activities. (A)MCF-7 cells transfected with control, wild-type or mu-tant MT1-MMP, MT2-MMP, or MT3-MMP constructs asindicated were labeled with fluorescent nanospheres(green), inoculated atop the CAM of 11-day-old chicksand cultured for 3 days in the absence or presence ofBB-2516. Cross-sections were prepared and examined byfluorescentmicroscopy.Red, type IVcollagen;blue,DAPIstaining for cell nuclei. (Scale bar, 50 �m.) (B–D) MCF-7cells transfected with wild-type or mutant MT1-MMP,MT2-MMP, or MT3-MMP constructs were inoculatedatop the CAM of 11-day-old chicks and cultured as de-scribed above for 3 days. Invasion, angiogenic activityand MCF-7 cell proliferative responses were monitoredas described in Methods. Results are expressed as themean � 1 SD of three or more experiments. (Scale bar, 50�m.)

20320 � www.pnas.org�cgi�doi�10.1073�pnas.0910962106 Ota et al.

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nisms (1, 13). Whereas well-differentiated carcinoma cell lines thatretain epithelial characteristics, such as MCF-7 cells, do not displayBM-invasive activity, Snail1 induces these cells to assume anaggressive phenotype characterized by BM remodeling, transmi-gration, angiogenesis and intravasation. Furthermore, MCF-7Snail1

cells display carcinomatous properties similar, if not identical, tothose exhibited by MDA-MB-231 cells, a de-differentiated breastcancer cell line known to express high levels of Snail1 as well asother EMT-inducing transcription factors, including ZEB1 (4, 24,28, 29). Importantly, both MCF-7Snail1 and MDA-MB-231 cellsdegrade and invade the chick BM by a process that was inhibitedcompletely by synthetic MMP inhibitors, raising the possibility thatcancer- or chick-cell-derived MMPs, acting alone or in combina-tion, drive tissue-invasive programs.

Multiple MMP family members are thought to participate in BMremodeling, but the ability of these enzymes to confer cancer cellswith BM-degradative or BM-invasive activity in an in vivo settingremains controversial and has not been examined previously (1). AsMT1-MMP, MT2-MMP, and MT3-MMP have recently beenshown to degrade acellular sheets of intact BM recovered from

animal or human tissues (13), we first sought to determine theimpact of MT-MMP expression on MCF-7-BM interactions in vivo.Remarkably, MT1-MMP- or MT2-MMP-expressing MCF-7 cellsnot only acquire the ability to degrade the underlying BM, but toalso mount a potent invasion program that coincides with theinduction of an angiogenic response, increased cancer cell prolif-eration and tumor cell intravasation. The broad impact of MT1-MMP and MT2-MMP expression on cancer cell phenotype isfurther confirmed by silencing endogenous levels of MT1-MMPand MT2-MMP in MDA-MB-231 cells. In the absence of these twoproteases, the ability of MDA-MB-231 cells to remodel or trans-migrate the BM, to initiate angiogenesis, accelerate proliferation orintravasate is blocked completely, and neither cancer cells nor chickhost cells are able to activate alternate pathways to re-engage thecancer invasion program. We considered the possibility that MT1-MMP or MT2-MMP might, in and of themselves, initiate EMTprograms (30), but MT1-MMP- or MT2-MMP-expressing MCF-7cells cultured in vitro maintain epithelial phenotypes and do notupregulate Snail1 levels (Fig. S5). More likely, in an in vivoenvironment, MCF-7 cells may be triggered to undergo EMT-

Fig. 3. MT1-MMP and MT2-MMP mediate MDA-MB-231 cell BM invasion and intravasation in vivo. (A) CAMinvasion by MDA-MB-231 cells electroporated with ascrambled siRNA control alone or siRNAs directedagainst MT1-MMP, MT2-MMP, MMP-1, or MMP-9. Inselected experiments, MDA-MB-231 cells electropo-rated with siRNAs directed against both MT1-MMPand MT2-MMP were transfected with mouse MT1-MMP or MT2-MMP expression vectors. Cancer cell in-vasion was visualized by fluorescent microscopy ofCAM cross-sections after a 3-day incubation period.The upper CAM BM is marked by arrowheads. Red,type IV collagen; blue, DAPI staining for cell nuclei.(Scale bar, 50 �m.) (B) RT-PCR analysis of MT1-MMPand MT2-MMP expression in MDA-MB-231 cells fol-lowing electroporation with scrambled, MT1-MMP- orMT2-MMP- specific siRNAs. (C–E) MDA-MB-231 cell in-vasion (C), angiogenic activity (D), and proliferative (E)indices were determined in CAM cross-sections. Re-sults are expressed as the mean � 1 SD of three or moreexperiments.

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mimetic programs by growth factors, chemokines or ECM degra-dation products generated as a consequence of MT1-MMP orMT2-MMP activity (Fig. S5) (1). Although earlier studies havesuggested that MT3-MMP might display activity similar to MT1-MMP or MT2-MMP (13), MT3-MMP plays neither sufficient nornecessary roles in BM transmigration in vivo. Apparently in thedynamic system of the live chick CAM, either MT3-MMP-dependent BM degradation did not outstrip the ability of the CAMepithelium to maintain ECM architecture, or alternatively, MT3-MMP activity was muted by chick-derived inhibitors that prefer-entially target the protease.

The ability of MT1-MMP or MT2-MMP to trigger cancer cellinvasion, angiogenesis, proliferation, and intravasation correlatesclosely with the ability of these proteases to degrade the underlyingBM. However, the secreted MMP, MMP-13 (collagenase-3), like-wise induced BM degradation, but was unable to support MCF-7cell invasion into the interstitial matrix or any of the other sequelaeassociated with MT1-MMP or MT2-MMP expression. In vitrostudies have failed to identify MMP-13 as a BM-degradativeprotease (13), raising the possibility that MMP-13 acts in collabo-ration with host cells or other proteinases expressed in vivo toremodel the BM. Nevertheless, while BM degradation has beensuggested as a process sufficient to elicit an EMT-linked invasionprogram (31, 32), MMP-13 alone is unable to initiate this morecomplex response. As the BM interfaces with a dense, underlyingECM comprised largely of types I and III collagen, and onlyMT1-MMP and MT2-MMP can support invasive activity in colla-

gen-rich interstitial tissues (8, 11, 23, 33), we posit that invasivecancer cells must not only be able to degrade the BM, but also toinfiltrate type I/III collagen-rich tissues to access the host vascula-ture or induce angiogenesis. Although other secreted MMPs suchas MMP-1, -2, -3, -7, or -9, have been linked indirectly to cancer cellinvasion programs or cancer cell-induced neovascularization (19,34), none of these proteinases played required roles in either BMdegradation, transmigration, angiogenesis, or intravasation in our invivo system.

Given the ability of MT1-MMP or MT2-MMP to phenocopyMCF-7Snail1 cell behavior, we examined directly the role of thesemembrane-anchored MMPs during the Snail1-dependent EMTprogram in vivo. In accordance with our predictions, Snail1 inducedthe expression of MT1-MMP and MT2-MMP not only in tandemwith BM degradation and invasion, but also angiogenesis andintravasation. At first glance, the ability of Snail1 to drive intrava-sation within a 72-h culture period in vivo is surprising. Yet, recentstudies suggest that BM invasion and intravasation may occur at theearliest steps of carcinoma formation (35, 36). Indeed, circulatingneoplastic cells have been shown to express either MT1-MMP orMT2-MMP in mouse models as well as human cancer (35, 37). Mostremarkably, despite the ability of Snail1 to alter the expression ofhundreds of gene products associated with motility, invasion, cellcycle control, apoptosis and angiogenesis (38–41), the ability ofMCF-7Snail1 cells to degrade and transmigrate the BM, invade localtissues, trigger an angiogenic response and intravasate were allinhibited following MT1-MMP and MT2-MMP silencing. Like-

Fig. 4. Snail1 induces an MT1-MMP/MT2-MMP-dependent BM invasion program. (A) MCF-7Snail1 cellswere electroporated with a scrambled siRNA controlor siRNAs directed against MT1-MMP, MT2-MMP, orboth MT1-MMP and MT2-MMP. In selected experi-ments, cells co-electroporated with MT1-MMP andMT2-MMP siRNAs were transfected with mouse MT1-MMP or MT2-MMP expression vectors. (Scale bar, 50�m.) Results are representative of three or more ex-periments performed. (B) RT-PCR analysis of MT1-MMP and MT2-MMP mRNA levels in non-induced andSnail1-induced MCF-7 cells cultured atop the CAM sur-face for 3 days as well as in induced cells whereinMT1-MMP, MT2-MMP, or both MT1-MMP and MT2-MMP were targeted with specific siRNAs. (C and D)CAM invasion (C) and angiogenic activity (D) werequantified as described. Results are the mean � 1 SD ofthree or more experiments performed. (E) Intravasa-tion by MCF-7Snail1 cells was monitored by Alu-sequence PCR as described following silencing of MT1-MMP and MT2-MMP in tandem or after rescue withmouse MT1-MMP or MT2-MMP. Results are represen-tative of three or more experiments performed.

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Page 6: Induction of a MT1-MMP and MT2-MMP-dependent basement ...Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1 Ichiro Otaa,b,

wise, we have previously reported that Snail1 silencing inhibitsMDA-MB-231 cell invasion in the CAM system although thedownstream proteolytic effectors were not identified (4). WhileMDA-MB-231 cells also express ZEB1 (24, 28, 29), the tandemknockdown of MT1-MMP and MT2-MMP expression similarlyabrogated the carcinomatous phenotype.

The mechanisms by which MT1-MMP or MT2-MMP can exertsuch global effects on cancer cell function remain to be established,but it is important to stress the fact that the substrate repertoire ofthese enzymes can extend far beyond the ECM itself to includehundreds of potential targets (42). While MT1-MMP can specifi-cally regulate cancer cell proliferation within the context of the 3-DECM by exerting collagenolytic activity (22), the growth promotingeffects observed atop the CAM surface more likely occurs as aconsequence of neovascularization. In this regard, MT1-MMPactivity (like Snail1) has been linked to pro-angiogenic responsesthrough largely uncharacterized processes that may involve VEGF,semaphorin secretion, or the generation of bioactive ECM products(38). MT2-MMP has not previously been shown to affect angio-genesis, intravasation, or cancer cell proliferation, but our resultsprovide evidence that a second MT-MMP family member caninduce an aggressive carcinoma-like phenotype in expressing cells.Finally, independent of the ability of MT1-MMP or MT2-MMP tohydrolyze substrates in their surrounding milieu, it is important tonote that MT1-MMP/MT2-MMP-dependent proteolysis may indi-rectly exert complex effects on gene expression in the cancer cellsthemselves by altering local ECM rigidity as well as cell shape (1,

38). With an increasing body of evidence supporting the earlyexpression of EMT-inducing transcription factors as well as MT1-MMP or MT2-MMP at the cancer cell invasion front, we posit thatthese two proteolytic enzymes, working alone or in concert, playcritical roles in orchestrating carcinoma cell behavior.

Materials and MethodsCell Culture and Transfection. MCF-7 and MDA-MB231 cells were routinelymaintained in complete DMEM supplemented with 10% FBS (FBS). Tet-on-Snail1MCF-7 cells were provided by E. Fearon.

CAM Invasion and Intravasation Assays. In vivo cancer cell invasion and intrava-sation assays were conducted using 11-day-old chick embryos wherein MCF-7,MDA-MB-231, or the transfected cells (105 cells labeled with Fluoresbrite carbox-ylatedpolystyrenenanospheresof45-nmdiameter;Polysciences) (8)wereseededatoptheCAMandincubatedfor72hasdescribedpreviously (8,18).TheCAMwasdropped without damaging the epithelial BM by applying gentle negative pres-sure at the air sac, and an opening of approximately 1 cm2 was cut in the shellabove the CAM with an electric drill. Tumor cell intravasation was detected ashuman-specific Alu-sequences by PCR on DNA extracted from the lower CAMafter a 3-day culture period (Fig. S1). Chick GAPDH (chGADPH) serves as theloading control (14).

Additional Details. For details of expression plasmids, siRNA electroporation andRT-PCR analysis, and histology and microscopy, see SI Materials and Methods.

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth Grants R01 CA116516, CA071699, and CA088308 and the Breast CancerResearch Foundation.

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