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MT1‐MMP proinvasive activity is regulated by a novel Rab8‐dependent exocytic pathway

MT1‐MMP proinvasive activity is regulated by a novel Rab8‐dependent exocytic pathway The EMBO Journal (2007) 26, 1499–1510 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway (Egeblad and Werb, 2002; Sato et al, 2005). MMPs are either Jose J Bravo-Cordero , Raquel Marrero- 1 1 2 secreted from the cell or anchored to the plasma membrane Diaz , Diego Megı´as , Laura Genı´s , 1 1 (PM) as integral proteins (membrane-type MMPs). Of these, Aranzazu Garcı´a-Grande , Maria A Garcı´a , 2 1, MT1-MMP has been widely studied as its expression is Alicia G Arroyo and Marı´a C Montoya * closely associated with invasiveness and malignancy of Confocal Microscopy and Cytometry Unit, Biotechnology Programme, tumors (Egeblad and Werb, 2002). Moreover, MT1-MMP over- Spanish Nacional Cancer Research Center (CNIO), Madrid, Spain and expression enhances invasive ability of cells and silencing Matrix metalloproteinases Group, Centro Nacional de Investigaciones MT1-MMP suppresses cell migration and invasion, demon- Cardiovasculares (CNIC), Madrid, Spain strating that this enzyme is one of the most critical factors MT1-matrix metalloproteinase (MT1-MMP) is one of the of the invasion machinery (Sato et al, 2005; Itoh and Seiki, most critical factors in the invasion machinery of tumor 2006). cells. Subcellular localization to invasive structures is key MT1-MMP is produced as an inactive precursor and is for MT1-MMP proinvasive activity. However, the mechan- proteolytically cleaved intracellularly by furin, being deliv- ism driving this polarized distribution remains obscure. ered to the PM in the active form as a type I transmembrane We now report that polarized exocytosis of MT1-MMP protein (Osenkowski et al, 2004). However, the exact me- occurs during MDA-MB-231 adenocarcinoma cell migra- chanism by which active MT1-MMP traffics to the PM is not tion into collagen type I three-dimensional matrices. known. Once in the PM, MT1-MMP can degrade a number of Polarized trafficking of MT1-MMP is triggered by b1 in- ECM macromolecules including type I, II and VI collagens, tegrin-mediated adhesion to collagen, and is required for gelatin, laminins 1 and 5, fibronectin, vitronectin, aggrecan, protease localization at invasive structures. Localization of fibrin and lumican. It also activates other proteases like MT1-MMP within VSV-G/Rab8-positive vesicles, but not pro-MMP2 and pro-MMP13 and cleaves several cell surface in Rab11/Tf/TfRc-positive compartment in invasive cells, proteins such as CD44, transglutaminase, low-density lipo- suggests the involvement of the exocytic traffic pathway. protein receptor-related protein, a integrin and syndecan Furthermore, constitutively active Rab8 mutants induce (Sato et al, 2005; Itoh and Seiki, 2006). Given the wide MT1-MMP exocytic traffic, collagen degradation and inva- array of substrates that can be irreversibly processed by sion, whereas Rab8- but not Rab11-knockdown inhibited MT1-MMP and the fact that the enzyme is expressed at the these processes. Altogether, these data reveal a novel PM as an active enzyme (Sato et al, 1994; Mazzone et al, pathway of MT1-MMP redistribution to invasive struc- 2004), it seems clear that MT1-MMP is a potentially harmful tures, exocytic vesicle trafficking, which is crucial for its enzyme and needs to be tightly regulated. Classical regula- role in tumor cell invasiveness. Mechanistically, MT1- tory mechanisms of MT1-MMP include transcriptional regu- MMP delivery to invasive structures, and therefore its lation, intracellular processing of the inactive zymogen (Sato proinvasive activity, is regulated by Rab8 GTPase. et al, 1994; Mazzone et al, 2004) and inhibition by endogen- The EMBO Journal (2007) 26, 1499–1510. doi:10.1038/ ous tissue inhibitors (TIMP-2, RECK or testican) (Will et al, sj.emboj.7601606; Published online 1 March 2007 1996; Nakada et al, 2001; Oh et al, 2001). Recently, more Subject Categories: cell & tissue architecture; molecular precise means of regulating MT1-MMP activity on the cell biology of disease surface, like internalization (Jiang et al, 2001; Uekita et al, Keywords: matrix metalloproteinases; membrane traffic; 2001; Galvez et al, 2002; Wang et al, 2004), recycling MT1-MMP; Rab8; tumor invasion (Remacle et al, 2003; Wang et al, 2004), autocatalytic proces- sing to an inactive degradation product (Stanton et al, 1998; Lehti et al, 2000; Tam et al, 2002; Toth et al, 2002), oligomer- ization (Itoh et al, 2001; Rozanov et al, 2001; Lehti et al, 2002; Galvez et al, 2005) and post-trasductional regulation (Wu Introduction et al, 2004) have been described. Subcellular localization of Key processes for tumor progression such as angiogenesis, MT1-MMP to invasive structures is another important aspect cell growth, invasion and metastasis are based on the ability of MT1-MMP regulation and constitutes a prerequisite for of endothelial and tumor cells to invade the surrounding exerting its proinvasive activity (Nakahara et al, 1997; Lehti tissue. Focused degradation of tissue barriers by matrix et al, 2000; Mori et al, 2002), although the mechanism driving metalloproteinases (MMPs) plays a critical role in invasion this polarized distribution remains to be elucidated. Expression of PM proteins is controlled by the ubiquitous *Corresponding author. Confocal Microscopy and Cytometry Unit, process of constitutive secretion, and can be slowly up- or Biotechnology Programme, Spanish National Cancer Research Center (CNIO), C/Melchor Ferna´ndez Almagro 3, Madrid E-28029, Spain. downregulated by synthesis de novo or degradation of exist- Tel.: þ 34 91 7328012; Fax: þ 34 91 2246980; ing protein. Additionally, cells can rapidly modulate the E-mail: [email protected] levels of surface expression of some receptors, channels and transporters by having a pool of ready synthesized Received: 26 June 2006; accepted: 24 January 2007; published online: 1 March 2007 molecules available for their rapid insertion into and retrieval &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1499 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al from the PM in a process called constitutive cycling regulate polarized membrane transport of newly synthesized (Royle and Murrell-Lagnado, 2003). This process involves proteins to PM protrusions, participating in remodelling the regulated exocytosis, which is the translocation of membrane cell shape in response to different signals (Huber et al, 1993; proteins from intracellular compartments to the PM as a Peranen et al, 1996; Hattula et al, 2002; Ang et al, 2003). consequence of cell stimulation (Chieregatti and Meldolesi, We herein report a novel regulatory mechanism of MT1- 2005). Polarized exocytosis towards the leading edge of MMP activity involving regulated exocytosis to the cell migrating cells has been suggested as a mechanism causing surface at invasive structures driven by integrin-mediated membrane extension and recycling of integrin molecules adhesive events that is controlled by Rab8 GTPase. The endocytosed at the rear of the cell (Lawson and Maxfield, importance of this regulatory mechanism is highlighted by 1995; Sesaki and Ogihara, 1997). Leading edge-directed the complete functional blockade of MT1-MMP-dependent exocytosis seems to transport both secretion and endocytic collagen degradation and invasion when Rab8 protein levels recycling membranes (Bretscher and Aguado-Velasco, are knocked down. 1998). MT1-MMP has been shown to reside intracellularly (Jiang et al, 2001; Uekita et al, 2001; Galvez et al, 2002; Results Remacle et al, 2003; Wang et al, 2004) and its expression at the cell surface is usually very weak in most cell types. There Live cell confocal imaging of MT1-MMP dynamic are clear evidences showing that this protein undergoes redistribution and activity at invasive structures endocytosis (Jiang et al, 2001; Uekita et al, 2001; Galvez To understand better MT1-MMP regulation during tumor cell et al, 2002) and recycling to the surface (Remacle et al, 2003; invasion, we transfected breast carcinoma MDA-MB-231 cells Wang et al, 2004) in stationary cells. However, there are with MT1-MMP-GFP and embedded them in three-dimen- no evidences so far describing regulation of MT1-MMP by sional matrices (3D-Col I). Live cell confocal imaging regulated exocytic processes. showed, for the first time, activity and dynamics of MT1- Rab8 was initially isolated as a transforming gene from a MMP during cell invasion (Figure 1A and Supplementary melanoma cell line (Nimmo et al, 1991). It belongs to the Rab Video 1). Fluorescence and reflection images, revealing MT1- family of Ras-related GTPases that play a crucial role in MMP-GFP localization and collagen fiber organization membrane traffic by determining the specificity of vesicle respectively, showed MT1-MMP dynamic redistribution to col- transport (Zerial and McBride, 2001). Although the traffic lagen fiber adhesion sites at the PM, and subsequent collagen route regulated by Rab8 is not established, it is known to fiber degradation (Figure 1A and Supplementary Video 1). Figure 1 MT1-MMP accumulates at the sites of active collagen degradation during invasion of 3D-Col I matrices. (A) Live cell confocal imaging of MT1-MMP-GFP expressed in MDA-MB-231 cells embedded into 3D-Col I. Overlay of MT1-MMP-GFP fluorescence (green) and collagen fiber reflection (blue) images obtained at the indicated time points during the course of a time-lapse experiment is shown (see Supplementary Video 1). (B, C) Localization of endogenous MT1-MMP revealed by immunofluorescence staining with Lem-2/15 mAb of MDA-MB-231 cells and endometrial carcinoma primary cultured cells embedded into 3D-Col I. (B) Overlay of MT1-MMP staining (green) and collagen fiber reflection (blue) images is shown. (C) Overlay of phase contrast and fluorescence images. Insets show membrane sites engaging bundles of collagen fibers. GFP concentration at invasive structures is pointed by arrowheads. 1500 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Cells overexpressing large amounts of MT1-MMP-GFP were Polarized vesicle traffic is responsible for the used only to monitor MT1-MMP activity during invasion accumulation of MT1-MMP at collagen contact sites as they produced a high extent of matrix destruction, thus Localization of MT1-MMP to invasive structures has been clearly showing that MT1-MMP-GFP retains protease activity. described previously (Nakahara et al, 1997; Lehti et al, 2000; However, subsequent live cell invasion studies were carried Mori et al, 2002), although the underlying mechanism out using cells expressing low amounts of MT1-MMP-GFP, responsible for its redistribution on the cell surface has not which behaved in a more physiological manner. This was been elucidated. MT1-MMP localization was analyzed in accomplished by choosing cells with dim GFP fluorescence MDA-MB-231 cells embedded into 3D-Col I. Interestingly, a producing punctual degradation of collagen fibers during the novel compartmentalization of MT1-MMP-positive vesicles invasive process. A non-linear pattern of MT1-MMP localiza- at submembranous pools in invasive structures was observed tion at membrane protrusions in contact with the underlying (Figure 2A). Clear evidence of recruitment of MT1-MMP- 3D matrix was also shown for endogenous MT1-MMP in carrying vesicles to invasive structures was obtained when MDA-MB-231 and primary carcinoma cells as revealed by monitoring the formation of a new membrane protrusion immunostaining with specific anti-MT1-MMP antibodies event (Figure 2A, pointed by arrowheads and Supplementary (Abs) (Figure 1B and C). Video 2). Cell adhesion to collagen fibers leading to PM Figure 2 MT1-MMP intracellular vesicle recruitment toward collagen contact sites at the PM of MDA-MB-231 cells. (A) Live cell imaging of MDA-MB-231 cells transfected with MT1-MMP-GFP and embedded into 3D-Col I. Overlay of fluorescence and phase-contrast images showing MT1-MMP-GFP localization (pink) and cell morphology/collagen fiber distribution respectively, acquired at different time points is shown (see Supplementary Video 2). Arrowheads point to a new contact established between the cell membrane and a meshwork of collagen fibers, where active vesicle recruitment is observed. (B) MDA-MB-231 cells expressing MT1-MMP-GFP (green), cultured on glass coverslips, were incubated with Col I- or BSA-coated beads for 1 h, fixed and imaged. Fluorescence image (showing GFP), phase-contrast image (showing cell morphology and bead localization) and their overlay are presented. (C) Beads coated with BSA, Fn, anti-b1 Ab (TS2/16) or Col I were allowed to interact for 1 h with MDA-MB-231 cells that had been previously treated with or without not with a blocking anti-b1 (Lia1/2) or control (BerEP4) Abs. Cells were then fixed and imaged by confocal microscopy. Bars represent relative fluorescence intensity at the bead surrounding area normalized for background fluorescence calculated at 10–15 beads for each of the three independent experiments performed. The statistical significance of relative bead fluorescence comparing the different bead coatings and control (BSA) values (*) and antibody-treated compared to isotype /### control values (#) was evaluated using Student’s t-test. (*Po0.05; *** Po0.001). &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1501 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al protrusion was accompanied by local recruitment of MT1- The biosynthetic transport of proteins to the cell surface MMP-positive intracellular vesicles to a submembranous area, occurs via the recycling endosomes (Futter et al, 1995; followed by the local accumulation of MT1-MMP at the Leitinger et al, 1995; Ang et al, 2004; Lock and Stow, 2005). protrusive membrane (Figure 2A and Supplementary Video Moreover, recycling has been proposed as a mechanism of 2). Visualization of vesicle trajectories by fast scanning con- MT1-MMP recruitment to the leading edge during cell migra- focal imaging revealed highly complex traffic going to and tion (Remacle et al, 2003; Wang et al, 2004). We therefore from the PM in different directions. Traffic from the cell center sought to determine the involvement of recycling in MT1- to the periphery is not obvious, although there is clear PM MMP-polarized exocytosis. We allowed MT1-MMP-GFP- transport from submembranous pools localized at the polar- transfected cells to uptake transferrin (Tf) and low-density ized areas (see Supplementary Figure 3 and Supplementary lipoprotein (LDL) to label recycling and lysosomal compart- Video 4–6). Polarized MT1-MMP vesicle traffic was also ments, and analyzed their colocalization with MT1-MMP- induced by adhered Col I-coated beads but not control BSA- GFP (Figure 4B and Supplementary Figure 10). Surprisingly, coated beads (Figure 2B). Dynamic live cell studies show very MT1-MMP showed almost negligible colocalization with Tf active vesicle recruitment to collagen-coated beads, where and a strong colocalization with LDL in 3D-Col I-embedded MT1-MMP is accumulated (Supplementary Figure 7 and cells, suggesting that MT1-MMP is absent from recycling Supplementary Video 8). To gain insight into the cues that compartment, and instead is being sorted to lysosome induced MT1-MMP vesicle recruitment, we allowed cells to degradation in invasive cells. However, when we used the interact with beads coated with different ECM matrix proteins. same experimental conditions as previous studies that Quantitative analysis showed MT1-MMP-specific mobilization demonstrated MT1-MMP recycling (Remacle et al, 2003; induced by Col I, Fn or b1 integrin clustering Abs, but not by Wang et al, 2004), that is, plating cells in coverslips, we BSA. Col I-induced MT1-MMP recruitment could be specifi- confirmed MT1-MMP localization at recycling compartments cally impaired by function blocking anti-b1 Abs (Figure 2C). (Figure 4B). In addition, primary tumor cells also showed Altogether, these results show that recruitment of MT1-MMP overlap of the TfRc/Rab11-positive recycling compartment vesicles induced by collagen engagement in MDA-MB-231 cells with endogenous MT1-MMP in cells grown on coverslips but is mediated by b1 integrin-dependent adhesive events. not in 3D-Col I-embedded cells (Figure 4C). Therefore, MT1- It has been proposed that MT1-MMP delivery to invasive MMP is confined within the biosynthetic, although it is structures is mediated by CD44-dependent membrane trans- absent from the recycling compartment in invasive cells. port, although our live cell studies prompted us to hypothesize that intracellular vesicle traffic was responsible for the accu- Rab8 but not Rab11 codistributes with MT1-MMP at mulation of MT1-MMP at invasive structures. We addressed exocytic vesicles, and is specifically mobilized by Col I this issue by combining fluorescence recovery after photo- Because Rab8 GTPase has been involved in polarized bleaching (FRAP) at the PM with fluorescence loss in membrane transport of PM proteins during the formation of photobleaching (FLIP) at the underlying submembranous membrane protrusions, we sought to determine its involve- compartment. Recovery of fluorescence monitored at the ment in MT1-MMP exocytic delivery to invasive structures. FRAP region quantitatively estimates the extent to which We found a strong colocalization of MT1-MMP and Rab-8 MT1-MMP membrane localization is dependent on membrane in intracellular vesicles (Figure 5A). Time-lapse confocal transport, independently of the contribution of vesicle income imaging revealed the presence of MT1-MMP in Rab8-positive from the intracellular compartment. In contrast to the lateral vesicles being transported to the invasive PM (Figure 5B PM (Figure 3A–F), no relocalization of fluorescent MT1-MMP and Supplementary Video 11), whereas colocalization of at the invasive PM was observed (Figure 3G–L) when the MT1-MMP with Rab11 was negligible (Figure 5C). Vesicles intracellular pool of vesicles was continuously bleached. recruited to Col I-coated beads also showed a strong coloca- Additional example is shown in Supplementary Figure 9. lization of Rab8 and MT1-MMP (Figure 5D). Interestingly, we Hence, at the invasive lamella, MT1-MMP membrane diffusion observed a striking colocalization of MT1-MMP and Rab8 is compromised and intracellular traffic is most likely the within membranes deposited at degraded matrix (Figure 5E). source of MT1-MMP accumulation at invading structures. Deposition of cell fragments within the extracellular matrix caused by exocytic release of vesicles has been related to rear retraction during tumor cell invasion (Friedl and Wolf, 2003; MT1-MMP is found in the biosynthetic, not the recycling Mayer et al, 2004), and MT1-MMP has been previously compartment in invasive MDA-MB-231 cells To explore the involvement of the biosynthetic pathway in shown to be released within these fragments in endothelial MT1-MMP-polarized exocytosis, we performed colocalization cells (Taraboletti et al, 2002). These results strongly suggest studies using a classical marker of this route, VSV-G, as a that traffic and fusion of exocytic vesicles carrying MT1-MMP reporter. MDA-MB-231 cells coexpressing VSV-G-YFP and to matrix degradation sites is regulated by Rab8. Moreover, collagen-coated beads specifically induced recruitment of MT1-MMP-mRFP were embedded into 3D-Col I; cells were Rab8- but not Rab11-positive vesicles (Figure 5F), indicating then incubated at 201C to allow accumulation at the TGN (Ang et al, 2003), where both proteins were found colocaliz- that Rab8-mediated traffic is induced by collagen interaction. ing (not shown). When shifting to 321C to allow rapid exit of VSV-G from the TGN, a number of vesicles displayed strong Rab8 regulates traffic of MT1-MMP to invasive colocalization of MT1-MMP and VSV-G, and were found structures, and MT1-MMP-dependent collagen to translocate to the PM at invasive sites (Figure 4A). degradative activity and invasion These results suggest that biosynthetic exocytic traffic is The involvement of Rab8 in the regulation of MT1-MMP involved in the recruitment of MT1-MMP to invasive activity was first evaluated by quantitative experiments of structures at the PM. MT1-MMP vesicle recruitment in cells expressing wtRab8, 1502 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 3 MT1-MMP FRAP/FLIP experiments reveal that intracellular vesicle traffic is responsible for the accumulation of MT1-MMP at the invasive PM. MT1-MMP-GFP expressing MDA-MB-231 cells embedded into 3D-Col I were subjected to FRAP-FLIP photobleaching experiments. Images showing prebleaching, bleaching and post-bleaching at the PM (FRAP region) during continuous photobleaching of the submem- branous compartment (FLIP region) at the lateral (A–E) and invading (G–K) PM. Fluorescence recovery quantification at the FRAP region is calculated at the lateral (F) and invading (L) PM and represented in the graph. Rab8-activated mutant (Rab8Q67L) or Rab8DC (inactive Rab8 overexpression and activation induced collagen degra- mutant with impaired membrane localization owing to loss of dative activity (Figure 6C). Function blocking anti-MT1-MMP the prenylation site). MT1-MMP-mRFP vesicle recruitment to Ab (Lem-2/15) significantly abrogated Rab8-induced inva- Col I-coated beads was significantly induced by overexpres- sion and collagen degradation (Figure 6B, C), thus indicating sion of Rab8-activated mutant, but not by Rab8DC control its endogenous MT1-MMP dependence. These studies de- (Figure 6A). Specificity of Rab8 effect was demonstrated by monstrate the involvement of Rab8 in regulating MT1-MMP examining CD44 recruitment to hyaluronic acid (HA)-coated delivery to the PM and its collagenolytic and proinvasive beads, which was unaffected by the expression of Rab8 activities. constructs (Supplementary Figure 12). Furthermore, trans- To demonstrate further the role of Rab8 in MT1-MMP well collagen invasion assays showed that similar to MT1- exocytic traffic to the PM, we performed gene silencing MMP overexpression, Rab8Q67L and wtRab8 induced an studies. Stable cell lines carrying short-hairpin RNA increase in cell invasion, whereas DC control was shown to (shRNA) targeted Rab8a and Rab11a showed protein deple- be ineffective (Figure 6B). Pericellular collagenolysis evalu- tions of approximately 80 and 60%, respectively, as assessed ated in cells expressing the different constructs revealed that by Western blotting analysis (Figure 7A and B). The possibi- &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1503 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 4 MT1-MMP colocalization with markers of the biosynthetic/recycling and degradative routes. (A) MDA-MB-231 cells cotransfected with MT1-MMP-mRFP and VSV-G-YFP were embedded into 3D-Col I. Cells were incubated overnight at 401C, then transferred to 201C for 2 h and finally shifted to 321C for 1 h. Cells were then fixed and imaged. Arrowheads point to vesicles positive for both VSV-G (green) and MT1- MMP (red). Overlay image shows colocalization (yellow) and fiber reflection (blue). (B) MDA-MB-231 cells transfected with MT1-MMP-GFP were either embedded into 3D-Col I (upper panel) or plated on coverslips (lower panel) and incubated with labelled Tf and LDL for 1 h at 371C to allow their internalization. Images show localization of MT1-MMP-GFP (green), Tf (blue), LDL (red), and their overlay. (C) Primary lung adenocarcinoma cells were either embedded into 3D-Col I (upper panel) or plated on coverslips (lower panel) and immunostained with specific Abs for TfRc or Rab11 (red), and MT1-MMP (green), as indicated. Insets show superimposed fluorescence images pseudocolored in green/red; arrowheads point colocalization vesicles (shown in yellow). lity of having off-target effects was ruled out by analyzing the (Figure 7D) and collagen degradation (Figure 7E) were levels of Rab11 protein in Rab8 knocked down cells and vice impaired in cells expressing shRNA for Rab8 but not Rab11. versa control and Rab8-silenced cells displayed similar levels Moreover, ectopic expression of Rab8 coding sequence tagged of surface MT1-MMP expression (10 and 9.3 mean fluores- with mRFP carrying four silent mutations in Rab8shRNA1 cence intensity) as revealed by flow cytometry analysis. targeting sequence reconstituted these functions (Supple- Thus, steady-state expression of MT1-MMP at the cell surface mentary Figure 13). Transiently transfected Rab8shRNA in was unaffected by Rab8 knockdown. However, endogenous mammalian expression vectors rendered similar effects MT1-MMP vesicle recruitment to Col I-coated beads (Supplementary Figure 14). CD44 recruitment to HA-coated (Figure 7C), MT1-MMP-induced tumor cell invasion beads was unaffected by Rab8shRNA, further demonstrating 1504 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 5 Rab8 but not Rab11 codistributes with MT1-MMP during vesicle transport to the PM. MDA-MB-231 transfected with MT1-MMP-mRFP and Rab8-GFP were embedded into 3D-Col I and analyzed by confocal imaging. (A) MT1-MMP-mRFP fluorescence (red), Rab8-GFP (green) and the superimposed images where colocalization can be seen in yellow, as well as the image showing exclusively colocalizing pixels (white) are shown. 2D colocalization histogram corresponding to these images obtained using Imaris software (Bitplane AG, Zurich, Switzerland) is also shown. Live cell imaging of 3D-Col I invading MDA-MB-231 cells transfected with MT1-MMP-mRFP and either Rab8-GFP (B) or Rab-11- GFP (C). Images acquired at the indicated time points show Rab8 or Rab11 (green) and MT1-MMP (red) localization during the course of the experiment (see Supplementary Video 11). (D) MDA-MB-231 cells expressing MT1-MMP-mRFP (red) and Rab8-GFP (green), cultured on glass coverslips, were incubated with Col I-coated beads for 1 h, then fixed and imaged. Overlay of fluorescence images is presented in inset. Asterisk indicates bead localization. (E) Confocal images of MT1-MMP-mRFP (red) and Rab8-GFP (green) fluorescence and collagen fiber reflection (blue) shows colocalization of MT1-MMP and Rab8 attached to degraded collagen fibers. (F) Beads coated with BSA or Col I were allowed to interact with Rab8-GFP- or Rab11-GFP-expressing MDA-MB-231 cells. Bars represent relative fluorescence intensity at the bead surrounding area normalized to background fluorescence calculated at 10–15 beads for each of the three independent experiments performed. that polarized distribution of other surface proteins is inde- to collagen engagement. Accordingly, integrin clustering pendent of Rab8 (Supplementary Figure 12). These results stimulates cell-surface expression of MT1-MMP (Ellerbroek clearly demonstrate that Rab8 GTPase specifically mediates et al, 2001), and coclustering of b1 integrins and MT1-MMP regulated, not constitutive, transport of MT1-MMP to the PM, has been shown in tumor cells invading 3D collagen matrices MT1-MMP-dependent collagen degradation and invasion. (Wolf et al, 2003), and endothelial cells adhered to collagen- coated surfaces, where the biochemical association of both MT1-MMP and b1 integrin was demonstrated (Galvez et al, Discussion 2002). MT1-MMP proinvasive activity requires its redistribution Focal degradation of the ECM barrier at the invading cell front to motility-related structures (Nakahara et al, 1997; Lehti is a key process in tumor invasion, and this is achieved by et al, 2000; Mori et al, 2002). Seiki and co-workers have localization of proteases at the leading edge of migrating suggested the interaction of MT1-MMP with CD44, and the cells. There are clear evidences that MT1-MMP localizes at invasive structures (Nakahara et al, 1997; Lehti et al, 2000; linkage of the latter to the actin cytoskeleton, as the mechan- Mori et al, 2002). However, how precisely the enzyme is ism driving the proteinase to the leading edge of migrating cells (Mori et al, 2002; Suenaga et al, 2005). Our data on 3D targeted to the the invasion sites remains to be determined. invasion models point out a completely novel mechanism, Three dimensional collagen matrices mimic the ‘in vivo’ regulated exocytosis of MT1-MMP vesicles, mediating MT1- environment encountered by tumor cells, and so provide a MMP recruitment to invasive structures. This hypothesis is surrogate of the tissue microenvironment, allowing us to perform live cell studies of tumor cell invasion. We herein based in several pieces of evidence: (1) dynamic visualization show for the first time dynamic redistribution and activity of of MT1-MMP vesicles being recruited to the cell surface from MT1-MMP at invasive structures, as visualized by live con- intracellular locations at collagen fiber attachment sites preceding membrane protrusion; (2) FRAP/FLIP experiments focal imaging of MDA-MB-231 adenocarcinoma cell invasion showing that submembranous vesicle pool rather than mem- of 3D-Col I. b1 Integrin-dependent adhesion was found to be brane diffusion is required for the accumulation of MT1-MMP the spatial cue leading to MT1-MMP recruitment in response &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1505 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 6 Rab8 activation induces recruitment of MT1-MMP vesicles, MT1-MMP-dependent collagen degradation and invasion. (A) MDA-MB- 231 cells were cotransfected with MT1-MMP-mRFP and either GFP, wtRab8-GFP, Rab8Q67L-GFP or Rab8DC-GFP, and, allowed to interact with Col I-coated beads for 1 h, then fixed and analyzed by confocal microscopy. Bars represent the percentage of MT1-MMP-mRFP fluorescence intensity around the bead calculated in 10–18 cells expressing the different GFP constructs from three independent experiments. (B) MDA-MB- 231 cells were transfected with GFP, wtRab8-GFP, Rab8Q67L-GFP, Rab8DC-GFP or MT1-MMP-GFP. Cells were then allowed to migrate for 48 h on transwell filters coated with 3D-Col I to FCS containing media in the presence of isotype control IgG (solid bars), or function blocking anti- MT1-MMP Ab (Lem-2/15) (open bars). Bars represent the percentage of invaded GFP-expressing cells quantified in seven independent experiments by counting four different fields for each experiment. (C) MDA-MB-231 cells transfected with GFP, wtRab8-GFP, Rab8Q67L-GFP, Rab8DC-GFP or MT1-MMP-GFP were cultured on 2D-Col I layers for 48 h in the presence or absence of function blocking anti-MT1-MMP Ab (Lem-2/15), then fixed and labelled with anti-Col I antibody to evaluate degradation. Representative overlay images of Col I staining (red) and expression of the different constructs (green) is shown. The statistical significance comparing expression of different constructs to control /# /## (GFP) values (*) and antibody-treated compared to isotype control values (#) was evaluated using Student’s t-test (* Po0.05; ** Po0.01; /### *** Po0.001). at the invasive PM; and (3) the requirement of active Rab8, complex to the PM that bypasses recycling endosomes, which a GTPase involved in exocytic traffic, for collagen-induced could be involved in the traffic of MT1-MMP to invasive MT1-MMP recruitment to the membrane, MT1-MMP-depen- structures. Our results showing that MT1-MMP intracellular dent collagen degradation and invasion. In agreement with compartmentalization depends on the extracellular context this hypothesis, an intracellular functional pool of MT1-MMP may provide a rationale for internalized MT1-MMP. MT1- available for trafficking to the cell surface upon stimulation of MMP will recycle when cells are not involved in ECM HT1080 cells with ConA has been reported (Zucker et al, degradation thus maintaining a controlled surface activity, 2002). while allowing intracellular pools to be stored for rapid The confinement of MT1-MMP within the biosynthetic and trafficking if necessary. In contrast, MT1-MMP will be mobi- its absence from recycling compartments seems contradictory lized to a degradative compartment when cells are actively as there is increasing evidence showing that biosynthetic involved in ECM proteolytic processing to prevent accumula- transport to the cell surface occurs via recycling endosomes tion of inactivated MT1-MMP (TIMP-2-inhibited or partially (Futter et al, 1995; Leitinger et al, 1995; Ang et al, 2004; Lock degraded molecules). We can, therefore, establish a strong and Stow, 2005). However, a number of live imaging studies parallelism between the homeostasis of MT1-MMP and the (Lippincott-Schwartz et al, 2000; Lock and Stow, 2005) sup- so-called constitutive cycling traffic reported for a number of port the existence of a direct delivery pathway from the Golgi membrane proteins (reviewed by Royle and Murrell-Lagnado, 1506 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 7 Rab8 but not Rab11 knockdown with shRNA decreases MT1-MMP vesicle recruitment, collagen degradation and invasion. MDA-MB- 231 cells stably expressing PMCSV Pig (control), or PMCSV carrying Rab8shRNA sequences 1 and 2 or Rab11shRNA sequences 1 and 2. (A) The levels of Rab8 protein was assessed by Western blot analysis. Control tubulin blotting is also shown. (B) Quantification of Rab8 protein levels normalized using tubulin as a loading control is represented in the bar diagram. Endogenous vesicle recruitment (C), transwell invasion (D) and collagen degradation (E) were evaluated as described in Figure 6. (E) Representative images show Col I staining (red) and GFP expression from PMCSV vector (green). Asterisks indicate statistical significance comparing the expression of the different shRNAs to control (PMCSV Pig) values. 2003). A good example is the glucose transporter GLUT4, the recycling compartment instead; (iii) the possibility that, which undergoes rapid constitutive internalization and sub- as reported for GLUT4 and Rab8, there is a transport loop sequent slow recycling back to the surface, and therefore between the storage compartment and recycling endosomes under basal conditions, exists predominantly within intra- is not excluded; and (iv) endocytosis targeted to lysosome cellular compartments (Dugani and Klip, 2005). Despite being degradation will most likely be the fate of surface MT1-MMP, engaged in a recycling loop, there is a more static secretory inactivated during the process of matrix degradation. This pool of GLUT4 storage vesicles ready to move directly to the model would keep a potentially harmful enzyme away from cell surface in response to insulin stimulation (Dugani and the PM, where it could exert unwanted side-effects, despite Klip, 2005). Accordingly, both MT1-MMP and GLUT4 have being an extremely sensitive system for rapid and localized been localized at Rab8-positive vesicles (our data and Miinea enzyme mobilization, avoiding the slow process of protein et al, 2005), and their transport to the membrane is dependent synthesis. on syntaxin 4 (Widberg et al, 2003; Miyata et al, 2004). Rab8 was first described as an oncogene isolated as a Our studies reveal a novel pathway in the regulation of transforming gene from a melanoma cell line (Nimmo et al, MT1-MMP and allow us to propose a model for MT1-MMP 1991), although its relevance in cancer has not been estab- homeostasis (Figure 8). In this model, different traffic path- lished yet. Notably, Rab8 search in Oncomine cancer profiling ways of MT1-MMP are highlighted: (i) Rab8-regulated exo- database (www.oncomine.org) showed its overexpression in cytic mobilization from an intracellular storage compartment tumoral versus normal tissues in different microarray data different from recycling endosomes would account for polar- sets. Rab8 belongs to the family of Ras-like small GTPases ized recruitment of MT1-MMP to the invasive PM engaged that are major regulators of membrane trafficking in eukar- in matrix degradation (i.e. this report); (ii) constitutive cycling yotic cells (Zerial and McBride, 2001). Although the traffic will be predominant in a stationary cell, where MT1-MMP is route regulated by Rab8 is still not clarified, there is however not involved in ECM proteolytic processing, being found in evidence that it is involved in the transport of PM proteins at &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1507 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 8 Model for MT1-MMP intracellular trafficking. The model depicts two main intracellular pathways (I) Rab8-regulated exocytic mobilization of MT1-MMP from a biosynthetic storage compartment induced by collagen engagement in invading cells (II) Constitutive cycling from recycling endosomes in a stationary cell involves MT1-MMP un-engaged in matrix degradation. Additional pathways could involve (III) transport loop between the biosynthetic storage and recycling compartments and (IV) endocytosis targeted to lysosome degradation of surface MT1-MMP involved in collagen degradative activity. et al, 2003). MT1-MMP-mRFP was obtained by subcloning mRFP membrane protrusions (Peranen et al, 1996; Hattula et al, into EGFP restriction sites. Ts045 VSV-G-YFP and Rab11-GFP were 2002; Ang et al, 2003). Our results clearly show the involve- kindly provided by Dr R Peppercok and Dr D Sheff, respectively. ment of Rab8 in the traffic of MT1-MMP to the PM and in Abs used include LEM-2/15 anti-MT1-MMP (Galvez et al, 2002) and MT1-MMP-dependent collagen degradation and invasion, TS2/16 and Lia1/2 anti-human b1 integrin, kindly provided by Dr Sa´nchez-Madrid. Mouse mAb anti-TfRc and rabbit polyclonal Ab which may help to explain Rab8-transforming activity. to Rab11 were from Zymed Laboratories (South San Francisco, Considering the importance of MT1-MMP in tumor cell CA, USA). Goat polyclonal anti-Rab8 Ab was from Santa Cruz invasion and angiogenesis, there is a great interest in target- Biotechnology (Santa Cruz, CA, USA) and mAb anti-Col I and anti- ing this enzyme with new inhibitors. Cancer therapeutics tubulin DM1a Ab were from Sigma (St Louis, MO, USA). Anti- human epithelial antigen BerEP4 was from DakoCytomation designed to target protease activity by synthetic MMP inhi- (Glostrup, Denmark). bitors have proven ineffective. According to our results, an alternative strategy based on blocking MT1-MMP delivery Immunofluorescence, Tf/LDL uptake and confocal microscopy to invasive structures by means of Rab8 targeting will be a Cells were either plated onto coverslips or embedded into 3D-Col I, more rational means of preventing invasion and metastasis fixed at 41C for 5 min with 4% paraformaldehyde and permeabi- mediated by MT1-MMP, without affecting the enzyme basal lized with 0.5% Triton X-100 and stained with the appropriate Abs. Tf/LDL uptake was monitored by incubating 1 h serum-starved cells homeostasis. Important future work involving animal models with Tf-Alexa 647 (20 mg/ml) and dil-LDL (low-density lipoprotein should first be undertaken to validate Rab8 as a therapeutic conjugated to 3,3 -dioctadecylindocarbocyanine) (10 mg/ml) for 1 h cancer target. at 371C. Cell imaging was performed using a Leica TSC SP2 AOBS and SP5-RS AOBS with a 63  Plan Apo 1.32 NA oil-immersion objective (Leica, Mannheim, Germany). Leica Confocal Software Materials and methods (LCS) was used for acquisition of images, which were later adjusted for contrast using Adobe Photoshop Software. Colocalization Cell culture, transfection and collagen inclusion analysis was performed with Imaris software (Bitplane AG, Zurich, Breast adenocarcinoma MDA-MB-231 cells were maintained in Switzerland). DMEM supplemented with 10% FBS. Primary carcinoma cells were purified from fresh human endometrial and lung carcinoma tumor Polystyrene bead assays samples by enzymatic digestion as described elsewhere (Allinen Polystyrene divinyl-benzene beads (5 mm) (Duke Scientific Corpora- et al, 2004) and cultured in HAMF10 medium supplemented with tion, Palo Alto, CA, USA) were incubated with 0.5% BSA, 100 mg/ml 10% FBS. Cell transfection was performed using lipofectamine 2000 Col I (Vitrogen Palo Alto, CA, USA), 20 mg/ml Fn, 1 mg/ml HA (Invitrogen, Carlsbag, CA, USA) according to the manufacturer’s (Sigma, St Louis, MO, USA) or TS2/16 Ab anti-b1 integrin culture instructions. At 24 h post-transfection, cells were trypsinized and supernatant. Cells expressing the different constructs were incu- mixed with readily prepared Col I solution (2,4 mg/ml bovine Col I bated for 1 h with coated beads at a cell to bead ratio of 1:40. For (Vitrogen, Palo Alto, CA, USA), 1  RPMI, 19 mM HEPES (Gibco), inhibition studies, cells were previously incubated with or without 0.19% sodium bicarbonate (Sigma) and 5% FBS) that was then 10 mg/ml of blocking anti-b1 integrin Ab Lia1/2 or control BerEP4 allowed to polymerize for 2 h at 371C (3D-Col I) or plated on rat tail Ab (anti-human epithelial antigen). Confocal images were analyzed Col I protein (Roche Diagnostics, Panzberg, Germany)-coated for MT1-MMP fluorescence in a region around the bead and surfaces (2D-Col I layers). normalized to the overall background MT1-MMP fluorescence determined in three regions at irrelevant membrane areas of the Constructs and antibodies cell. Relative bead fluorescence represents quantified bead fluor- EGFP-tagged MT1-MMP, Rab8wt, Rab8Q67L and Rab8DC con- escence\background fluorescence  100 scored in at least 10 beads structs have been described previously (Galvez et al, 2002; Ang for each experimental condition. 1508 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Confocal photobleaching experiments the amphotropic vector pCL-Ampho, retrovirus packaging vector. After 48 h, transfection retrovital supernatants were used as retro- A combination of both FRAP and FLIP techniques was developed on a Leica TSC SP2 AOBS microscope using Leica Confocal Software viral stock for transduction of MDA-MB 231 cells. Cells expressing the (Leica, Mannheim, Germany). Live MT1-MMP-GFP expressing cells different shRNA constructs were selected with puromycin (0.5 mg/ embedded into 3D-Col I gels were exposed to a bleaching regime ml) for 5 days and GFP-expressing cells were sorted by flow consisting of (1) prebleach recording (scanning three images with cytometry to obtain stable shRNA-expressing cell lines. Only shRNA laser AOTF 20%), (2) bleaching and scanning at two different Rab8 (1), shRNA Rab8 (2), shRNA Rab11 (1) and shRNA Rab11 (2) regions of interest (membrane and submembranous compartments) showed significant depletion of Rab8 and Rab11 and were used for using bleaching laser excitation settings (100% AOTF) in both subsequent analysis. For shRNA rescue assays, four silent mutations regions and regular imaging scanning settings (20% AOTF) for the were introduced to the shRNA Rab8 1 targeting sequence (nucleo- rest of the field and (3) post-bleaching recording. During the post- tides 165–183). The final mutated Rab8 sequence (aggattaagttgcaaa- mut bleaching phase, the PM (FRAP region) was excited with regular ta) was obtained by PCR and subcloned into mRFP vector. Rab8 - imaging settings (20% AOTF,) whereas continuous bleaching mRFP was transiently transfected into shRNA Rab8 (1) -expressing settings (100% AOTF) were used at the FLIP region. The relative stable cell line. Rab8 and Rab11 protein levels were analyzed by loss of intensity and recovery of fluorescence was calculated at Western blotting. Alexa Fluor 680-conjugated secondary Abs were the FRAP region after background subtraction using Siggia normal- used to visualize and quantify the blots using the Odyssey Infrared ization (Siggia et al, 2000). Imaging System (Li-COr, Biosciences). Collagen degradation and cell invasion assays Statistical analysis MDA-MB231 cells were transfected with the different constructs. All numerical values reported represent mean7s.e. The statistical At 24 h after transfection, cells were plated onto 2D-Col I layers and significance comparing differences between the experimental and incubated for additional 48 h, fixed and immunostained for Col I. control (GFP/BSA) values (*) and Ab-treated compared with MDA-MB-231 cell invasion assays were performed in 8-mm pore 3D- isotype control values (#) was evaluated using Student’s t-test. Col I gel-coated transwell chambers (Costar). Cells were transfected Po0.05 was taken as the limits of statistical significance /# /## /### with the different constructs and after 24 h, resuspended in serum- (* Po0.05; ** Po0.01; *** Po0.001). free medium and seeded at 5 10 cells/well. Cells were allowed to transmigrate to 10% FBS media for 48 h and then counted at the top Supplementary data and bottom of the chamber using Image J software (NIH, Bethesda, Supplementary data are available at The EMBO Journal Online USA). Bars represent the percentage of invasive cells referred to the (http://www.embojournal.org). total number of cells considering only GFP or mRFP/GFP expressing cells. Acknowledgements Rab8 gene silencing with shRNAs Three different siRNA sequences were designed for silen- We thank Drs MA del Pozo and MA Alonso for helpful advice and cing Rab8a and Rab11a with the help of web-based algorithms critical reading of the manuscript, Dr Rivera for help with biochem- (http://side.bioinfo.ochoa.fib.es/) and (www.Invitrogen.com) (Rab8 ical studies, Dr M Malumbres for help with shRNA design and 0 0 (1) : 5 -GAGAATTAAACTGCAGATA, Rab8 (2) : 5 -GGAACTGGATTCG the Genomics Unit for help with shRNA cloning. Drs Mellman, 0 0 0 CAACATTG-3 and Rab8 (3) : 5 -GCTCGATGGCAAGAGAATTAA-3 ), Sa´nchez-Madrid, Pepperkok, Sheff and Tsien are acknowledged for 0 0 0 (Rab11 (1) : 5 -AAGAGCACCATTGGAGTAGAGTT-3 , Rab11 (2) : 5 -G providing us with reagents. Tumour cell samples were provided by 0 0 TACGACTACCTCTTTAAA-3 and Rab11 (3) : 5 -GCAACAATGTGGTT the CNIO Tumour Bank Unit. This work was supported by a grant CCTATTC-3 ). shRNAs were cloned into the retroviral vector MSCV from Fondo de Investigaciones Sanitarias (FIS PI031324) to MCM. Pig, a modified version of MSCV-puro (Clontech), which contains JJ B-C and R M-D are funded by the Ministry of Science and GFP to report shRNA expression. HEK-293T cells were cotransfected Technology of Spain (MCYT) and Fondo de Investigaciones with 10 mg of the plasmid containing the different shRNA and 10 mgof Sanitarias (FIS), respectively. 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MT1‐MMP proinvasive activity is regulated by a novel Rab8‐dependent exocytic pathway

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Abstract

The EMBO Journal (2007) 26, 1499–1510 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway (Egeblad and Werb, 2002; Sato et al, 2005). MMPs are either Jose J Bravo-Cordero , Raquel Marrero- 1 1 2 secreted from the cell or anchored to the plasma membrane Diaz , Diego Megı´as , Laura Genı´s , 1 1 (PM) as integral proteins (membrane-type MMPs). Of these, Aranzazu Garcı´a-Grande , Maria A Garcı´a , 2 1, MT1-MMP has been widely studied as its expression is Alicia G Arroyo and Marı´a C Montoya * closely associated with invasiveness and malignancy of Confocal Microscopy and Cytometry Unit, Biotechnology Programme, tumors (Egeblad and Werb, 2002). Moreover, MT1-MMP over- Spanish Nacional Cancer Research Center (CNIO), Madrid, Spain and expression enhances invasive ability of cells and silencing Matrix metalloproteinases Group, Centro Nacional de Investigaciones MT1-MMP suppresses cell migration and invasion, demon- Cardiovasculares (CNIC), Madrid, Spain strating that this enzyme is one of the most critical factors MT1-matrix metalloproteinase (MT1-MMP) is one of the of the invasion machinery (Sato et al, 2005; Itoh and Seiki, most critical factors in the invasion machinery of tumor 2006). cells. Subcellular localization to invasive structures is key MT1-MMP is produced as an inactive precursor and is for MT1-MMP proinvasive activity. However, the mechan- proteolytically cleaved intracellularly by furin, being deliv- ism driving this polarized distribution remains obscure. ered to the PM in the active form as a type I transmembrane We now report that polarized exocytosis of MT1-MMP protein (Osenkowski et al, 2004). However, the exact me- occurs during MDA-MB-231 adenocarcinoma cell migra- chanism by which active MT1-MMP traffics to the PM is not tion into collagen type I three-dimensional matrices. known. Once in the PM, MT1-MMP can degrade a number of Polarized trafficking of MT1-MMP is triggered by b1 in- ECM macromolecules including type I, II and VI collagens, tegrin-mediated adhesion to collagen, and is required for gelatin, laminins 1 and 5, fibronectin, vitronectin, aggrecan, protease localization at invasive structures. Localization of fibrin and lumican. It also activates other proteases like MT1-MMP within VSV-G/Rab8-positive vesicles, but not pro-MMP2 and pro-MMP13 and cleaves several cell surface in Rab11/Tf/TfRc-positive compartment in invasive cells, proteins such as CD44, transglutaminase, low-density lipo- suggests the involvement of the exocytic traffic pathway. protein receptor-related protein, a integrin and syndecan Furthermore, constitutively active Rab8 mutants induce (Sato et al, 2005; Itoh and Seiki, 2006). Given the wide MT1-MMP exocytic traffic, collagen degradation and inva- array of substrates that can be irreversibly processed by sion, whereas Rab8- but not Rab11-knockdown inhibited MT1-MMP and the fact that the enzyme is expressed at the these processes. Altogether, these data reveal a novel PM as an active enzyme (Sato et al, 1994; Mazzone et al, pathway of MT1-MMP redistribution to invasive struc- 2004), it seems clear that MT1-MMP is a potentially harmful tures, exocytic vesicle trafficking, which is crucial for its enzyme and needs to be tightly regulated. Classical regula- role in tumor cell invasiveness. Mechanistically, MT1- tory mechanisms of MT1-MMP include transcriptional regu- MMP delivery to invasive structures, and therefore its lation, intracellular processing of the inactive zymogen (Sato proinvasive activity, is regulated by Rab8 GTPase. et al, 1994; Mazzone et al, 2004) and inhibition by endogen- The EMBO Journal (2007) 26, 1499–1510. doi:10.1038/ ous tissue inhibitors (TIMP-2, RECK or testican) (Will et al, sj.emboj.7601606; Published online 1 March 2007 1996; Nakada et al, 2001; Oh et al, 2001). Recently, more Subject Categories: cell & tissue architecture; molecular precise means of regulating MT1-MMP activity on the cell biology of disease surface, like internalization (Jiang et al, 2001; Uekita et al, Keywords: matrix metalloproteinases; membrane traffic; 2001; Galvez et al, 2002; Wang et al, 2004), recycling MT1-MMP; Rab8; tumor invasion (Remacle et al, 2003; Wang et al, 2004), autocatalytic proces- sing to an inactive degradation product (Stanton et al, 1998; Lehti et al, 2000; Tam et al, 2002; Toth et al, 2002), oligomer- ization (Itoh et al, 2001; Rozanov et al, 2001; Lehti et al, 2002; Galvez et al, 2005) and post-trasductional regulation (Wu Introduction et al, 2004) have been described. Subcellular localization of Key processes for tumor progression such as angiogenesis, MT1-MMP to invasive structures is another important aspect cell growth, invasion and metastasis are based on the ability of MT1-MMP regulation and constitutes a prerequisite for of endothelial and tumor cells to invade the surrounding exerting its proinvasive activity (Nakahara et al, 1997; Lehti tissue. Focused degradation of tissue barriers by matrix et al, 2000; Mori et al, 2002), although the mechanism driving metalloproteinases (MMPs) plays a critical role in invasion this polarized distribution remains to be elucidated. Expression of PM proteins is controlled by the ubiquitous *Corresponding author. Confocal Microscopy and Cytometry Unit, process of constitutive secretion, and can be slowly up- or Biotechnology Programme, Spanish National Cancer Research Center (CNIO), C/Melchor Ferna´ndez Almagro 3, Madrid E-28029, Spain. downregulated by synthesis de novo or degradation of exist- Tel.: þ 34 91 7328012; Fax: þ 34 91 2246980; ing protein. Additionally, cells can rapidly modulate the E-mail: [email protected] levels of surface expression of some receptors, channels and transporters by having a pool of ready synthesized Received: 26 June 2006; accepted: 24 January 2007; published online: 1 March 2007 molecules available for their rapid insertion into and retrieval &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1499 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al from the PM in a process called constitutive cycling regulate polarized membrane transport of newly synthesized (Royle and Murrell-Lagnado, 2003). This process involves proteins to PM protrusions, participating in remodelling the regulated exocytosis, which is the translocation of membrane cell shape in response to different signals (Huber et al, 1993; proteins from intracellular compartments to the PM as a Peranen et al, 1996; Hattula et al, 2002; Ang et al, 2003). consequence of cell stimulation (Chieregatti and Meldolesi, We herein report a novel regulatory mechanism of MT1- 2005). Polarized exocytosis towards the leading edge of MMP activity involving regulated exocytosis to the cell migrating cells has been suggested as a mechanism causing surface at invasive structures driven by integrin-mediated membrane extension and recycling of integrin molecules adhesive events that is controlled by Rab8 GTPase. The endocytosed at the rear of the cell (Lawson and Maxfield, importance of this regulatory mechanism is highlighted by 1995; Sesaki and Ogihara, 1997). Leading edge-directed the complete functional blockade of MT1-MMP-dependent exocytosis seems to transport both secretion and endocytic collagen degradation and invasion when Rab8 protein levels recycling membranes (Bretscher and Aguado-Velasco, are knocked down. 1998). MT1-MMP has been shown to reside intracellularly (Jiang et al, 2001; Uekita et al, 2001; Galvez et al, 2002; Results Remacle et al, 2003; Wang et al, 2004) and its expression at the cell surface is usually very weak in most cell types. There Live cell confocal imaging of MT1-MMP dynamic are clear evidences showing that this protein undergoes redistribution and activity at invasive structures endocytosis (Jiang et al, 2001; Uekita et al, 2001; Galvez To understand better MT1-MMP regulation during tumor cell et al, 2002) and recycling to the surface (Remacle et al, 2003; invasion, we transfected breast carcinoma MDA-MB-231 cells Wang et al, 2004) in stationary cells. However, there are with MT1-MMP-GFP and embedded them in three-dimen- no evidences so far describing regulation of MT1-MMP by sional matrices (3D-Col I). Live cell confocal imaging regulated exocytic processes. showed, for the first time, activity and dynamics of MT1- Rab8 was initially isolated as a transforming gene from a MMP during cell invasion (Figure 1A and Supplementary melanoma cell line (Nimmo et al, 1991). It belongs to the Rab Video 1). Fluorescence and reflection images, revealing MT1- family of Ras-related GTPases that play a crucial role in MMP-GFP localization and collagen fiber organization membrane traffic by determining the specificity of vesicle respectively, showed MT1-MMP dynamic redistribution to col- transport (Zerial and McBride, 2001). Although the traffic lagen fiber adhesion sites at the PM, and subsequent collagen route regulated by Rab8 is not established, it is known to fiber degradation (Figure 1A and Supplementary Video 1). Figure 1 MT1-MMP accumulates at the sites of active collagen degradation during invasion of 3D-Col I matrices. (A) Live cell confocal imaging of MT1-MMP-GFP expressed in MDA-MB-231 cells embedded into 3D-Col I. Overlay of MT1-MMP-GFP fluorescence (green) and collagen fiber reflection (blue) images obtained at the indicated time points during the course of a time-lapse experiment is shown (see Supplementary Video 1). (B, C) Localization of endogenous MT1-MMP revealed by immunofluorescence staining with Lem-2/15 mAb of MDA-MB-231 cells and endometrial carcinoma primary cultured cells embedded into 3D-Col I. (B) Overlay of MT1-MMP staining (green) and collagen fiber reflection (blue) images is shown. (C) Overlay of phase contrast and fluorescence images. Insets show membrane sites engaging bundles of collagen fibers. GFP concentration at invasive structures is pointed by arrowheads. 1500 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Cells overexpressing large amounts of MT1-MMP-GFP were Polarized vesicle traffic is responsible for the used only to monitor MT1-MMP activity during invasion accumulation of MT1-MMP at collagen contact sites as they produced a high extent of matrix destruction, thus Localization of MT1-MMP to invasive structures has been clearly showing that MT1-MMP-GFP retains protease activity. described previously (Nakahara et al, 1997; Lehti et al, 2000; However, subsequent live cell invasion studies were carried Mori et al, 2002), although the underlying mechanism out using cells expressing low amounts of MT1-MMP-GFP, responsible for its redistribution on the cell surface has not which behaved in a more physiological manner. This was been elucidated. MT1-MMP localization was analyzed in accomplished by choosing cells with dim GFP fluorescence MDA-MB-231 cells embedded into 3D-Col I. Interestingly, a producing punctual degradation of collagen fibers during the novel compartmentalization of MT1-MMP-positive vesicles invasive process. A non-linear pattern of MT1-MMP localiza- at submembranous pools in invasive structures was observed tion at membrane protrusions in contact with the underlying (Figure 2A). Clear evidence of recruitment of MT1-MMP- 3D matrix was also shown for endogenous MT1-MMP in carrying vesicles to invasive structures was obtained when MDA-MB-231 and primary carcinoma cells as revealed by monitoring the formation of a new membrane protrusion immunostaining with specific anti-MT1-MMP antibodies event (Figure 2A, pointed by arrowheads and Supplementary (Abs) (Figure 1B and C). Video 2). Cell adhesion to collagen fibers leading to PM Figure 2 MT1-MMP intracellular vesicle recruitment toward collagen contact sites at the PM of MDA-MB-231 cells. (A) Live cell imaging of MDA-MB-231 cells transfected with MT1-MMP-GFP and embedded into 3D-Col I. Overlay of fluorescence and phase-contrast images showing MT1-MMP-GFP localization (pink) and cell morphology/collagen fiber distribution respectively, acquired at different time points is shown (see Supplementary Video 2). Arrowheads point to a new contact established between the cell membrane and a meshwork of collagen fibers, where active vesicle recruitment is observed. (B) MDA-MB-231 cells expressing MT1-MMP-GFP (green), cultured on glass coverslips, were incubated with Col I- or BSA-coated beads for 1 h, fixed and imaged. Fluorescence image (showing GFP), phase-contrast image (showing cell morphology and bead localization) and their overlay are presented. (C) Beads coated with BSA, Fn, anti-b1 Ab (TS2/16) or Col I were allowed to interact for 1 h with MDA-MB-231 cells that had been previously treated with or without not with a blocking anti-b1 (Lia1/2) or control (BerEP4) Abs. Cells were then fixed and imaged by confocal microscopy. Bars represent relative fluorescence intensity at the bead surrounding area normalized for background fluorescence calculated at 10–15 beads for each of the three independent experiments performed. The statistical significance of relative bead fluorescence comparing the different bead coatings and control (BSA) values (*) and antibody-treated compared to isotype /### control values (#) was evaluated using Student’s t-test. (*Po0.05; *** Po0.001). &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1501 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al protrusion was accompanied by local recruitment of MT1- The biosynthetic transport of proteins to the cell surface MMP-positive intracellular vesicles to a submembranous area, occurs via the recycling endosomes (Futter et al, 1995; followed by the local accumulation of MT1-MMP at the Leitinger et al, 1995; Ang et al, 2004; Lock and Stow, 2005). protrusive membrane (Figure 2A and Supplementary Video Moreover, recycling has been proposed as a mechanism of 2). Visualization of vesicle trajectories by fast scanning con- MT1-MMP recruitment to the leading edge during cell migra- focal imaging revealed highly complex traffic going to and tion (Remacle et al, 2003; Wang et al, 2004). We therefore from the PM in different directions. Traffic from the cell center sought to determine the involvement of recycling in MT1- to the periphery is not obvious, although there is clear PM MMP-polarized exocytosis. We allowed MT1-MMP-GFP- transport from submembranous pools localized at the polar- transfected cells to uptake transferrin (Tf) and low-density ized areas (see Supplementary Figure 3 and Supplementary lipoprotein (LDL) to label recycling and lysosomal compart- Video 4–6). Polarized MT1-MMP vesicle traffic was also ments, and analyzed their colocalization with MT1-MMP- induced by adhered Col I-coated beads but not control BSA- GFP (Figure 4B and Supplementary Figure 10). Surprisingly, coated beads (Figure 2B). Dynamic live cell studies show very MT1-MMP showed almost negligible colocalization with Tf active vesicle recruitment to collagen-coated beads, where and a strong colocalization with LDL in 3D-Col I-embedded MT1-MMP is accumulated (Supplementary Figure 7 and cells, suggesting that MT1-MMP is absent from recycling Supplementary Video 8). To gain insight into the cues that compartment, and instead is being sorted to lysosome induced MT1-MMP vesicle recruitment, we allowed cells to degradation in invasive cells. However, when we used the interact with beads coated with different ECM matrix proteins. same experimental conditions as previous studies that Quantitative analysis showed MT1-MMP-specific mobilization demonstrated MT1-MMP recycling (Remacle et al, 2003; induced by Col I, Fn or b1 integrin clustering Abs, but not by Wang et al, 2004), that is, plating cells in coverslips, we BSA. Col I-induced MT1-MMP recruitment could be specifi- confirmed MT1-MMP localization at recycling compartments cally impaired by function blocking anti-b1 Abs (Figure 2C). (Figure 4B). In addition, primary tumor cells also showed Altogether, these results show that recruitment of MT1-MMP overlap of the TfRc/Rab11-positive recycling compartment vesicles induced by collagen engagement in MDA-MB-231 cells with endogenous MT1-MMP in cells grown on coverslips but is mediated by b1 integrin-dependent adhesive events. not in 3D-Col I-embedded cells (Figure 4C). Therefore, MT1- It has been proposed that MT1-MMP delivery to invasive MMP is confined within the biosynthetic, although it is structures is mediated by CD44-dependent membrane trans- absent from the recycling compartment in invasive cells. port, although our live cell studies prompted us to hypothesize that intracellular vesicle traffic was responsible for the accu- Rab8 but not Rab11 codistributes with MT1-MMP at mulation of MT1-MMP at invasive structures. We addressed exocytic vesicles, and is specifically mobilized by Col I this issue by combining fluorescence recovery after photo- Because Rab8 GTPase has been involved in polarized bleaching (FRAP) at the PM with fluorescence loss in membrane transport of PM proteins during the formation of photobleaching (FLIP) at the underlying submembranous membrane protrusions, we sought to determine its involve- compartment. Recovery of fluorescence monitored at the ment in MT1-MMP exocytic delivery to invasive structures. FRAP region quantitatively estimates the extent to which We found a strong colocalization of MT1-MMP and Rab-8 MT1-MMP membrane localization is dependent on membrane in intracellular vesicles (Figure 5A). Time-lapse confocal transport, independently of the contribution of vesicle income imaging revealed the presence of MT1-MMP in Rab8-positive from the intracellular compartment. In contrast to the lateral vesicles being transported to the invasive PM (Figure 5B PM (Figure 3A–F), no relocalization of fluorescent MT1-MMP and Supplementary Video 11), whereas colocalization of at the invasive PM was observed (Figure 3G–L) when the MT1-MMP with Rab11 was negligible (Figure 5C). Vesicles intracellular pool of vesicles was continuously bleached. recruited to Col I-coated beads also showed a strong coloca- Additional example is shown in Supplementary Figure 9. lization of Rab8 and MT1-MMP (Figure 5D). Interestingly, we Hence, at the invasive lamella, MT1-MMP membrane diffusion observed a striking colocalization of MT1-MMP and Rab8 is compromised and intracellular traffic is most likely the within membranes deposited at degraded matrix (Figure 5E). source of MT1-MMP accumulation at invading structures. Deposition of cell fragments within the extracellular matrix caused by exocytic release of vesicles has been related to rear retraction during tumor cell invasion (Friedl and Wolf, 2003; MT1-MMP is found in the biosynthetic, not the recycling Mayer et al, 2004), and MT1-MMP has been previously compartment in invasive MDA-MB-231 cells To explore the involvement of the biosynthetic pathway in shown to be released within these fragments in endothelial MT1-MMP-polarized exocytosis, we performed colocalization cells (Taraboletti et al, 2002). These results strongly suggest studies using a classical marker of this route, VSV-G, as a that traffic and fusion of exocytic vesicles carrying MT1-MMP reporter. MDA-MB-231 cells coexpressing VSV-G-YFP and to matrix degradation sites is regulated by Rab8. Moreover, collagen-coated beads specifically induced recruitment of MT1-MMP-mRFP were embedded into 3D-Col I; cells were Rab8- but not Rab11-positive vesicles (Figure 5F), indicating then incubated at 201C to allow accumulation at the TGN (Ang et al, 2003), where both proteins were found colocaliz- that Rab8-mediated traffic is induced by collagen interaction. ing (not shown). When shifting to 321C to allow rapid exit of VSV-G from the TGN, a number of vesicles displayed strong Rab8 regulates traffic of MT1-MMP to invasive colocalization of MT1-MMP and VSV-G, and were found structures, and MT1-MMP-dependent collagen to translocate to the PM at invasive sites (Figure 4A). degradative activity and invasion These results suggest that biosynthetic exocytic traffic is The involvement of Rab8 in the regulation of MT1-MMP involved in the recruitment of MT1-MMP to invasive activity was first evaluated by quantitative experiments of structures at the PM. MT1-MMP vesicle recruitment in cells expressing wtRab8, 1502 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 3 MT1-MMP FRAP/FLIP experiments reveal that intracellular vesicle traffic is responsible for the accumulation of MT1-MMP at the invasive PM. MT1-MMP-GFP expressing MDA-MB-231 cells embedded into 3D-Col I were subjected to FRAP-FLIP photobleaching experiments. Images showing prebleaching, bleaching and post-bleaching at the PM (FRAP region) during continuous photobleaching of the submem- branous compartment (FLIP region) at the lateral (A–E) and invading (G–K) PM. Fluorescence recovery quantification at the FRAP region is calculated at the lateral (F) and invading (L) PM and represented in the graph. Rab8-activated mutant (Rab8Q67L) or Rab8DC (inactive Rab8 overexpression and activation induced collagen degra- mutant with impaired membrane localization owing to loss of dative activity (Figure 6C). Function blocking anti-MT1-MMP the prenylation site). MT1-MMP-mRFP vesicle recruitment to Ab (Lem-2/15) significantly abrogated Rab8-induced inva- Col I-coated beads was significantly induced by overexpres- sion and collagen degradation (Figure 6B, C), thus indicating sion of Rab8-activated mutant, but not by Rab8DC control its endogenous MT1-MMP dependence. These studies de- (Figure 6A). Specificity of Rab8 effect was demonstrated by monstrate the involvement of Rab8 in regulating MT1-MMP examining CD44 recruitment to hyaluronic acid (HA)-coated delivery to the PM and its collagenolytic and proinvasive beads, which was unaffected by the expression of Rab8 activities. constructs (Supplementary Figure 12). Furthermore, trans- To demonstrate further the role of Rab8 in MT1-MMP well collagen invasion assays showed that similar to MT1- exocytic traffic to the PM, we performed gene silencing MMP overexpression, Rab8Q67L and wtRab8 induced an studies. Stable cell lines carrying short-hairpin RNA increase in cell invasion, whereas DC control was shown to (shRNA) targeted Rab8a and Rab11a showed protein deple- be ineffective (Figure 6B). Pericellular collagenolysis evalu- tions of approximately 80 and 60%, respectively, as assessed ated in cells expressing the different constructs revealed that by Western blotting analysis (Figure 7A and B). The possibi- &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1503 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 4 MT1-MMP colocalization with markers of the biosynthetic/recycling and degradative routes. (A) MDA-MB-231 cells cotransfected with MT1-MMP-mRFP and VSV-G-YFP were embedded into 3D-Col I. Cells were incubated overnight at 401C, then transferred to 201C for 2 h and finally shifted to 321C for 1 h. Cells were then fixed and imaged. Arrowheads point to vesicles positive for both VSV-G (green) and MT1- MMP (red). Overlay image shows colocalization (yellow) and fiber reflection (blue). (B) MDA-MB-231 cells transfected with MT1-MMP-GFP were either embedded into 3D-Col I (upper panel) or plated on coverslips (lower panel) and incubated with labelled Tf and LDL for 1 h at 371C to allow their internalization. Images show localization of MT1-MMP-GFP (green), Tf (blue), LDL (red), and their overlay. (C) Primary lung adenocarcinoma cells were either embedded into 3D-Col I (upper panel) or plated on coverslips (lower panel) and immunostained with specific Abs for TfRc or Rab11 (red), and MT1-MMP (green), as indicated. Insets show superimposed fluorescence images pseudocolored in green/red; arrowheads point colocalization vesicles (shown in yellow). lity of having off-target effects was ruled out by analyzing the (Figure 7D) and collagen degradation (Figure 7E) were levels of Rab11 protein in Rab8 knocked down cells and vice impaired in cells expressing shRNA for Rab8 but not Rab11. versa control and Rab8-silenced cells displayed similar levels Moreover, ectopic expression of Rab8 coding sequence tagged of surface MT1-MMP expression (10 and 9.3 mean fluores- with mRFP carrying four silent mutations in Rab8shRNA1 cence intensity) as revealed by flow cytometry analysis. targeting sequence reconstituted these functions (Supple- Thus, steady-state expression of MT1-MMP at the cell surface mentary Figure 13). Transiently transfected Rab8shRNA in was unaffected by Rab8 knockdown. However, endogenous mammalian expression vectors rendered similar effects MT1-MMP vesicle recruitment to Col I-coated beads (Supplementary Figure 14). CD44 recruitment to HA-coated (Figure 7C), MT1-MMP-induced tumor cell invasion beads was unaffected by Rab8shRNA, further demonstrating 1504 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 5 Rab8 but not Rab11 codistributes with MT1-MMP during vesicle transport to the PM. MDA-MB-231 transfected with MT1-MMP-mRFP and Rab8-GFP were embedded into 3D-Col I and analyzed by confocal imaging. (A) MT1-MMP-mRFP fluorescence (red), Rab8-GFP (green) and the superimposed images where colocalization can be seen in yellow, as well as the image showing exclusively colocalizing pixels (white) are shown. 2D colocalization histogram corresponding to these images obtained using Imaris software (Bitplane AG, Zurich, Switzerland) is also shown. Live cell imaging of 3D-Col I invading MDA-MB-231 cells transfected with MT1-MMP-mRFP and either Rab8-GFP (B) or Rab-11- GFP (C). Images acquired at the indicated time points show Rab8 or Rab11 (green) and MT1-MMP (red) localization during the course of the experiment (see Supplementary Video 11). (D) MDA-MB-231 cells expressing MT1-MMP-mRFP (red) and Rab8-GFP (green), cultured on glass coverslips, were incubated with Col I-coated beads for 1 h, then fixed and imaged. Overlay of fluorescence images is presented in inset. Asterisk indicates bead localization. (E) Confocal images of MT1-MMP-mRFP (red) and Rab8-GFP (green) fluorescence and collagen fiber reflection (blue) shows colocalization of MT1-MMP and Rab8 attached to degraded collagen fibers. (F) Beads coated with BSA or Col I were allowed to interact with Rab8-GFP- or Rab11-GFP-expressing MDA-MB-231 cells. Bars represent relative fluorescence intensity at the bead surrounding area normalized to background fluorescence calculated at 10–15 beads for each of the three independent experiments performed. that polarized distribution of other surface proteins is inde- to collagen engagement. Accordingly, integrin clustering pendent of Rab8 (Supplementary Figure 12). These results stimulates cell-surface expression of MT1-MMP (Ellerbroek clearly demonstrate that Rab8 GTPase specifically mediates et al, 2001), and coclustering of b1 integrins and MT1-MMP regulated, not constitutive, transport of MT1-MMP to the PM, has been shown in tumor cells invading 3D collagen matrices MT1-MMP-dependent collagen degradation and invasion. (Wolf et al, 2003), and endothelial cells adhered to collagen- coated surfaces, where the biochemical association of both MT1-MMP and b1 integrin was demonstrated (Galvez et al, Discussion 2002). MT1-MMP proinvasive activity requires its redistribution Focal degradation of the ECM barrier at the invading cell front to motility-related structures (Nakahara et al, 1997; Lehti is a key process in tumor invasion, and this is achieved by et al, 2000; Mori et al, 2002). Seiki and co-workers have localization of proteases at the leading edge of migrating suggested the interaction of MT1-MMP with CD44, and the cells. There are clear evidences that MT1-MMP localizes at invasive structures (Nakahara et al, 1997; Lehti et al, 2000; linkage of the latter to the actin cytoskeleton, as the mechan- Mori et al, 2002). However, how precisely the enzyme is ism driving the proteinase to the leading edge of migrating cells (Mori et al, 2002; Suenaga et al, 2005). Our data on 3D targeted to the the invasion sites remains to be determined. invasion models point out a completely novel mechanism, Three dimensional collagen matrices mimic the ‘in vivo’ regulated exocytosis of MT1-MMP vesicles, mediating MT1- environment encountered by tumor cells, and so provide a MMP recruitment to invasive structures. This hypothesis is surrogate of the tissue microenvironment, allowing us to perform live cell studies of tumor cell invasion. We herein based in several pieces of evidence: (1) dynamic visualization show for the first time dynamic redistribution and activity of of MT1-MMP vesicles being recruited to the cell surface from MT1-MMP at invasive structures, as visualized by live con- intracellular locations at collagen fiber attachment sites preceding membrane protrusion; (2) FRAP/FLIP experiments focal imaging of MDA-MB-231 adenocarcinoma cell invasion showing that submembranous vesicle pool rather than mem- of 3D-Col I. b1 Integrin-dependent adhesion was found to be brane diffusion is required for the accumulation of MT1-MMP the spatial cue leading to MT1-MMP recruitment in response &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1505 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 6 Rab8 activation induces recruitment of MT1-MMP vesicles, MT1-MMP-dependent collagen degradation and invasion. (A) MDA-MB- 231 cells were cotransfected with MT1-MMP-mRFP and either GFP, wtRab8-GFP, Rab8Q67L-GFP or Rab8DC-GFP, and, allowed to interact with Col I-coated beads for 1 h, then fixed and analyzed by confocal microscopy. Bars represent the percentage of MT1-MMP-mRFP fluorescence intensity around the bead calculated in 10–18 cells expressing the different GFP constructs from three independent experiments. (B) MDA-MB- 231 cells were transfected with GFP, wtRab8-GFP, Rab8Q67L-GFP, Rab8DC-GFP or MT1-MMP-GFP. Cells were then allowed to migrate for 48 h on transwell filters coated with 3D-Col I to FCS containing media in the presence of isotype control IgG (solid bars), or function blocking anti- MT1-MMP Ab (Lem-2/15) (open bars). Bars represent the percentage of invaded GFP-expressing cells quantified in seven independent experiments by counting four different fields for each experiment. (C) MDA-MB-231 cells transfected with GFP, wtRab8-GFP, Rab8Q67L-GFP, Rab8DC-GFP or MT1-MMP-GFP were cultured on 2D-Col I layers for 48 h in the presence or absence of function blocking anti-MT1-MMP Ab (Lem-2/15), then fixed and labelled with anti-Col I antibody to evaluate degradation. Representative overlay images of Col I staining (red) and expression of the different constructs (green) is shown. The statistical significance comparing expression of different constructs to control /# /## (GFP) values (*) and antibody-treated compared to isotype control values (#) was evaluated using Student’s t-test (* Po0.05; ** Po0.01; /### *** Po0.001). at the invasive PM; and (3) the requirement of active Rab8, complex to the PM that bypasses recycling endosomes, which a GTPase involved in exocytic traffic, for collagen-induced could be involved in the traffic of MT1-MMP to invasive MT1-MMP recruitment to the membrane, MT1-MMP-depen- structures. Our results showing that MT1-MMP intracellular dent collagen degradation and invasion. In agreement with compartmentalization depends on the extracellular context this hypothesis, an intracellular functional pool of MT1-MMP may provide a rationale for internalized MT1-MMP. MT1- available for trafficking to the cell surface upon stimulation of MMP will recycle when cells are not involved in ECM HT1080 cells with ConA has been reported (Zucker et al, degradation thus maintaining a controlled surface activity, 2002). while allowing intracellular pools to be stored for rapid The confinement of MT1-MMP within the biosynthetic and trafficking if necessary. In contrast, MT1-MMP will be mobi- its absence from recycling compartments seems contradictory lized to a degradative compartment when cells are actively as there is increasing evidence showing that biosynthetic involved in ECM proteolytic processing to prevent accumula- transport to the cell surface occurs via recycling endosomes tion of inactivated MT1-MMP (TIMP-2-inhibited or partially (Futter et al, 1995; Leitinger et al, 1995; Ang et al, 2004; Lock degraded molecules). We can, therefore, establish a strong and Stow, 2005). However, a number of live imaging studies parallelism between the homeostasis of MT1-MMP and the (Lippincott-Schwartz et al, 2000; Lock and Stow, 2005) sup- so-called constitutive cycling traffic reported for a number of port the existence of a direct delivery pathway from the Golgi membrane proteins (reviewed by Royle and Murrell-Lagnado, 1506 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 7 Rab8 but not Rab11 knockdown with shRNA decreases MT1-MMP vesicle recruitment, collagen degradation and invasion. MDA-MB- 231 cells stably expressing PMCSV Pig (control), or PMCSV carrying Rab8shRNA sequences 1 and 2 or Rab11shRNA sequences 1 and 2. (A) The levels of Rab8 protein was assessed by Western blot analysis. Control tubulin blotting is also shown. (B) Quantification of Rab8 protein levels normalized using tubulin as a loading control is represented in the bar diagram. Endogenous vesicle recruitment (C), transwell invasion (D) and collagen degradation (E) were evaluated as described in Figure 6. (E) Representative images show Col I staining (red) and GFP expression from PMCSV vector (green). Asterisks indicate statistical significance comparing the expression of the different shRNAs to control (PMCSV Pig) values. 2003). A good example is the glucose transporter GLUT4, the recycling compartment instead; (iii) the possibility that, which undergoes rapid constitutive internalization and sub- as reported for GLUT4 and Rab8, there is a transport loop sequent slow recycling back to the surface, and therefore between the storage compartment and recycling endosomes under basal conditions, exists predominantly within intra- is not excluded; and (iv) endocytosis targeted to lysosome cellular compartments (Dugani and Klip, 2005). Despite being degradation will most likely be the fate of surface MT1-MMP, engaged in a recycling loop, there is a more static secretory inactivated during the process of matrix degradation. This pool of GLUT4 storage vesicles ready to move directly to the model would keep a potentially harmful enzyme away from cell surface in response to insulin stimulation (Dugani and the PM, where it could exert unwanted side-effects, despite Klip, 2005). Accordingly, both MT1-MMP and GLUT4 have being an extremely sensitive system for rapid and localized been localized at Rab8-positive vesicles (our data and Miinea enzyme mobilization, avoiding the slow process of protein et al, 2005), and their transport to the membrane is dependent synthesis. on syntaxin 4 (Widberg et al, 2003; Miyata et al, 2004). Rab8 was first described as an oncogene isolated as a Our studies reveal a novel pathway in the regulation of transforming gene from a melanoma cell line (Nimmo et al, MT1-MMP and allow us to propose a model for MT1-MMP 1991), although its relevance in cancer has not been estab- homeostasis (Figure 8). In this model, different traffic path- lished yet. Notably, Rab8 search in Oncomine cancer profiling ways of MT1-MMP are highlighted: (i) Rab8-regulated exo- database (www.oncomine.org) showed its overexpression in cytic mobilization from an intracellular storage compartment tumoral versus normal tissues in different microarray data different from recycling endosomes would account for polar- sets. Rab8 belongs to the family of Ras-like small GTPases ized recruitment of MT1-MMP to the invasive PM engaged that are major regulators of membrane trafficking in eukar- in matrix degradation (i.e. this report); (ii) constitutive cycling yotic cells (Zerial and McBride, 2001). Although the traffic will be predominant in a stationary cell, where MT1-MMP is route regulated by Rab8 is still not clarified, there is however not involved in ECM proteolytic processing, being found in evidence that it is involved in the transport of PM proteins at &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 6 2007 1507 | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Figure 8 Model for MT1-MMP intracellular trafficking. The model depicts two main intracellular pathways (I) Rab8-regulated exocytic mobilization of MT1-MMP from a biosynthetic storage compartment induced by collagen engagement in invading cells (II) Constitutive cycling from recycling endosomes in a stationary cell involves MT1-MMP un-engaged in matrix degradation. Additional pathways could involve (III) transport loop between the biosynthetic storage and recycling compartments and (IV) endocytosis targeted to lysosome degradation of surface MT1-MMP involved in collagen degradative activity. et al, 2003). MT1-MMP-mRFP was obtained by subcloning mRFP membrane protrusions (Peranen et al, 1996; Hattula et al, into EGFP restriction sites. Ts045 VSV-G-YFP and Rab11-GFP were 2002; Ang et al, 2003). Our results clearly show the involve- kindly provided by Dr R Peppercok and Dr D Sheff, respectively. ment of Rab8 in the traffic of MT1-MMP to the PM and in Abs used include LEM-2/15 anti-MT1-MMP (Galvez et al, 2002) and MT1-MMP-dependent collagen degradation and invasion, TS2/16 and Lia1/2 anti-human b1 integrin, kindly provided by Dr Sa´nchez-Madrid. Mouse mAb anti-TfRc and rabbit polyclonal Ab which may help to explain Rab8-transforming activity. to Rab11 were from Zymed Laboratories (South San Francisco, Considering the importance of MT1-MMP in tumor cell CA, USA). Goat polyclonal anti-Rab8 Ab was from Santa Cruz invasion and angiogenesis, there is a great interest in target- Biotechnology (Santa Cruz, CA, USA) and mAb anti-Col I and anti- ing this enzyme with new inhibitors. Cancer therapeutics tubulin DM1a Ab were from Sigma (St Louis, MO, USA). Anti- human epithelial antigen BerEP4 was from DakoCytomation designed to target protease activity by synthetic MMP inhi- (Glostrup, Denmark). bitors have proven ineffective. According to our results, an alternative strategy based on blocking MT1-MMP delivery Immunofluorescence, Tf/LDL uptake and confocal microscopy to invasive structures by means of Rab8 targeting will be a Cells were either plated onto coverslips or embedded into 3D-Col I, more rational means of preventing invasion and metastasis fixed at 41C for 5 min with 4% paraformaldehyde and permeabi- mediated by MT1-MMP, without affecting the enzyme basal lized with 0.5% Triton X-100 and stained with the appropriate Abs. Tf/LDL uptake was monitored by incubating 1 h serum-starved cells homeostasis. Important future work involving animal models with Tf-Alexa 647 (20 mg/ml) and dil-LDL (low-density lipoprotein should first be undertaken to validate Rab8 as a therapeutic conjugated to 3,3 -dioctadecylindocarbocyanine) (10 mg/ml) for 1 h cancer target. at 371C. Cell imaging was performed using a Leica TSC SP2 AOBS and SP5-RS AOBS with a 63  Plan Apo 1.32 NA oil-immersion objective (Leica, Mannheim, Germany). Leica Confocal Software Materials and methods (LCS) was used for acquisition of images, which were later adjusted for contrast using Adobe Photoshop Software. Colocalization Cell culture, transfection and collagen inclusion analysis was performed with Imaris software (Bitplane AG, Zurich, Breast adenocarcinoma MDA-MB-231 cells were maintained in Switzerland). DMEM supplemented with 10% FBS. Primary carcinoma cells were purified from fresh human endometrial and lung carcinoma tumor Polystyrene bead assays samples by enzymatic digestion as described elsewhere (Allinen Polystyrene divinyl-benzene beads (5 mm) (Duke Scientific Corpora- et al, 2004) and cultured in HAMF10 medium supplemented with tion, Palo Alto, CA, USA) were incubated with 0.5% BSA, 100 mg/ml 10% FBS. Cell transfection was performed using lipofectamine 2000 Col I (Vitrogen Palo Alto, CA, USA), 20 mg/ml Fn, 1 mg/ml HA (Invitrogen, Carlsbag, CA, USA) according to the manufacturer’s (Sigma, St Louis, MO, USA) or TS2/16 Ab anti-b1 integrin culture instructions. At 24 h post-transfection, cells were trypsinized and supernatant. Cells expressing the different constructs were incu- mixed with readily prepared Col I solution (2,4 mg/ml bovine Col I bated for 1 h with coated beads at a cell to bead ratio of 1:40. For (Vitrogen, Palo Alto, CA, USA), 1  RPMI, 19 mM HEPES (Gibco), inhibition studies, cells were previously incubated with or without 0.19% sodium bicarbonate (Sigma) and 5% FBS) that was then 10 mg/ml of blocking anti-b1 integrin Ab Lia1/2 or control BerEP4 allowed to polymerize for 2 h at 371C (3D-Col I) or plated on rat tail Ab (anti-human epithelial antigen). Confocal images were analyzed Col I protein (Roche Diagnostics, Panzberg, Germany)-coated for MT1-MMP fluorescence in a region around the bead and surfaces (2D-Col I layers). normalized to the overall background MT1-MMP fluorescence determined in three regions at irrelevant membrane areas of the Constructs and antibodies cell. Relative bead fluorescence represents quantified bead fluor- EGFP-tagged MT1-MMP, Rab8wt, Rab8Q67L and Rab8DC con- escence\background fluorescence  100 scored in at least 10 beads structs have been described previously (Galvez et al, 2002; Ang for each experimental condition. 1508 The EMBO Journal VOL 26 NO 6 2007 &2007 European Molecular Biology Organization | | MT1-MMP polarized exocytosis mediated by Rab8 JJ Bravo-Cordero et al Confocal photobleaching experiments the amphotropic vector pCL-Ampho, retrovirus packaging vector. After 48 h, transfection retrovital supernatants were used as retro- A combination of both FRAP and FLIP techniques was developed on a Leica TSC SP2 AOBS microscope using Leica Confocal Software viral stock for transduction of MDA-MB 231 cells. Cells expressing the (Leica, Mannheim, Germany). Live MT1-MMP-GFP expressing cells different shRNA constructs were selected with puromycin (0.5 mg/ embedded into 3D-Col I gels were exposed to a bleaching regime ml) for 5 days and GFP-expressing cells were sorted by flow consisting of (1) prebleach recording (scanning three images with cytometry to obtain stable shRNA-expressing cell lines. Only shRNA laser AOTF 20%), (2) bleaching and scanning at two different Rab8 (1), shRNA Rab8 (2), shRNA Rab11 (1) and shRNA Rab11 (2) regions of interest (membrane and submembranous compartments) showed significant depletion of Rab8 and Rab11 and were used for using bleaching laser excitation settings (100% AOTF) in both subsequent analysis. For shRNA rescue assays, four silent mutations regions and regular imaging scanning settings (20% AOTF) for the were introduced to the shRNA Rab8 1 targeting sequence (nucleo- rest of the field and (3) post-bleaching recording. During the post- tides 165–183). The final mutated Rab8 sequence (aggattaagttgcaaa- mut bleaching phase, the PM (FRAP region) was excited with regular ta) was obtained by PCR and subcloned into mRFP vector. Rab8 - imaging settings (20% AOTF,) whereas continuous bleaching mRFP was transiently transfected into shRNA Rab8 (1) -expressing settings (100% AOTF) were used at the FLIP region. The relative stable cell line. Rab8 and Rab11 protein levels were analyzed by loss of intensity and recovery of fluorescence was calculated at Western blotting. Alexa Fluor 680-conjugated secondary Abs were the FRAP region after background subtraction using Siggia normal- used to visualize and quantify the blots using the Odyssey Infrared ization (Siggia et al, 2000). Imaging System (Li-COr, Biosciences). Collagen degradation and cell invasion assays Statistical analysis MDA-MB231 cells were transfected with the different constructs. All numerical values reported represent mean7s.e. The statistical At 24 h after transfection, cells were plated onto 2D-Col I layers and significance comparing differences between the experimental and incubated for additional 48 h, fixed and immunostained for Col I. control (GFP/BSA) values (*) and Ab-treated compared with MDA-MB-231 cell invasion assays were performed in 8-mm pore 3D- isotype control values (#) was evaluated using Student’s t-test. Col I gel-coated transwell chambers (Costar). Cells were transfected Po0.05 was taken as the limits of statistical significance /# /## /### with the different constructs and after 24 h, resuspended in serum- (* Po0.05; ** Po0.01; *** Po0.001). free medium and seeded at 5 10 cells/well. Cells were allowed to transmigrate to 10% FBS media for 48 h and then counted at the top Supplementary data and bottom of the chamber using Image J software (NIH, Bethesda, Supplementary data are available at The EMBO Journal Online USA). Bars represent the percentage of invasive cells referred to the (http://www.embojournal.org). total number of cells considering only GFP or mRFP/GFP expressing cells. Acknowledgements Rab8 gene silencing with shRNAs Three different siRNA sequences were designed for silen- We thank Drs MA del Pozo and MA Alonso for helpful advice and cing Rab8a and Rab11a with the help of web-based algorithms critical reading of the manuscript, Dr Rivera for help with biochem- (http://side.bioinfo.ochoa.fib.es/) and (www.Invitrogen.com) (Rab8 ical studies, Dr M Malumbres for help with shRNA design and 0 0 (1) : 5 -GAGAATTAAACTGCAGATA, Rab8 (2) : 5 -GGAACTGGATTCG the Genomics Unit for help with shRNA cloning. Drs Mellman, 0 0 0 CAACATTG-3 and Rab8 (3) : 5 -GCTCGATGGCAAGAGAATTAA-3 ), Sa´nchez-Madrid, Pepperkok, Sheff and Tsien are acknowledged for 0 0 0 (Rab11 (1) : 5 -AAGAGCACCATTGGAGTAGAGTT-3 , Rab11 (2) : 5 -G providing us with reagents. Tumour cell samples were provided by 0 0 TACGACTACCTCTTTAAA-3 and Rab11 (3) : 5 -GCAACAATGTGGTT the CNIO Tumour Bank Unit. This work was supported by a grant CCTATTC-3 ). shRNAs were cloned into the retroviral vector MSCV from Fondo de Investigaciones Sanitarias (FIS PI031324) to MCM. Pig, a modified version of MSCV-puro (Clontech), which contains JJ B-C and R M-D are funded by the Ministry of Science and GFP to report shRNA expression. HEK-293T cells were cotransfected Technology of Spain (MCYT) and Fondo de Investigaciones with 10 mg of the plasmid containing the different shRNA and 10 mgof Sanitarias (FIS), respectively. 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Journal

The EMBO Journal – Springer Journals

Published: Mar 21, 2007

Keywords: matrix metalloproteinases; membrane traffic; MT1‐MMP; Rab8; tumor invasion

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