Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-␤ and promotes tumor invasion and angiogenesis Qin Yu and Ivan Stamenkovic1 Molecular Pathology Unit, Massachusetts General Hospital, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02129 USA We have uncovered a novel functional relationship between the hyaluronan receptor CD44, the matrix metalloproteinase-9 (MMP-9) and the multifunctional cytokine TGF-␤ in the control of tumor-associated tissue remodeling. CD44 provides a cell surface docking receptor for proteolytically active MMP-9 and we show here that localization of MMP-9 to cell surface is required for its ability to promote tumor invasion and angiogenesis. Our observations also indicate that MMP-9, as well as MMP-2, proteolytically cleaves latent TGF-␤, providing a novel and potentially important mechanism for TGF-␤ activation. In addition, we show that MMP-9 localization to the surface of normal keratinocytes is CD44 dependent and can activate latent TGF-␤. These observations suggest that coordinated CD44, MMP-9, and TGF-␤ function may provide a physiological mechanism of tissue remodeling that can be adopted by malignant cells to promote tumor growth and invasion. [Key Words: MMP-9; CD44; TGF-␤; tumor angiogenesis; tumor invasion] Received September 10, 1999; revised version accepted December 7, 1999. The ability of tumor cells to establish a functional rela- nals transduced by integrins following engagement by tionship with their host tissue microenvironment and to ligands may provide tumor cells with critical survival utilize the local resources to their advantage are key fac- signals within the host tissue microenvironment (Frisch tors in determining tumor cell survival, local tumor de- and Ruoslahti 1997). Ligand engagement of CD44 can velopment, and subsequent tumor invasion. Interaction also promote survival of some types of tumor cells in between tumor cells and the host tissue stroma results vivo (Yu et al. 1997), and numerous studies have pro- in local tissue remodeling that, on the one hand, reflects vided evidence that CD44 may play an important role in a reaction of the tissue to the invading tumor cells and, promoting tumor invasion and metastasis (Gunthert et on the other hand, serves to facilitate and promote tumor al. 1991; Sy et al. 1991; Seiter et al. 1993; Bartolazzi et al. development. Similar to inflammation and repair, tu- 1994; Lamb et al. 1997; Yu et al. 1997). Although the mor-associated tissue remodeling relies on a complex in- ability of CD44 to bind and internalize hyaluronan has terplay of at least three classes of molecules, that is, been shown to correlate with tumor cell invasiveness adhesion receptors that mediate physical interactions (Bartolazzi et al. 1994; Culty et al. 1994; Yu et al. 1997), between tumor cells and host tissue stromal cells and the mechanisms underlying CD44-mediated enchance- extracellular matrix (ECM), matrix metalloproteinases ment of tumor growth have remained largely obscure. (MMPs) that degrade ECM proteins, and cytokines/ We have shown recently that CD44 can localize proteo- growth factors that promote tumor cell survival and lytically active matrix metalloproteinase-9 (MMP-9) to growth. the surface of TA3 mammary carcinoma and MC mela- Experimental evidence indicates that integrin (for re- noma cells (Yu and Stamenkovic 1999). Hyaluronan-me- view, see Varner and Cheresh 1996) and hyaluronan re- diated cross-linking of CD44 induces coclustering of ceptor CD44 (Bartolazzi et al. 1994; Gunthert et al. 1991; CD44 and MMP-9 and promotes MMP-9 proteolytic ac- Sy et al. 1991)-mediated adhesion are required for tumor tivity, which correlates with TA3 cell invasiveness in cell migration as well as for growth and dissemination of vivo and in vitro and enhances tumor growth in vivo (Yu a variety of tumor types. Recent work suggests that sig- and Stamenkovic 1999). Compromising CD44 function by overexpression of a dominant-negative soluble CD44 1Corresponding author. disrupts cell surface CD44–MMP-9 cluster formation, re- E-MAIL [email protected]; FAX (617) 726-5684. duces cell surface MMP-9 activity, and inhibits invasion GENES & DEVELOPMENT 14:163–176 © 2000 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/00 $5.00; www.genesdev.org 163 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Yu and Stamenkovic and growth of the tumor cells (Yu and Stamenkovic growth as well. We also demonstrate that MMP-9, as 1999). Thus, one mechanism by which CD44 may pro- well as its relative, MMP-2, cleave latent transforming mote tumor growth and invasion is by regulating MMP-9 growth factor-␤ (TGF-␤), which constitutes a novel function on the cell surface. mechanism of TGF-␤ activation. Finally, we provide evi- Matrix metalloproteinases are thought to play a key dence that CD44-dependent cell surface localization of role in promoting tumor invasion and tissue remodeling proteolytically active MMP-9, and the corresponding la- by inducing proteolysis of several ECM components tent TGF-␤ activation, occur in normal as well as in (Stetler-Stevenson et al. 1993; Werb 1997). Recent evi- malignant cells. dence indicates that MMPs may promote angiogenesis (Brooks et al. 1996; 1998; Vu et al. 1998), and augment Results genetic instability in tumor cells (Sternlicht et al. 1999). CD44 is implicated in tumor angiogenesis MMP expression is induced in stromal cells at sites of injury and repair as well as at sites of tumor invasion, Using TA3 murine mammary carcinoma cells, which and many tumors produce their own MMPs as a part of constitutively express several CD44 isoforms and ex- their invasive machinery. However, the mechanisms by hibit CD44-dependent HA binding, up-take and degrada- which MMPs promote tumor invasion and angiogenesis tion, we have shown previously that expression of domi- are still poorly understood. For example, the role that nant-negative soluble CD44 inhibits tumor invasion and MMP activity may play in the release and activation of growth in syngeneic mice (Yu and Stamenkovic 1999; ECM-sequestered growth and angiogenic factors, which Fig. 1 A–C). We now show that in addition to impaired may be essential for tumor development, is largely un- invasiveness and reduced size, solid tumors derived from known. Furthermore, the majority of MMPs whose pro- the soluble CD44-transfected TA3 cells (TA3sCD44) are teolytic activity is associated with tissue remodeling are less vascularized than tumors derived from TA3 cells soluble and are efficiently inhibited by tissue inhibitors transfected with expression vector only (TA3wt, Fig. of metalloproteinases (TIMPs), which are abundant in 1A,B). To compare angiogenesis in the solid TA3wt and the ECM. Despite significant steps toward understand- TA3sCD44 tumors, we performed immunohistochemis- ing the complexities of MMP activation (for review, see try on sections of tumors derived from subcutaneously Nagase 1997), it remains unclear how MMPs might es- injected TA3wt and TA3sCD44 cells using anti-von Wil- cape or overcome TIMP inhibition to promote ECM lebrand factor antibody (anti-vWF), which stains vascu- breakdown and tumor invasion. lar endothelial cells. Solid TA3sCD44 tumors were ob- In the present study, we show that cell surface local- served to display less vascular endothelial cell staining ization of MMP-9 is an important factor in its ability to than their TA3wt counterparts (Fig. 1D,E). Blood vessel promote not only tumor invasion, but angiogenesis and quantification by averaging the number of capillary Figure 1. CD44 promotes tumor growth and angiogenesis. (A, B) Gross appearance of solid tumors derived from TA3wt (A)and TA3sCD44 cells (B). Bar, 7.5mm. (C) A total of 2 × 106 viable cells were injected subcutaneously into syngeneic A/Jax mice. The tumor specimens were collected 3 weeks after injection, and tumor weight was assessed. In each experiment, six mice were injected with each transfectant. Weight of tumors derived from TA3 transfectants is expressed relative to that of TA3wt 1 ±S.D.(D, E) Immuno- histochemistry with anti-vWF antibody (Dako) on tissue sections of solid tumors derived from subcutaneously injected TA3wt (D)or TA3sCD44 cells (E). The average number of the capillary blood vessels, as indicated by anti-vWF antibody staining, per 10× power microscopic field from 10 randomly chosen fields is shown (F). Bar, 160 µm. 164 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press MMP-9 activates TGF-␤ and promotes tumor invasion blood vessels positively stained by anti-vWF antibody in (Yu and Stamenkovic 1999). To investigate whether the 10 randomly picked microscopic fields revealed an ap- observed invasiveness is dependent on cell surface local- proximately threefold reduction in the number of blood ization of the proteolytic activity of MMP-9, TA3sCD44 vessels in the solid tumors derived from TA3sCD44 cells cells were stably transfected with a chimeric construct compared with tumors derived from TA3wt cells (Fig. composed of sequences encoding MMP-9 and the trans- 1F). Thus, reduced angiogenesis in solid tumors derived membrane and intracellular domains of CD44 (MMP-9/ from TA3sCD44 cells appeared to correlate with im- CD44fp, Fig. 2A). On expression in mammalian cells, paired CD44 function. the resulting fusion protein was tethered to the cell sur- face. As a control, TA3sCD44 cells were transfected with carboxy-terminal v5-epitope-tagged soluble MMP-9 (MMP-9v5, Fig. 2A). Expression of endogenous, and Localization of MMP-9 to the cell surface augments its soluble, transfected, CD44 were not affected by the in- ability to promote tumor invasion and angiogenesis troduction of MMP-9/CD44fp and MMP-9v5 cDNAs In previous studies we showed that CD44 localizes the into TA3sCD44 cells, as assessed by Western blot analy- proteolytic form of MMP-9 on the surface of TA3wt sis (Fig. 2Ba and b).