Abstract
Association of matrix metalloprotease 9 (MMP9) to the cell membrane is considered important in tumor growth and angiogenesis. To dissect this regulatory mechanism, we generated raft and non-raft MMP9 chimeras to force membrane expression in the MCF-7 human breast carcinoma cell line. MMP9 targeting to non-raft cell surface domains rendered a constitutive active membrane MMP9 form, suggesting a contribution by the lipid environment in MMP activation. We generated human breast cancer xenograft models using MCF-7 cells overexpressing secreted and membrane-anchored MMP9. The non-raft MMP9 chimera was constitutively active at the cell membrane in xenografts, but this activation did not correlate with an increase in MMP9-induced angiogenesis. Capillary number and vessel perimeter were specifically increased only in tumors overexpressing wild-type MMP9 (the secreted form); this increase was inhibited when tumors were induced in doxycycline-treated mice. Xenografts from tumor cells overexpressing wild-type MMP9 showed increased vascular endothelial growth factor (VEGF)/VEGFR2 receptor association, which was also dependent on MMP9 activity. These observations indicate that membrane location can influence MMP9 activity in vitro and in vivo, and confirm the relevance of stromal-associated, but not tumor-bound MMP9 in mediating tumor-induced angiogenesis.
Highlights
Increasing evidence suggests that matrix metalloproteases (MMP), a family of multidomain, zinc-containing neutral endopeptidases, contribute to the formation of a microenvironment that promotes tumor growth during early stages of tumorigenesis (Egeblad and Werb, 2002; Overall and López-Otín, 2002)
matrix metalloprotease 9 (MMP9) targeting to raft and non-raft membranes To force MMP9 expression at the cell membrane, we fused a GPI consensus sequence (MMP9-GPI) or the transmembrane low-density lipoprotein receptor (LDLR) domain (MMP9-LDL) to the MMP9 C terminus (Fig. 1A)
A fraction of MMP9-GPI was recovered in the conditioned medium, it represented less than 0.1% of the total chimera expressed
Summary
Increasing evidence suggests that matrix metalloproteases (MMP), a family of multidomain, zinc-containing neutral endopeptidases, contribute to the formation of a microenvironment that promotes tumor growth during early stages of tumorigenesis (Egeblad and Werb, 2002; Overall and López-Otín, 2002). The MMPs have traditionally been considered proteolytic enzymes for extracellular matrix (ECM) components, it is recognized that MMPs cleave ‘non-matrix’ molecules, including growth factors, growth factor binding proteins, cytokines, chemokines, adhesion and death receptors as well as other proteinases, modifying their biological activity (Mañes et al, 1997; Mañes et al, 1999a; Wilson et al, 1999; Bergers et al, 2000; McQuibban et al, 2000; Yu and Stamenkovic, 2000; Fingleton et al, 2001; Rodríguez-Manzaneque et al, 2001; McQuibban et al, 2002) These MMP degradative activities may augment or reduce proliferation, survival and migration of both tumor and stromal cells. It has been proposed that membrane-bound MMP9 represents only a very small fraction of the enzyme and that the MMP9 biologically relevant for tumor development is the secreted form (Ramos-DeSimone et al, 1999)
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