Processing of the laminin-5 (Ln-5) ␥2 chain by membrane-type-1 matrix metalloproteinases (MT1-MMP) promotes migration and invasion of epithelial and tumor cells. We previously demonstrated that MT1-MMP cleaves the rat ␥2 chain at two sites, producing two major C-terminal fragments of 100 (␥2) and 80 (␥2x) kDa and releasing a 30-kDa fragment containing epidermal growth factor (EGF)-like motifs (domain III (DIII) fragment). The DIII fragment bound the EGF receptor (EGF-R) and stimulated cell scattering and migration. However, it is not yet clear whether human Ln-5 is processed in a similar fashion to rat Ln-5 because one of the two MT1-MMP cleavage sites present in rat ␥2 is not found in human ␥2. To identify the exact cleavage site for MT1-MMP in human Ln-5, we purified both the whole molecule as well as a monomeric form of human ␥2 that is frequently expressed by malignant tumor cells. Like rat Ln-5, both the monomer of ␥2, as well as the ␥2 derived from intact Ln-5, were cleaved by MT1-MMP in vitro, generating C-terminal ␥2 (100 kDa) and ␥2x (85 kDa) fragments and releasing DIII fragments (25 and 27k Da). In addition to the conserved first cleavage site used to generate ␥2, two adjacent cleavage sites (Gly 559 -Asp 560 and Gly 579 -Ser 580 ) were found that could generate the ␥2x and DIII fragments. Two of the three EGF-like motifs present in the rat DIII fragment are present in the 27-kDa human fragment, and like the rat DIII, this fragment can promote breast carcinoma cell migration by engaging the EGF-R. These results suggest that MT1-MMP processing of Ln-5 in human tumors may stimulate the EGF-R, resulting in increased tumor cell scattering and migration that could possibly increase their metastatic potential.Laminin-5 (Ln-5), 1 a major component of the basement membrane, is a heterotrimer composed of ␣3, ␤3, and ␥2 subunits (1, 2). Migration and scattering of epithelial and tumor cells are induced by proteolytic processing of the ␥2 chain of Ln-5. The ␥2 chain is a 140-kDa polypeptide and forms a triple helix with the other subunits at its C-terminal (see Fig. 1A) (3, 4). Processing of the ␥2 chain occurs at the N terminus generating two major C-terminal fragments of 100 (␥2Ј) and 80 (␥2x) kDa (see Fig. 1A), and this processing has been observed in different species including humans and rodents (3, 4). Because of the limited availability of purified Ln-5, most biochemical studies in this area have been carried out using the rat protein. MT1-MMP and MMP-2 were identified as the proteases responsible for the second cleavage of the N terminus generating the ␥2x fragment (3, 4). In contrast, only MT1-MMP cleaved the first site to generate the ␥2Ј fragment (5). The two cleavage sites on the rat ␥2 chain were identified as Gly 434 -Asp 435 for ␥2Ј and Ala 586 -Leu 587 for ␥2x (3, 6). Processing of the rat ␥2 chain at these two sites releases an internal fragment containing three of the four EGF-like motifs in domain III (DIII) (7). Although Ln-5 does not stimulate the EGF receptor (EGF-R), the DIII fragment rele...

Eph receptor tyrosine kinases are considered candidate therapeutic targets in cancer, but they can exert opposing effects on cell growth. In presence of its ligands, Eph receptor EphA2 suppresses signaling by other growth factor receptors, including ErbB, whereas ligand-independent activation of EphA2 augments ErbB signaling. To deploy EphA2-targeting drugs effectively in tumors, the anti-oncogenic ligand-dependent activation state of EphA2 must be discriminated from its oncogenic ligand-independent state. Since the molecular basis for the latter is little understood, we investigated how the activation state of EphA2 can be switched in tumor tissue. We found that ligand-binding domain of EphA2 is cleaved frequently by the membrane metalloproteinase MT1-MMP, a powerful modulator of the pericellular environment in tumor cells. EphA2 immunostaining revealed a significant loss of the N-terminal portion of EphA2 in areas of tumor tissue that expressed MT1-MMP. Moreover, EphA2 phosphorylation patterns that signify ligand-independent activation were observed specifically in these areas of tumor tissue. Mechanistic experiments revealed that processing of EphA2 by MT1-MMP promoted ErbB signaling, anchorage-independent growth, and cell migration. Conversely, expression of a proteolysis-resistant mutant of EphA2 prevented tumorigenesis and metastasis of human tumor xenografts in mice. Overall, our results showed how the proteolytic state of EphA2 in tumors determines its effector function and influences its status as a candidate biomarker for targeted therapy.

Epidermal growth factor (EGF) receptors (ErbB) and EGF family members represent promising targets for cancer therapy. Heparin-binding EGF (HB-EGF) is a member of the EGF family and is an important target for therapy in some types of human cancers. Processing of HB-EGF by proprotein convertases, and successively, by ADAM family proteases, generates a soluble growth factor that requires heparin as a cofactor. Although heparin potentiates HB-EGF activity in vitro, it is not clear how the heparin-binding activity of HB-EGF is regulated. Here, we show that membrane type 1-matrix metalloproteinase (MT1-MMP; MMP14), a potent invasion-promoting protease, markedly enhances HB-EGF-dependent tumor formation in mice. MT1-MMP additionally cleaves HB-EGF and removes the NH 2 -terminal 20 amino acids that are important for binding heparin. Consequently, the processing of HB-EGF by MT1-MMP converts HB-EGF into a heparin-independent growth factor with enhanced mitogenic activity, and thereby, expression of both proteins costimulates tumor cell growth in vitro and in vivo. The ErbB family of receptors expressed in human gastric carcinoma cells play a role in mediating enhanced HB-EGF activity by MT1-MMP during invasive cell growth in collagen. Thus, we shed light on a new mechanism whereby HB-EGF activity is regulated that should be considered when designing HB-EGF-targeted cancer therapy. Cancer Res; 70(14); 6093-103. ©2010 AACR.