The multifaceted ability of decorin to retard in vivo tumor growth and metastatic spreading has a mechanistic explanation in decorin’s ability to down-regulate multiple signaling pathways. We show for the first time that decorin is a novel endogenous antagonistic ligand of the Met receptor. Signaling mediated by HGF/Met axis promotes multiple biological activities, including survival, proliferation, motility/invasion, and angiogenesis (Trusolino and Comoglio, 2002
; Birchmeier et al., 2003
). Deregulation of the Met signaling pathway leads to uncontrolled growth and transformation, as shown by the TPR-Met, an oncogene that exhibits constitutive tyrosine kinase activation, and by activating mutations of Met intracellular domain in both hereditary and sporadic cancers (Gentile et al., 2008
). Our results indicate that decorin is an inhibitor of multiple RTKs, insofar as it down-regulates the Met receptor as well as ErbB family members. The unique activity of decorin as a Met antagonist is manifested by a rapid induction of both Met receptor shedding and internalization with consequent downstream degradation of β-catenin, which is required for cell survival.
To date, there is only one known mammalian ligand of the Met receptor (i.e., HGF) and one bacterial protein, internalin B, which is synthesized and partly secreted by Lysteria monocytogenes
. Internalin B, a protein containing seven leucine-rich tandem repeats with homology to decorin, mimics HGF-induced receptor trafficking (Li et al., 2005
) and causes sustained activation of the Met receptor (Shen et al., 2000
), leading to bacterial internalization into host cells (Shen et al., 2000
; Ireton, 2007
; Disson et al., 2008
). The 213–amino acid leucine-rich repeat portion of internalin B is sufficient for entry into mammalian cells (Braun et al., 1999
). The recent cocrystallization of internalin B with the ectodomain of the Met receptor has shown that internalin B complexes with the first Ig domain of the receptor (Niemann et al., 2007
; Niemann et al., 2008
). This interaction keeps Met in an active configuration while maintaining the flexibility in the Met semaphorin domain, where HGF binds with low affinity (Stamos et al., 2004
). The interaction interface includes the concave part of the leucine-rich domain of internalin B and a loop that protrudes from the first Ig-like domain of the Met receptor (Niemann et al., 2008
). Notably, several key aromatic amino acids within the concave face of internalin B are required for Met binding and internalization of the bacteria (Machner et al., 2003
). These results have been confirmed in the aforementioned cocrystallization study (Niemann et al., 2007
). Mutation of each of these residues (Fig. S5 A
) abolishes binding to the Met receptor (Machner et al., 2003
). Very importantly, a specific sequence of internalin B, encoding Y170 (required for Met binding) and surrounding residues, is highly analogous to a sequence of mammalian decorin (Fig. S5 B). This highly conserved motif suggests that both proteins have evolved to fulfill a common function, i.e., interacting with the Met receptor, albeit with divergent outcomes.
In contrast to internalin B, HGF binds with low affinity to the Met semaphorin domain (Stamos et al., 2004
) and with high affinity to the terminal Ig3-4 (Basilico et al., 2008
). This is in agreement with early biochemical experiments demonstrating that internalin B and HGF do not substantially compete for receptor occupancy (Shen et al., 2000
). We discovered that decorin is readily displaced by HGF (IC50
= ~2.3 nM) from binding to the immobilized Met ectodomain fused to the dimerizing Fc fragment. In contrast, internalin B was much less efficient in displacing decorin binding to Met-Fc because it required >50-fold higher concentrations (IC50
= ~120 nM). These findings suggest that decorin binds to a similar location of the Met ectodomain where HGF binds with additional secondary sites overlapping with internalin B binding.
In spite of the fact that decorin mode of binding to the Met ectodomain is apparently similar to that of HGF, decorin evokes a profound antagonistic effect on the receptor signaling by inducing both physical and functional receptor down-regulation and by triggering apoptosis via induction of caspase-3/7 activity. Moreover, decorin causes Met-mediated down-regulation of β-catenin levels and transcriptional activity. It is well established that the Met receptor not only physically interacts with β-catenin on the cell surface but upon HGF binding, also phosphorylates β-catenin and triggers its translocation into the nucleus and consequent transcription of genes vital for cell proliferation and migration (Monga et al., 2002
; Müller et al., 2002
; Herynk et al., 2003
; Ishibe et al., 2006
; Rasola et al., 2007
). Importantly, the Met receptor and β-catenin are engaged in a positive feedback loop that sustains tumor growth and invasion, where β-catenin drives Met receptor expression (Rasola et al., 2007
). β-Catenin is a key player in Wnt signaling and plays a central role in cancer development (Clevers, 2006
). For instance, β-catenin regulates both differentiation and proliferation of intestinal epithelial cells by enhancing the expression of genes, such as cyclin D1 and D4, associated with tumor progression. The ability of exogenous decorin to suppress β-catenin levels and transcriptional activity, coupled with the decorin-evoked translocation of β-catenin from the perinuclear to plasmalemmal compartments, suggests that decorin signaling affects the β-catenin pathway. Our data show that this effect is mediated through the Met pathway. A recent study using decorin-deficient mice has shown that ~30% of these mutant mice develop intestinal tumors, a process that is accelerated and amplified when the decorin-deficient animals are subjected to a high risk diet (Bi et al., 2008
). Notably, the endogenous β-catenin levels were markedly increased in the intestinal epithelium of the decorin-null mice, suggesting that lack of decorin is permissive for tumorigenesis, as we hypothesized previously (Iozzo et al., 1999a
), thereby providing in vivo evidence that β-catenin might be regulated by extracellular signaling events evoked by decorin.
How does decorin induce protracted Met degradation? In the case of the EGFR, EGF but not TGF-α induces efficient receptor internalization and degradation. EGF remains closely linked to its receptor during clathrin-dependent endocytosis, whereas TGF-α rapidly dissociates from the receptor in the acidic microenvironment of early endosomes, resulting in receptor recycling (Schlessinger, 2000
). Decorin causes a caveolar-mediated endocytosis of the EGFR, and even after 30 min, decorin and EGFR colocalize within late endocytic compartments and subsequently within lysosomes (Zhu et al., 2005
). This mechanism might explain the lower levels of EGFR after decorin treatment due in part to a reduced receptor recycling to the surface. A similar scenario could occur with the Met receptor, although we have not formally shown that Met internalization and degradation occur via a caveolar-mediated endocytosis. This idea is supported by a recent study, which has shown that both internalin B and the leucine-rich repeats of internalin B, the region that shares analogy with decorin, are properly internalized and remain associated with Met during transit through early and late endosomes when provided as soluble ligands to HeLa cells (Gao et al., 2009
). Thus, one possibility is that HGF/internalin B, as agonistic ligands for Met, are internalized via a clathrin-mediated pathway and in analogy with EGF/EGFR, clathrin-mediated internalization has been shown to be essential for sustained receptor signaling (Sigismund et al., 2008
). In contrast, antagonistic ligands such as decorin could induce internalization via a caveolar-mediated pathway, leading to attenuated signaling and intracellular proteolysis of the receptor.
The ability of decorin to differentially phosphorylate Met receptor Tyr residues is fascinating. More investigation into this novel decorin mechanism of action will be needed in the future, and most likely, more information regarding the peculiar Met conformation induced by decorin binding will shed light onto the phosphorylation events described in this study. Notably, coactivation of RTKs affects the response of tumor cells to targeted therapies (Stommel et al., 2007
), and amplification of the Met-encoding gene promotes drug resistance in ErbB-driven cancers (Engelman et al., 2007
). Although in the past main efforts were aimed at developing highly specific inhibitors acting on single RTKs, more recently there has been a general consensus that molecules interfering simultaneously with multiple RTKs might be more effective than single target agents (Knudsen and Vande Woude, 2008
). In this perspective, the activity of decorin, and perhaps of other molecules harboring leucine-rich repeats, might represent a novel therapeutic modality against metastatic cancer.