The Antitumor Properties of the α3(IV)-(185-203) Peptide from the NC1 Domain of Type IV Collagen (Tumstatin) Are Conformation-dependent

Abstract

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the ␣3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to ␣ v ␤ 3 integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 ␣3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 ␣3(IV)-(185-203) peptides show a ␤-turn at the YSNS (188 -191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the ␤-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the ␤-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of ␣ v ␤ 3 integrin in melanoma progression.Tumor growth and metastasis require cross-talk between tumor cells and host stromal cells as well as interactions between tumor cells and the environmental extracellular matrix (ECM), 1 especially basement membranes (1). Basement membranes are thin layers of a specialized ECM which not only provide a supporting structure for various cell types but also largely influence their physiologic behavior. Basement membrane components can also regulate neovascularization, an important process of tumor growth (2, 3). During tumor invasion, the reinforced interactions of tumor cells with basement membranes lead to a sequential proteolytic degradation of their components (4).The degradation of basement membrane macromolecules involves various proteolytic cascades, such as matrix metalloproteinases (MMPs) and the plasminogen activation system. MMPs are a large family of at least 25 members of zinccontaining endopeptidases that degrade ECM macromolecules. Among them, gelatinase A (MMP-2), gelatinase B (MMP-9), and stromelysin (MMP-3) have widely been shown to play a crucial role in the degradation of basement membranes during melanoma progression (5-7). MMPs are secreted as inactive zymogens. Their activation, a major step of tumor invasion, results from an imbalance between levels of tissue inhibitors of metalloproteinases (TIMPs) and the MMP activators, such as urokinase, plasmin, or membrane-type MMPs (MT-MMPs). MT-MMPs constitute a new subgroup of MMPs containing an additional domain for anchorage to th...

An Endostatin-derived Peptide Interacts with Integrins and Regulates Actin Cytoskeleton and Migration of Endothelial Cells

“…In addition, studies using peptide fragments from another basement membrane-derived angiogenesis inhibitor, tumstatin, suggest that the length of the peptide is a critical determinant of biological activity. This is most likely due to differences in secondary structure of peptides of different lengths (38). Furthermore, the peptides with anti-angiogenic activity (37) do not contain the specific arginine residues found to be important for the biological activity of endostatin, suggesting that additional novel domains could be involved in the activity of endostatin.…”

Endogenous Inhibitors of Angiogenesis

“…The antiangiogenic activity is localized to a distinct region of the tumstatin molecule that is separate from the region responsible for the antitumor cell activity (90). Tumstatin has two binding sites for a v h 3 integrin, one in the NH 2 -terminal end of the molecule (containing amino acids that is associated with the antiangiogenic properties and the other in the COOHterminal end (containing amino acids [185][186][187][188][189][190][191][192][193][194][195][196][197][198][199][200][201][202][203] that is associated with the antitumor cell activity (89,91,93). The tumstatin fragment containing amino acids 54 to 132 binds to both endothelial cells and melanoma cells but only inhibits proliferation of endothelial cells, with no effect on tumor cell proliferation.…”

Structural Basis for the Functions of Endogenous Angiogenesis Inhibitors

“…Tumstatin has two binding sites for αvβ3 integrin, one in the amino-terminal end of the molecule (residues 54-132) that is associated with antiangiogenic activity, and the other in the carboxy-terminal end (residues 185-203) that is associated with antitumor cell activity (Shahan et al 1999b;Maeshima et al 2000b;Floquet et al 2004). The presence of cyclic RGD peptides does not compete for the αVβ3 integrin-dependent activity of tumstatin, suggesting unique αVβ3 integrin-mediated mechanisms governing these two distinct antiangiogenic and antitumor activities (Maeshima et al 2000a).…”

“…The 183-205 peptide does not affect endothelial cells, but rather demonstrates an anti-melanoma cell activity (Maeshima et al 2000b). Specifically, the 183-205 fragment of tumstatin binds both endothelial and melanoma cells but only inhibits the proliferation of melanoma cells (Shahan et al 1999b;Maeshima et al 2000b;Floquet et al 2004).…”