Inhibition of tumor cell invasion by the a3 chain of type IV collagen

Pasco Sylvie1, Ramont Laurent 1, Kieffer Nelly2, Maquart Francois-Xavier1, Monboisse Jean-Claude1

1Laboratoire de Biochimie Médicale et de Biologie Moléculaire CNRS FRE 2260, Faculté de Médecine, Université Reims-Champagne-Ardenne, Reims, France. 2 Laboratoire Franco-Luxembourgeois de Recherche Biomédicale CNRS/CRP-Santé, Centre Universitaire, L-1511 Luxembourg, Grand-Duchy of Luxembourg

Tumor cell invasion and metastasis are complex multistep processes that involve cell attachment to basement membrane or extracellular matrix proteins, degradation of adhesive proteins and migration through the proteolytically modified tumor cell microenvironment. The aim of our studies is to demonstrate the inhibitory effect of the a3 chain of type IV collagen on invasive properties of tumor cells through its a3(IV)185-203 peptide sequence (Han J. et al, J. Biol. Chem., 272, 20395-20401, 2000 ; Pasco S. et al, Cancer Research, 60, 467-473, 2000). By liquid phase receptor capture assay, we showed that the a3(IV) peptide specifically interact with the b3 subunit of integrin avb3 independently of CD47. Incubation of melanoma cells with the a3(IV) peptide induced de novo exposure of LIBS epitopes on the b3 subunit similar to those triggered by RGDS stimulation. However, RGDS did not prevent melanoma cell attachment and spreading on immobilized a3(IV) peptide, suggesting that the a3(IV) binding domain is distinct from the RGD recognition site. The a3(IV) peptide stimulated tyrosine phosphorylation in a time dependent manner. Two major phospho-proteins of 120-130 and 85 kDa were identified as FAK and PI3-kinase. Furthermore, a direct involvement of PI3-kinase in a3(IV)-dependent *3 integrin signaling could be documented since pretreatment of melanoma cells with wortmannin, a PI3-kinase inhibitor, reverted the known inhibitory effect of a3(IV) on tumor cell proliferation as well as MT1-MMP gene expression. These results provide evidence that the a3(IV) peptide, by directly interacting with the *3 subunit of *v*3, activates a signaling cascade involving FAK and PI3-kinase. In order to check the inhibitory effect of the a3 peptide on tumor invasion in vivo, we have subcutaneously injected B16F1 melanoma cells into syngenic C57BL6 mice, and demonstrated a significant decrease of tumor growth. Collectively, these results support the a3(IV) peptide as a potential candidate to limit tumor progression.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Prevention.

This presentation received an honorable mention in our poster contest and was recognized with the Symposium Presidents' Award for Scientific Excellence.