Inhibition of αvβ3 integrin induces loss of cell directionality of oral squamous carcinoma cells (OSCC)

by Cyntia F. Montenegro, Bruna C. Casali, Rafael L. B. Lino, Bianca C. Pachane, Patty K. Santos, Alan R. Horwitz, Heloisa S. Selistre-de-Araujo, Marcelo L. Lamers

The connective tissue formed by extracellular matrix (ECM) rich in fibronectin and collagen consists a barrier that cancer cells have to overpass to reach blood vessels and then a metastatic site. Cell adhesion to fibronectin is mediated by α_vβ₃ and α₅β₁ integrins through an RGD motif present in this ECM protein, thus making these receptors key targets for cell migration studies. Here we investigated the effect of an RGD disintegrin, DisBa-01, on the migration of human fibroblasts (BJ) and oral squamous cancer cells (OSCC, SCC25) on a fibronectin-rich environment. Time-lapse images were acquired on fibronectin-coated glass-bottomed dishes. Migration speed and directionality analysis indicated that OSCC cells, but not fibroblasts, showed significant decrease in both parameters in the presence of DisBa-01 (1μM and 2μM). Integrin expression levels of the α₅, α_v and β₃ subunits were similar in both cell lines, while β1 subunit is present in lower levels on the cancer cells. Next, we examined whether the effects of DisBa-01 were related to changes in adhesion properties by using paxillin immunostaining and total internal reflection fluorescence TIRF microscopy. OSCCs in the presence of DisBa-01 showed increased adhesion sizes and number of maturing adhesion. The same parameters were analyzed usingβ3-GFP overexpressing cells and showed that β3 overexpression restored cell migration velocity and the number of maturing adhesion that were altered by DisBa-01. Surface plasmon resonance analysis showed that DisBa-01 has 100x higher affinity for α_vβ₃ integrin than forα5β1 integrin. In conclusion, our results suggest that the α_vβ₃ integrin is the main receptor involved in cell directionality and its blockage may be an interesting alternative against metastasis.

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