ADAM9 (A Disintegrin And Metalloproteinase 9) is a member of the ADAM protein family which contains a disintegrin domain. This protein family plays key roles in many physiological processes, including fertilization, migration, and cell survival. The ADAM proteins have also been implicated in various diseases, including cancer. Specifically, ADAM9 has been suggested to be involved in metastasis. To address this question, we generated ADAM9 knockdown clones of MDA-MB-231 breast tumor cells using silencing RNAs that were tested for cell adhesion, proliferation, migration and invasion assays. In RNAi-mediated ADAM9 silenced MDA-MB-231 cells, the expression of ADAM9 was lower from the third to the sixth day after silencing and inhibited tumor cell invasion in matrigel by approximately 72% when compared to control cells, without affecting cell adhesion, proliferation or migration. In conclusion, the generation of MDA-MB-231 knockdown clones lacking ADAM9 expression inhibited tumor cell invasion in vitro, suggesting that ADAM9 is an important molecule in the processes of invasion and metastasis.

Integrin αvβ3 is most likely the foremost modulator of angiogenesis among all known integrins. Recombinant disintegrin DisBa-01, originally obtained from snake venom glands, binds to αvβ3, thereby significantly inhibiting adhesion and generating in vivo anti-metastatic ability. However, its function in mediator production is not clear. Here, we observed that the mediators VEGF-A, IL-8, and TGF-β are not produced by human umbilical vein endothelial cells (HUVEC cell line) or monocyte/macrophage cells (SC cell line) when cells adhered to vitronectin. However, when exposed to DisBa-01, HUVECs produced higher levels of TGF-β, and SC cells produced higher levels of VEGF-A. Nonetheless, HUVECs also showed an enhancement of apoptosis after losing adherence when exposed to disintegrin, which is a characteristic of anoikis. We propose that disintegrin DisBa-01 could be used to modulate integrin αvβ3 functions.

Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood and evasive resistance of antiangiogenic therapy has been reported. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin mediated downstream signaling cascades and cell migration. Recently, we reported that DisBa-01 strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. Here, DisBa-01 was submitted to adhesion under flow and transmigration assays, both using different DisBa-01 concentrations and β3-siMDA-MB-231 cells. In the first assay, labeled tumor cells incubated with Disba-01 perfused for 5 minutes through a chamber containing endothelial cells as coat. In the second, labeled tumor cells incubated with DisBa-01 were attracted by complete medium and transmigrated through an endothelial cell coat. Individually, assays were photographed and the cells quantified. Results show that DisBa-01 dose-dependently inhibited adhesion of MDA-MB-231 cells to human endothelial cells under flow conditions, similarly to the effect observed by β3-siMDA-MB-231 cells. In addition, the disintegrin or β3-silencing inhibited the transmigration of tumor cells through endothelial cells in vitro. These results demonstrate that endothelial αvβ3 integrin has a key role in tumor cell adhesion and transmigration through the endothelium under flow conditions. Support: Fapesp (grant no. 1998/14138-2), CNPq, and CAPES (Brazil) Citation Format: Cyntia Freitas Montenegro, Araceli Cristina Durante, Kelli Cristina Micocci, Antonio Carlos Manucci Pereira, Jr., Heloisa Sobreiro Selistre-de-Araujo. A role for endothelial αvβ3 integrin in breast tumor cell migration. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B44.