Abstract 1642: The role of αv integrin in melanoma cell migration and invasion.
Authors: Sangeet Lal, Y. Jeffrey Wu, Leslie L. Muldoon, Edward A. Neuwelt
Journal: Cancer Research (2013)

Abstract Melanoma, the most aggressive form of skin cancer, has a high incidence of forming metastases with a poor prognosis and median survival of less than 12 months. Tumor metastasis depends on cell interaction with vascular endothelial cells and the extracellular matrix (ECM) regulated by adhesion molecules such as integrin transmembrane receptors. Integrin αv (vitronectin receptor) has been implicated in the metastatic cascade of many cancers, and blocking αv integrin with intetumumab (fully humanized monoclonal antibody) decreases the formation of breast cancer brain metastases and inhibited human melanoma tumor growth in a rat xenograft model. The purpose of this study is to test the hypothesis that αv integrins are crucial for the cell migration and invasion characteristics of melanoma cells that are involved in metastasis. Human A2058 melanoma cells pretreated with hexadimethrine bromide were transduced with lentiviral clones of anti-αv integrin shRNAs. Stable cell lines were selected with puromycin showing 75% knockdown (A2058-75KD) or 40% knockdown (A2058-40KD) of the αv-integrin protein as determined with western blotting. Using the Cell Titer-Glo luminescence cell viability assay, we determined that the A2058-75KD cells have a 60% decreased rate of proliferation compared to control cells, and grow as large cell aggregates in suspension unlike adherent monolayer of control cells. The A2058-75KD cells demonstrate an ECM-dependent growth pattern. In a 3-dimensional matrix of Matrigel, the cell clumps dissociate and grow as infiltrating single cells, however, after harvesting from the Matrigel, cells revert back to non-adherent suspension clumps under 2-dimensional culture conditions. In comparison, the A2058-40KD cells with 40% αv integrin knockdown showed no difference in the rate of proliferation compared to control cells and also grow as adherent monolayer under standard culture conditions. A transwell invasion assay was performed to assess migration through a barrier of Matrigel (2mg/ml) ECM for 72h using 10% fetal bovine serum media as the chemoattractant. A2058-40KD cells showed 50% decreased invasion compared to control cells. Using an ibidi 2-dimensional cell migration assay, we found that A2058-40KD cells showed markedly reduced migration at 6 days after plating, which was significantly reduced compared to control A2058 cells. Blocking αv integrin with intetumumab decreased the migration of the control A2058 cells. Further experiments are underway to investigate the effect of αv integrin knockdown on cell adhesion under dynamic fluid flow conditions and the specific β integrins involved in αv-mediated effects in melanoma. We conclude that αv integrins play a crucial role in the invasiveness of metastatic melanoma cells. Citation Format: Sangeet Lal, Y. Jeffrey Wu, Leslie L. Muldoon, Edward A. Neuwelt. The role of αv integrin in melanoma cell migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1642. doi:10.1158/1538-7445.AM2013-1642