Endothelial-Rac1 Is Not Required for Tumor Angiogenesis unless avb3-Integrin Is Absent Gabriela D’Amico1*¤, Stephen D. Robinson 1 , Mitchel Germain1, Louise E. Reynolds1, Gareth J. Thomas2, George Elia1, Garry Saunders1, Marcus Fruttiger3, Victor Tybulewicz4, Georgia Mavria5, Kairbaan M. Hodivala-Dilke1* 1 Adhesion and Angiogenesis Laboratory, Institute of Cancer, Bart’s and The London, Queen Mary’s School of Medicine and Dentistry, London, United Kingdom, 2 Centre for Tumour Biology, Institute of Cancer, Bart’s and The London, Queen Mary’s School of Medicine and Dentistry, London, United Kingdom, 3 Institute of Ophthalmology, University College London, London, United Kingdom, 4 Division of Immune Cell Biology, National Institute for Medical Research, London, United Kingdom, 5 Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, London, United Kingdom Abstract Endothelial cell migration is an essential aspect of tumor angiogenesis. Rac1 activity is needed for cell migration in vitro implying a requirement for this molecule in angiogenesis in vivo. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. Here we show that depletion of endothelial Rac1 expression in adult mice, unexpectedly, has no effect on tumor growth or tumor angiogenesis. In addition, repression of Rac1 expression does not inhibit VEGF-mediated angiogenesis in vivo or ex vivo, nor does it affect chemotactic migratory responses to VEGF in 3-dimensions. In contrast, the requirement for Rac1 in tumor growth and angiogenesis becomes important when endothelial b3-integrin levels are reduced or absent: the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in b3-null mice are all Rac1-dependent. These data indicate that in the presence of avb3-integrin Rac1 is not required for tumor angiogenesis. Citation: D’Amico G, Robinson SD, Germain M, Reynolds LE, Thomas GJ, et al. (2010) Endothelial-Rac1 Is Not Required for Tumor Angiogenesis unless avb3- Integrin Is Absent. PLoS ONE 5(3): e9766. doi:10.1371/journal.pone.0009766 Editor: Erik H. j. Danen, Leiden University, Netherlands Received December 1, 2009; Accepted March 1, 2010; Published March 22, 2010 Copyright: 2010 D’Amico et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by Cancer Research UK, the Medical Research Council, Breast Cancer Campaign, The Association for International Cancer Research, and The Health Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gabriela.damico@helsinki.fi (GD); k.hodivala-dilke@qmul.ac.uk (KMH-D) ¤ Current address: Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Introduction Tumor growth and metastasis are both dependent on the development of a tumor vasculature in a process termed angiogenesis. This process involves changes in the migration and proliferation of endothelial cells to form capillaries which invade into the growing tumor mass to supply it with nutrients and oxygen [1]. The list of molecular co-ordinators of angiogenesis is vast and includes some members of the family of extracellular adhesion molecules, integrins, and several growth factors and their receptors such as vascular endothelial growth factor (VEGF) and VEGF-receptor2 (VEGFR2) [2]. Blood vascular endothelial cells express several integrins including avb3, a predominant vitronec- tin receptor. Inhibitors of avb3 have been developed as anti- angiogenic agents for the treatment of cancer [2]. Indeed avb3- and VEGF-inhibitors, used in combination with chemotherapy, are both in Phase III clinical trials, the former for the treatment of glioblastoma and the latter especially for colorectal cancer [3,4,5,6,7]. However, neither anti-integrin nor anti-VEGF treat- ments are effective enough at blocking angiogenesis to completely halt cancer progression and, at best, can extend mean patient survival by a few months [8,9,10,11]. In contrast to the inhibition studies, global genetic ablation of b3-integrin results in viable, fertile animals which display enhanced pathological angiogenesis due in part to the up-regulation of VEGFR2 [12], whereas in a transgenic model, where the cytoplasmic tail of b3-integrin is mutated, pathological angiogenesis is impaired [13]. Taken together these data demonstrate that the molecular mechanisms by which these molecules regulate angiogenesis are complex and in need of further investigation. There are several examples where the levels of b3-integrin have been reported to be undetectable in developing blood vessels. Apart from the lack of b3-integrin in Glanzmann thromabastenic patients, there are several physiological examples where b3 levels are low or undetectable: (1) during embryonic development, b3- integrin expression is barely detectable until mouse embryonic day E15.5 onwards, before which, most developmental angiogenesis is already well established [14]; (2) b3-integrin expression is not observed in the vasculature of healing liver [15]; (3) the vasculature of angiosarcomas has undetectable levels of b3-integrin [16]; (4) the vasculature of lung metastases originated from colorectal cancer also lack b3-integrin [17,18]; (5) the blood vessels in neuroendocrine tumors also show no significant change in b3- integrin levels between normal and tumor tissue [19]; (6) in Lewis cell lung carcinomas, b3-integrin expression is absent until the tumors reach a relatively large size of 500 mm3 or more suggesting that the initial stages of angiogenesis lack b3-integrin [15]. Taken together, these types of data suggest that screening for b3-integrin levels, will be important in better detecting which tumor types will be responsive to b3-specific anti-angiogenic therapy. Indeed the PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9766