Oncogene (2011) 30, 1183–1193 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Fibroblast growth factor 2 regulates endothelial cell sensitivity to sunitinib JC Welti1, M Gourlaouen1, T Powles2, SC Kudahetti2, P Wilson3, DM Berney2 and AR Reynolds1 1Tumour Angiogenesis Group, The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK; 2Centre for Molecular Oncology, Queen Mary University of London, Barts & The London School Of Medicine & Dentistry, Institute of Cancer, John Vane Science Centre, Charterhouse Square, London, UK and 3Department of Medical Oncology, West Smithfield, London, UK The vascular endothelial growth factor (VEGF) receptor variety of human tumours (Ellis and Hicklin, 2008). tyrosine kinase inhibitor sunitinib has been approved for VEGF-A drives angiogenesis by signalling through two first-line treatment of patients with metastatic renal cognate receptors on local endothelial cells, VEGFR1 cancer and is currently being trialled in other cancers. and VEGFR2 (Ferrara et al., 2003; Olsson et al., 2006). However, the effectiveness of this anti-angiogenic agent Binding of VEGF-A to VEGFR2 activates key intra- is limited by the presence of innate and acquired drug cellular signalling molecules including ERK1/2 and resistance. By screening a panel of candidate growth PLCg. Pathways such as these coordinate the biological factors we identified fibroblast growth factor 2 (FGF2) processes necessary for VEGF to induce the formation as a potent regulator of endothelial cell sensitivity to of new vessels (Olsson et al., 2006). sunitinib. We show that FGF2 supports endothelial Numerous inhibitors of the VEGF signalling axis proliferation and de novo tubule formation in the presence have been developed, including the VEGF-neutralising of sunitinib and that FGF2 can suppress sunitinib-induced antibody, bevacizumab, and VEGF receptor tyrosine retraction of tubules. Importantly, these effects of FGF2 kinase inhibitors, such as sunitinib (Ferrara et al., 2004; were ablated by PD173074, a small molecule inhibitor of Ferrara and Kerbel, 2005; Ellis and Hicklin, 2008; FGF receptor signalling. We also show that FGF2 can Kerbel, 2008). These drugs are particularly effective in stimulate pro-angiogenic signalling pathways in endothe- VEGF-driven tumours such as clear-cell renal cancer. lial cells despite the presence of sunitinib. Finally, analysis Indeed sunitinib monotherapy doubles progression-free of clinical renal-cancer samples demonstrates that a large survival and extends overall survival in this disease and proportion of renal cancers strongly express FGF2. We is now standard first-line therapy (Motzer et al., 2007, suggest that therapeutic strategies designed to simulta- 2009). Despite these impressive results, variation in neously target both VEGF and FGF2 signalling may response between individuals occurs and, more impor- prove more efficacious than sunitinib in renal cancer tantly, resistance to therapy invariably develops (Motzer patients whose tumours express FGF2. et al., 2009; Rini and Atkins, 2009). Determinants of Oncogene (2011) 30, 1183–1193; doi:10.1038/onc.2010.503; sunitinib response and the mechanisms that mediate the published online 8 November 2010 onset of resistance to sunitinib are poorly understood. This presents a significant barrier to the development of Keywords: angiogenesis; growth factors; sunitinib; renal individualized therapy. cancer; resistance The tumour microenvironment contains numerous soluble factors, besides VEGF, that possess pro- angiogenic properties. These include growth factors that signal through tyrosine kinase receptors, such as Introduction members of the fibroblast growth factor (FGF), placental growth factor (PLGF) and platelet-derived Tumour angiogenesis (the growth of new blood vessels growth factor (PDGF) families and factors that into tumours) is a key driver of tumour progression and signal through non-tyrosine kinase receptors, such as has received considerable attention as a therapeutic interleukin-8 (IL-8) and transforming growth-factor-b target (Hanahan and Folkman, 1996; Kerbel and (Relf et al., 1997; Ferrara, 2002; Presta et al., 2005). Folkman, 2002; Ferrara and Kerbel, 2005). One of the These factors may have an important role in the most highly studied pro-angiogenic growth factors is induction of tumour angiogenesis and may also be vascular endothelial growth factor (VEGF), particularly involved in altering the responsiveness of tumour blood the VEGF-A isoform, which is highly expressed in a vessels to inhibitors of the VEGF signalling axis (Bergers and Hanahan, 2008). In support of this latter concept, Correspondence: Dr AR Reynolds, Tumour Angiogenesis Group, FGF2 is upregulated in a mouse tumour model of acquired The Breakthrough Breast Cancer Research Centre, The Institute of resistance to DC101 (a VEGFR2 inhibitory antibody) Cancer Research, 237 Fulham Road, London SW3 6JB, UK. and inhibition of FGF2 in combination with DC101 led E-mail: [email protected] Received 21 June 2010; revised 5 August 2010; accepted 3 September to improved anti-tumour responses (Casanovas et al., 2010; published online 8 November 2010 2005). Moreover, inhibition of PLGF or PDGF has been FGF2 regulates sunitinib sensitivity JC Welti et al 1184 reported to enhance the anti-tumour efficacy of VEGF absence of sunitinib (Figure 1c). These data therefore pathway inhibitors in some murine tumour models identify FGF1, and more strikingly FGF2, as candidate (Bergers et al., 2003; Fischer et al., 2007; Crawford et al., factors that may be potent mediators of endothelial cell 2009). Another recent study demonstrated that IL-8 is resistance to sunitinib. upregulated in sunitinib-resistant tumour xenografts and that inhibition of IL-8 can re-sensitize these resistant tumours to sunitinib (Huang et al., 2010b). FGF2 suppresses the anti-angiogenic activity of sunitinib Sunitinib is an orally available tyrosine kinase inhibitor in a 3-dimensional assay of endothelial tube formation that potently inhibits several angiogenesis-related tyrosine As FGF2 was identified as the most potent factor able to kinase receptors that is, VEGF receptors (VEGFR1, drive resistance to sunitinib in an endothelial cell VEGFR2 and VEGFR3), PDGF receptors (PDGFRa proliferation assay (Figure 1c), we proceeded to validate and PDGFRb), and also the KIT receptor (Abrams this activity within an in vitro model of angiogenesis. et al., 2003; Mendel et al., 2003; O’Farrell et al., 2003). Human umbilical vein endothelial cells (HUVEC)- A recent study that examined the activity of sunitinib coated beads were embedded in a fibrinogen gel and against a panel of 317 kinases (representing 450% of then incubated with VEGF and different concentrations the known kinome) revealed that sunitinib has relatively of sunitinib. After 7 days, endothelial tubules had high affinity for at least 40 other kinases, including sprouted from VEGF alone-treated beads, but tubule myosin light-chain kinase, MEK1 and MEK2 (Karaman formation was significantly inhibited by low concentra- et al., 2008). As activation of multiple kinase-signalling tions (10–100 nM) of sunitinib (Figures 2a and b) pathways is a requirement for growth factor-mediated confirming that sunitinib can inhibit VEGF-stimulated stimulation of angiogenesis, the relatively broad kinase tubule formation in this assay. Importantly, when the substrate specificity of sunitinib might be expected to assay was repeated in the presence of VEGF and FGF2, limit the potential for other growth factors to drive the number of tubules was enhanced 3–4 fold compared resistance to sunitinib through a mechanism that with beads cultured with VEGF alone (Figure 2c). involves direct stimulation of endothelial cells. These data are in agreement with previous reports Here we identify FGF2 to be a potent mediator of showing that FGF2 can enhance VEGF-mediated endothelial cell resistance to sunitinib. We further angiogenesis (Pepper et al., 1992; Giavazzi et al., demonstrate that FGF2 can suppress the anti-angio- 2003). Sunitinib was also able to suppress tubule genic activity of sunitinib by directly stimulating pro- formation in the presence of VEGF and FGF2 angiogenic signalling in endothelial cells. We also show (Figure 2c). However, tubule formation in the presence that FGF2 is expressed in a subset of human renal of VEGF, FGF2 and sunitinib was still significantly cancers. These data provide strong support for simulta- enhanced compared with tubule formation in the neous targeting of VEGF and FGF signalling as a presence of VEGF and sunitinib (Figure 2c). These therapeutic strategy in renal cancer. data confirm that FGF2 can act to suppress the anti- angiogenic effects of sunitinib. Having shown that FGF2 can suppress the effects of sunitinib on de novo tubule formation, we then Results addressed whether FGF2 could suppress the effects of sunitinib on pre-formed tubules. HUVEC-coated beads A screen of pro-angiogenic growth factors identifies FGF2 embedded in a fibrinogen gel were incubated from the as a mediator of endothelial cell resistance to sunitinib start of the assay with VEGF in the presence or absence Sunitinib is a potent inhibitor of VEGFR2 tyrosine kinase of FGF2. After 7 days, when tubules were clearly activity. In agreement with previous reports (Mendel visible, the medium was then additionally supplemented et al., 2003), nanomolar concentrations (10–100 nM)of with either vehicle or 10 nM sunitinib. The