Fibroblast Growth Factor Receptor-Dependent and -Independent Paracrine Signaling by Sunitinib-Resistant Renal Cell Carcinoma

Abstract

Antiangiogenic therapies, such as sunitinib, have revolutionized renal cell carcinoma (RCC) treatment. However, a precarious understanding of how resistance emerges and a lack of tractable experimental systems hinder progress. We evaluated the potential of primary RCC cultures (derived from tumors and tumor grafts) to signal to endothelial cells (EC) and fibroblasts in vitro and to stimulate angiogenesis ex vivo in chorioallantoic membrane (CAM) assays. From 65 patients, 27 primary cultures, including several from patients with sunitinib-resistant RCC, were established. RCC cells supported EC survival in coculture assays and induced angiogenesis in CAM assays. RCC-induced EC survival was sensitive to sunitinib in half of the tumors and was refractory in tumors from resistant patients. Sunitinib sensitivity correlated with vascular endothelial growth factor (VEGF) production. RCC induced paracrine extracellular signal-regulated kinase (ERK) activation in EC which was inhibited by sunitinib in sensitive but not in resistant tumors. As determined by fibroblast growth factor receptor substrate 2 (FRS2) phosphorylation in fibroblasts, RCC broadly induced low-level fibroblast growth factor receptor (FGFR) signaling. Whereas ERK activation in EC was uniformly inhibited by combined VEGF/platelet-derived growth factor (PDGF)/FGF receptor inhibitors, paracrine ERK activation in fibroblasts was blocked in only a fraction of tumors. Our data show that RCC activates EC through VEGF-dependent and -independent pathways, that sunitinib sensitivity correlates with VEGF-mediated ERK activation, and that combined inhibition of VEGF/PDGF/FGF receptors is sufficient to inhibit mitogenic signaling in EC but not in fibroblasts.

ACKNOWLEDGMENTS

We thank patients and their families for graciously donating samples for this study. We thank members of the Brugarolas laboratory for helpful discussions and reading of the manuscript, Payal Kapur for taking IHC pictures, William G. Kaelin for RCC3 and WT8 cell lines, and Osamu Ogawa for Caki-1 cells. We also thank Shane Alexander for generating 786-O cells with EV or VHL.

This work was supported by a T32CA124334 and subsequently a F32CA1360872 fellowship to T.A.T. and by the following grants to J.B.: RO1CA129387, RSG115739, RP160440, and RP101075. Tumor samples were collected through the UTSW Tissue resource, supported in part through grant 1P30CA142543. J.B. is a Virginia Murchison Linthicum Scholar in Medical Research at UT Southwestern.

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We declare that we have no conflicts of interest.