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.T herapies against tumors may be directed at tumor cells or nonmalignant components. Drugs like imatinib or gefitinib were developed to target aberrantly activated growth factor signaling kinases in tumor cells such as Abl or epidermal growth factor receptor (EGFR). In contrast, sunitinib (Su) was developed to inhibit growth factor receptor tyrosine kinases in endothelial cells (EC) and pericytes, which are implicated in angiogenesis.The most common malignant kidney tumors are renal cell carcinomas (RCCs), and the most common subtype is clear cell renal cell carcinoma (ccRCC). In ccRCC, angiogenesis is directly activated as a consequence of mutations in the tumor cells. ccRCC is characterized by von Hippel-Lindau gene (VHL) inactivation, which is found in over 80% of tumors (1, 2). The VHL protein (pVHL) is a component of a multisubunit ubiquitin ligase complex (3-9) that targets hypoxia-inducible factor alpha (HIF␣) subunits for degradation (10-12). The loss of pVHL in tumors stabilizes HIF␣, leading to constitutive HIF activation and the upregulation of HIF target genes, including the vascular endothelial growth factor gene (VEGF), encoding an important angiogenic factor (13). This may account, at least in part, for the vascular nature of ccRCC and may explain the unusual sensitivity of this tumor type to anti...

Modeling Renal Cell Carcinoma in Mice: Bap1 and Pbrm1 Inactivation Drive Tumor Grade

“…The tumor cell line was generated as described in Tran et al (62). Briefly, tumor tissue was dissected from the kidney in cold PBS, placed in MEM with Earle’s salts, 2 mM glutamine, 50 μg/ml of gentamicin [Life Technologies], 1% Pen/Strep, and 1% amphotericin B [Life Technologies], and minced into small pieces.…”

Predictive Biomarkers for Molecularly Targeted Therapies in Renal Cell Carcinoma

“…Interestingly, dovitinib, an inhibitor of angiogenesis that also targets fibroblast growth factor receptors, blocked endothelial cell activation by sunitinib-resistant RCCs. 18 Dovitinib was not better than sorafenib in a phase III trial, however. 19 One potential explanation for these results is the differential effect of dovitinib on endothelial cells and fibroblasts.…”

“…Recently, we showed that primary cultures from RCC activate mitogenic signaling in endothelial cells in coculture experiments. 18 Endothelial cell activation could be blocked by sunitinib. However, primary cultures derived from patients with sunitinib-resistant tumors retained the ability to induce mitogenic signaling in endothelial cells despite sunitinib.…”

“…Although dovitinib effectively inhibits endothelial cell activation by RCC cells, it fails to consistently inhibit fibroblasts, a cell type that may contribute to tumorigenesis. 18 Thus, failure to inhibit other cell types may explain the modest activity of dovitinib against RCC. Like dovitinib, lenvatinib (which was recently approved in combination with everolimus 20 ) also targets fibroblast growth factor receptors, but how it would affect signaling by RCC is unknown.…”

“…However, VEGF expression in tumors may more accurately reflect tumor angiogenesis, and in co-culture experiments, sunitinib sensitivity correlates with VEGF production. 18 Notably, VEGF production may differ not only across tumors from different patients but also among metastases within the same patient. This is evidenced by an elegant, albeit small, study using 89 Zr-bevacizumab PET.…”