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.

FIG 1

Sunitinib does not inhibit RCC cell proliferation or signaling. (A) Proliferation assays of the indicated RCC cell lines treated with vehicle (Ve) or sunitinib (Su; 1 μM). Data are means ± standard errors of the means (SEM); n = 4. RLU, relative luminescence units. (B and C) Western blot analysis of RCC cell lines treated with Ve, rapamycin (Ra; 25 nM), or Su (10 μM) overnight (ON) (B) or of the indicated tumor grafts in mice treated with Ve, Ra, or Su for 3 days (C). (D) Hematoxylin and eosin (H&E) staining and IHC analysis of the same tumors as those used for Western blotting in panel C (×100 magnification).

FIG 2

Sunitinib inhibits endothelial cell (EC) proliferation and signaling. (A) Proliferation assay of EC treated with vehicle (Ve) or sunitinib (Su; 100 nM) (VEGF, 100 ng/ml). (B) Western blot analysis of EC pretreated with Ve or Su and stimulated with VEGF for the indicated periods of time. (C) Tumor graft sections from tumors treated with Ve, Su, or Ra and stained for CD31, DAPI, and phospho-S6 (×400 magnification). (D) Quantification of phospho-S6- or phospho-ERK-positive EC in tumor grafts treated with Ve, Su, or Ra. Ratios refer to the numbers of EC that stained positive for either phospho-S6 or phospho-ERK divided by the total EC number. Data are means ± SEM. n = 2. **, P < 0.01; ***, P < 0.001.

FIG 3

786-O cells support EC survival in a VHL gene-dependent and sunitinib-sensitive manner. (A) Coculture assay schematic. (B) Histogram of EC numbers in cocultures with the indicated cell lines. #, EC number in cultures supplemented with recombinant VEGF (100 ng/ml). (C) Histogram of the effects of VHL gene reconstitution on EC numbers and VEGF levels using RCC3 (VHL gene deficient) or WT8 (VHL gene reconstituted) (n = 4). (D) Histogram of the effects of sunitinib (Su; 100 nM) on EC numbers and VEGF levels in cocultures of 786-O cells. Data are means ± SEM. *, P < 0.05; **, P < 0.01, ***, P < 0.001. In panel C, n = 21 for 786-O, n = 8 for RCC3, n = 13 for WT8, and n = 10 for HEK293; in panel D, n = 21 for EC numbers and n = 11 for VEGF levels.

FIG 4

786-O cells form tumors on the chorioallantoic membrane (CAM) of chicken embryos in a VHL gene- and sunitinib-dependent manner. (A) Fluorescence image of a tumor generated from 786-O cells (labeled with GFP) implanted on CAM. Green, tumor cells; red, Alexa Fluor 555-dextran labeling blood vessels. (B) Bright-field images of tumors on CAM using 786-O reconstituted with empty vector (Deficient) or VHL gene (Reconstituted). (C) Tumor take rates of VHL gene-deficient and reconstituted 786-O cells. (D) Bright-field and fluorescence images of 786-O-derived tumors on CAM treated with vehicle (Ve) or sunitinib (Su). (E) Tumor take rates of 786-O cells treated with Ve or Su. (F) Area of 786-O tumors treated with Ve or Su. Data are means ± SEM (n = 11 for Ve and n = 6 for Su). *, P < 0.05; **, P < 0.01.

FIG 5

Evaluation of the effects of sunitinib on EC survival in coculture with primary RCC. (A) CAM5.2 and PAX-2 staining of 786-O and indicated primary RCC cells; ×200 magnification. (B) Tumor-induced EC survival correlates with secreted VEGF levels; R = 0.57 and P = 0.0028. (C) EC survival in cocultures. Data are means ± SEM. n = 13 for TG206, n = 3 for TG250, n = 4 for TG158, n = 3 for T285, n = 3 for T283, n = 4 for TG185, n = 10 for TG127, n = 4 for T258, n = 6 for T241, n = 8 for TG121, n = 4 for PF22, and n = 6 for T239. *, P < 0.05; **, P < 0.01, ***, P < 0.001.

FIG 6

Testing sunitinib sensitivity using CAM assay. (A) Bright-field and fluorescence images of sunitinib-sensitive and -resistant tumors. Green, tumor cells. (B) Tumor take rates of indicated primary RCC cells treated with vehicle or sunitinib. (C) Bright-field and fluorescence images of sunitinib-sensitive and -resistant tumors following treatment with vehicle (Ve) or sunitinib (Su). *, P < 0.05.

FIG 7

VEGF dependency for mitogenic signaling in EC from sunitinib-sensitive but not sunitinib-resistant tumors. (A) Correlation of VEGF levels and sunitinib sensitivity (R = −0.73 and P = 0.0008). (B) Western blot analysis of HUVEC pretreated with Ve, Su (100 nM), dovitinib (Do; 500 nM), or PD173074 (PD; 50 nM) and then stimulated with conditioned media from the indicated RCC. “(S)” and “(R)” indicate sunitinib-sensitive and -resistant RCC lines, respectively. −, cells in the same base medium that was used to collect RCC-conditioned media.

FIG 8

FGF2 restores EC proliferation and signaling despite the presence of sunitinib, but FGF2 production does not correlate with sunitinib resistance in RCC. (A) Western blot analysis of HUVEC or HDMEC pretreated with Ve or Su (100 nM) and stimulated with VEGF (50 ng/ml) or VEGF plus FGF2 (50 ng/ml). (B) Proliferation curves of HUVEC and HDMEC treated with Ve or Su in the absence or presence of VEGF (100 ng/ml) or FGF2 (50 ng/ml) or both. (C) Neutralizing assay using anti-FGF2 antibodies to suppress the effect of FGF2. Data are means ± SEM. n = 4 (for panels B and C). (D) FGF2 levels in coculture supernatant from sensitive and resistant RCC lines. Horizontal bars indicate medians. (E) Correlation between FGF2 levels and sunitinib sensitivity (R = 0.035 and P = 0.91).

FIG 9

FGFR activation by RCC. (A) Proliferation of HUVEC and HDMEC treated with Ve, Su (100 nM), or Do (500 nM) in the absence or presence of VEGF (100 ng/ml) or FGF2 (50 ng/ml) or both. Data are means ± SEM. n = 4. (B) Western blot analysis of MEFs pretreated with Ve, Su, Do, or PD and stimulated with VEGF (50 ng/ml), FGF2 (25 ng/ml), or insulin (300 nM). (C) Western blot analysis of MEFs pretreated with Ve, Su, Do, or PD and stimulated with conditioned media (CM) obtained from the indicated RCC cells. Su was used at 200 nM, Do at 1 μM, and PD at 100 nM for MEFs. (S), sunitinib sensitive; (R), sunitinib resistant; −, cells in the same base medium that was used to collect RCC-conditioned media.

FIG 10

Expression of detectable FGFs, IL-8, and HGF in primary RCC cell lines. Results of qRT-PCR analysis of indicated growth factors normalized to PPIB. Data are means ± SEM (n = 2). PPIB, protein coding, peptidylprolyl isomerase B.

FIG 11

Suppression of RCC-mediated EC survival and tumor formation by dovinitib treatment. (A) Coculture assay testing the ability of dovitinib to suppress EC survival by the different RCC lines. Data are means ± SEM. n = 4 for TG144, n = 4 for TG158, n = 7 for TG206, n = 4 for TG127, n = 6 for TG169, n = 12 for TG121, n = 4 for T256, n = 3 for T241, n = 4 for T258, n = 4 for PF22, n = 4 for PF114, and n = 2 for T239. (B) Tumor take rates of sunitinib-resistant tumors upon dovitinib treatment. See Table 7 for more details. (C) Bright-field and fluorescence images of a sunitinib-resistant RCC line (T258) after treatment with Ve, Su, or Do. *, P < 0.05; **, P < 0.01, ***, P < 0.001.