Potent inhibition of angiogenesis by the IGF-1 receptor-targeting antibody SCH717454 is reversed by IGF-2

Abstract

Previously, we reported that a predominant action of a type-1 insulin-like growth factor receptor (IGF-1R)-targeted antibody was through inhibiting tumor-derived VEGF, and indirectly, angiogenesis. Here, we examined the direct antiangiogenic activity of the IGF-1R-targeted antibody SCH717454 that inhibits ligand-receptor binding and the mechanism by which tumors circumvent its antiangiogenic activity. Inhibition of ligand-stimulated activation of IGF-1R, insulin receptor (IN-R), or downstream signaling [phosphorylation of Akt (Ser473)] was determined by receptor-specific immunoprecipitation and immunoblotting. Inhibition of angiogenesis was determined by proliferation and tube formation using human umbilical vein endothelial cells (HUVEC) in vitro and in Matrigel plugs implanted in mice. SCH717454 blocked IGF-1-stimulated but not IGF-2-stimulated phosphorylation of Akt in sarcoma cells. Immunoprecipitation using anti-IGF-1R and anti-IN-R antibodies revealed that SCH717454 equally blocked IGF-1-stimulated and IGF-2-stimulated IGF-1R phosphorylation, but not IGF-2-stimulated phosphorylation of IN-R. SCH717454 completely blocked VEGF-stimulated proliferation and tube formation of HUVECs, but exogenous IGF-2 and insulin circumvented these inhibitory effects. Coculture of HUVECs with IGF-2-secreting tumor cells completely abrogated SCH717454 inhibition of VEGF-stimulated HUVEC tube formation. In mice, SCH717454 inhibited angiogenesis in VEGF-infused Matrigel plugs, but had no inhibitory activity when plugs contained both VEGF + IGF-2. These results reveal for the first time, a role for IGF-1R signaling in VEGF-mediated angiogenesis in vitro and indicate direct antiangiogenic activity of SCH717454. Both in vitro and in vivo IGF-2 circumvented these effects through IN-R signaling. Many childhood cancers secrete IGF-2, suggesting that tumor-derived IGF-2 in the microenvironment maintains angiogenesis in the presence of IGF-1R-targeted antibodies allowing tumor progression.

Figure 1

SCH717454 inhibits IGF-1 but not IGF-2 stimulation of Akt phosphorylation. A. Sarcoma cells were grown for 24 hr under serum-free conditions, then incubated for 5 – 60 min with SCH717454 (10μg/ml), or 5 min without antibody, and stimulated for 5 min with IGF-1 (10 ng/ml) or IGF-2 (50 ng/ml). Cells were harvested 5 min after IGF-stimulation at the times shown and phosphorylation of Akt (Ser473) and total Akt determined by immunoblotting. B. IGF-2 signals through IN-R in the presence of SCH717454. EW-8 cells were serum-starved for 24 hr, incubated with or without SCH717454 (10μg/ml) for 24 hr and left unstimulated (control) or stimulated 5 min with either IGF-1 or IGF-2. Cell lysates were immunoprecipitated with antibodies against IGF-1R or IN-R, or an irrelevant isotype control (IgG) and phosphorylated receptors and total receptor levels determined by immunoblotting.

Figure 2

SCH717454 inhibits angiogenesis in vitro. A. HUVECs were grown under serum-deficient conditions and not stimulated (PBS) or stimulated with VEGF (20 ng/ml) in the absence or presence of SCH717454 (5 or 10 μg/ml). Proliferation/viability was determined after 2 days by AB staining (top left). Migration was determined using the wound-healing assay as described in Materials and Methods (top right). Invasion was determined using Matrigel coated membranes (photomicrographs show representative fields) and is quantified (lower right panel). Each data set represents the mean ± SE for at least 3 independent experiments. B. SCH717454 inhibits HUVEC tube formation. HUVECs were grown as in (A), and incubated with PBS or VEGF (10 ng/ml) for 18–20 hr in the absence or presence of SCH717454 (5 or 10 μg/ml). Tube formation was quantified as described in Materials and Methods.

Figure 3

IGF-2 and insulin circumvent SCH717454 inhibition. HUVECs were grown as in Fig. 3B, then stimulated with VEGF in the absence or presence of SCH717454 (10 μg/ml), or antibody plus IGF-1 (10 ng/ml), IGF-2 (50 ng/ml) or insulin (Ins; 50 ng/ml). Tube formation was quantified as described in Materials and Methods. Each result is the mean ± SE for 3 independent experiments.

Figure 4

Sarcoma cells expressing IGF-2 circumvent the anti-angiogenic activity of SCH717454. A. Rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cells were grown for 24 hr under serum-free conditions, and levels of IGFs in the medium were determined by ELISA. Results are mean ± SD (n= 2). B. HUVECs were cultured on Matrigel in the presence of PBS or VEGF (10 ng/ml) without or with SCH717454 (10μg/ml) in the lower chamber of transwell plates. Sarcoma cells (1 × 10⁶) expressing IGF-2 (Rh30) or IGF-1 but low IGF-2 (ES1, EW8) were placed in the upper chamber, and tube formation determined after 20 hr (Rh30) or 30 hr (ES1, EW8), photomicrographs of stained HUVECs. C, quantitation of tube formation under each experimental condition. Mean ± SE for 3 independent experiments.

Figure 5

SCH717454 inhibits murine angiogenesis in the absence of IGF-2. A. Matrigel plugs containing with PBS, or VEGF (100 ng/ml) were implanted subcutaneously in mice that were or were not treated with SCH717454 (20 mg/kg day 0 and day 3). The Matrigel plugs were excised on day 7, fixed with formalin and 5-μm sections were stained with H&E, Messon Trichrome and CD34 staining. The numbers of CD34 positive vessels in HPF (magnification, 200X) were counted for each experimental condition. Right panel: Results are mean (n =4) ± SE. *P <0.05; **P <0.01 versus VEGF alone. B. Matrigel plugs containing PBS (control), VEGF (100 ng/ml) or VEGF (100 ng/ml) + IGF-2 (50 ng/ml) were implanted into mice that received SCH717454 (20 mg/kg day 0 and day 3) or no treatment. C. Matrigel plugs containing PBS (control), VEGF (100 ng/ml) or VEGF (100 ng/ml) + IGF-1 (10 ng/ml) were implanted into mice that received SCH717454 (20 mg/kg day 0 and day 3) or no treatment. Matrigel plugs were excised after 7 days and processed for CD34 staining. Left panels, photomicrographs showing CD34 staining; Right panel, quantification of vessels. Mean ± SE. (Six High Power Fields counted).