Epidermal growth factor (EGF)-enhanced vascular cell adhesion molecule-1 (VCAM-1) expression promotes macrophage and glioblastoma cell interaction and tumor cell invasion

Abstract

Activated EGF receptor (EGFR) signaling plays an instrumental role in glioblastoma (GBM) progression. However, how EGFR activation regulates the tumor microenvironment to promote GBM cell invasion remains to be clarified. Here, we demonstrate that the levels of EGFR activation in tumor cells correlated with the levels of macrophage infiltration in human GBM specimens. This was supported by our observation that EGFR activation enhanced the interaction between macrophages and GBM cells. In addition, EGF treatment induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression in a PKCε- and NF-κB-dependent manner. Depletion of VCAM-1 interrupted the binding of macrophages to GBM cells and inhibited EGF-induced and macrophage-promoted GBM cell invasion. These results demonstrate an instrumental role for EGF-induced up-regulation of VCAM-1 expression in EGFR activation-promoted macrophage-tumor cell interaction and tumor cell invasion and indicate that VCAM-1 is a potential molecular target for improving cancer therapy.

Keywords: Epidermal Growth Factor Receptor (EGFR); Glioblastoma; Invasion; Macrophages; Migration.

FIGURE 1.

EGFR activation is correlated with macrophage infiltration in human GBM tumors. IHC staining of 40 human GBM specimens with anti-CD68 and anti-phospho-EGFR Tyr-1172 (EGFR pY1172) antibodies was performed. A, representative images of two tumor sections. B, the sections were quantitatively scored according to the percentage of positive cells and staining intensity as described under “Experimental Procedures” (Pearson product-moment correlation test; r = 0.59, p < 0.01). Note that some of the dots on the graph represent more than one specimen (i.e. some scores overlapped).

FIGURE 2.

EGF stimulation increases macrophage binding to GBM cells. Representative images of THP-1 cell binding to GBM cells are shown. The ratios of adherent THP-1 cells to GBM cells were quantified. THP-1 cells were labeled with 0.1 μg/ml BCECF/AM (green), and their nuclei were stained with Hoechst 33342 (blue). The data represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; N.S., not significant. A, U251 cells were treated with or without EGF (100 ng/ml) for the indicated periods. B, U251 cells were treated with or without AG1478 (1 μm) for 30 min before being treated with or without EGF (100 ng/ml) for 24 h. C, D54 cells were treated with or without AG1478 (1 μm) for 30 min before being treated with or without EGF (100 ng/ml) for 24 h. D, A172 cells were treated with or without AG1478 (1 μm) for 30 min before being treated with or without EGF (100 ng/ml) for 24 h.

FIGURE 3.

EGFR activation results in PKCϵ- and NF-κB-dependent VCAM-1 expression. Immunoblot analyses were performed using the indicated antibodies. The data represent the mean ± S.D. from three independent experiments. A, U251 cells were treated with or without AG1478 (1 μm) for 30 min before being treated with or without EGF (100 ng/ml) for the indicated periods. WB, Western blot. B, D54 cells were treated with or without AG1478 (1 μm) for 30 min before being treated with or without EGF (100 ng/ml) for 24 h. C, A172 cells were treated with or without AG1478 (1 μm) for 30 min before being treated with or without EGF (100 ng/ml) for 24 h. D and E, IHC staining with anti-phospho-EGFR Tyr-1172 (EGFR pY1172) and anti-VCAM-1 antibodies was performed on 40 GBM specimens. D, representative images of two tumors are shown. E, we quantitatively scored the tissue of GBM according to the percentage of positive cells and staining intensity as described under “Experimental Procedures” (Pearson product-moment correlation test; r = 0.75, p < 0.01). Note that some of the dots on the graph represent more than one specimen (i.e. some scores overlapped). F, U251 cells were treated with or without a NF-κB inhibitor (10 μm) for 30 min before being treated with or without EGF (100 ng/ml) for 24 h. G, U251 cells with or without p65 shRNA expression were treated with or without EGF (100 ng/ml) for 24 h. H, U251 cells stably transfected with pGIPZ expressing control or PKCϵ shRNA (left panels) were treated with or without EGF (100 ng/ml) for 24 h (right panels).

FIGURE 4.

VCAM-1 mediates binding of THP-1 cells to GBM cells. A, U251 cells with or without VCAM-1 shRNA expression were treated with or without EGF (100 ng/ml) for 24 h. Immunoblot analyses were performed using the indicated antibodies. WB, Western blot. B, U251 cells with or without VCAM-1 shRNA expression were treated with or without EGF (100 ng/ml) for 24 h. The ratio of adherent THP-1 cells to U251 cells was quantified. The data represent the mean ± S.D. from three independent experiments. **, p < 0.01; N.S., not significant. C and D, IHC staining of 40 GBM specimens with anti-CD68 and anti-VCAM-1 antibodies was performed. C, representative images of two tumor sections are shown. D, the sections were quantitatively scored according to the percentage of positive cells and staining intensity as described under “Experimental Procedures.” Note that some of the dots on the graph represent more than one specimen (i.e. some scores overlapped).

FIGURE 5.

VCAM-1 expression is required for EGF-induced and THP-1 cell-mediated GBM cell invasion. A, U251 cells (1 × 10⁵) were added to a Matrigel-coated Transwell insert for 1 h. THP-1 cells (2 × 10⁵) were then loaded on top of the U251 cells for 1 h, followed by gentle washing twice with a medium to remove non-adherent THP-1 cells. The data represent the mean ± S.D. from three independent experiments. Abs, absorbance. *, p < 0.05. B, U251 cells (1 × 10⁵) with or without VCAM-1 shRNA expression were added to a Matrigel-coated Transwell insert for 1 h, followed by treatment with or without EGF (100 ng/ml) for 8 h. THP-1 cells (2 × 10⁵) were then loaded on top of the U251 cells for 1 h, followed by gentle washing twice with a medium to remove non-adherent THP-1 cells. The data represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; N.S., not significant.