Granulocyte colony-stimulating receptor promotes β 1-integrin-mediated adhesion and invasion of bladder cancer cells

Abstract

Objectives To determine whether granulocyte colony-stimulating factor receptor (G-CSFR) autocrine signaling promotes endothelial cell adhesion and invasion of bladder cancer cells through a β 1-integrin-mediated pathway. A significant fraction of invasive bladder carcinomas express both G-CSF and G-CSFR. Bladder carcinoma cell line 5637 constitutively secretes G-CSF but lacks G-CSFR expression. Thus, we studied the effects of G-CSFR expression on cell adhesion and invasion in this unique model system. Methods Flow cytometry and adhesion assay were performed to detect expression of β 1-integrin in G-CSFR-expressing 5637 cells and adhesion of these cells to human umbilical vein endothelial cell, respectively. Furthermore, an invasion chamber assay was done with the 5637 cells. Next, we used the G-CSF-specific antibody, siRNA, and a truncated version of G-CSFR (GR19) to block G-CSFR autocrine loop in these cells. We also used a β 1-integrin-specific neutralizing antibody in the adhesion and invasion assays with the 5637 cells. Results G-CSFR-mediated increased expression (approximately threefold) of β 1-integrin is significantly abrogated by G-CSF specific antibody or siRNA in 5637 cells. GR19 also completely blocked β 1-integrin expression. G-CSFR signaling increased adhesion (∼2.5-fold) of 5637 cells to human umbilical vein endothelial cells, which are potently blocked by β 1-integrin-specific antibody. G-CSF/G-CSFR autocrine signaling significantly increased the invasiveness of 5637 cells (∼10-fold), which was reduced by either attenuating G-CSF production (G-CSF-specific antibody and siRNA) or interfering with G-CSFR signaling (GR19). Furthermore, β 1-integrin-specific antibody completely blocked G-CSFR-mediated invasion of 5637 cells. Conclusions Autocrine G-CSF/G-CSFR signaling in bladder cancer can significantly contribute to cancer cell adhesion and invasion in a β 1-integrin-dependent manner.