Objectives: A significant fraction of invasive bladder carcinomas express both granulocyte colony-stimulating factor (G-CSF) and granulocyte colony-stimulating factor receptor (G-CSFR). We sought to determine whether G-CSF/G-CSFR signaling promotes survival and growth of bladder cancer cells. The bladder carcinoma cell line 5637 constitutively secretes G-CSF but lacks G-CSFR expression. In contrast, TCC-SUP lacks expression of both G-CSF and G-CSFR. Using these bladder cancer cell lines as our model systems, we studied the effects of G-CSFR expression on cell proliferation, survival, and growth in vivo.
Methods: The TCC-SUP and 5637 cells were transiently transfected with either empty vector (3.1) or G-CSFR (GR). Cell proliferation was assessed with or without G-CSF by MTT assay in TCC-SUP-3.1 and TCC-SUP-GR cells. Apoptosis was examined by flow cytometry in 5637-GR with or without anti-G-CSF antibody and in TCC-SUP-GR in the presence of increasing concentrations of G-CSF. We examined the effects of STAT3 (signal transducer and activator of transcription 3) dominant-negative expression on G-CSF/G-CSFR-mediated STAT3 phosphorylation by Western blotting in TCC-SUP-3.1 and TCC-SUP-GR cells. We characterized the effects of STAT3-dominant-negative expression on G-CSF/G-CSFR-mediated survivin expression by flow cytometry in TCC-SUP-3.1 and TCC-SUP-GR cells. We also examined tumor growth using 5637-3.1 and 5637-GR in the nude mice xenograft model.
Results: The G-CSF/G-CSFR loop significantly increased proliferation in TCC-SUP-GR cells. Anti-G-CSF antibody significantly increased apoptosis in serum-starved 5637-GR cells, G-CSF abrogated apoptosis in serum-starved TCC-SUP-GR cells in a dose-dependent manner. STAT3-dominant-negative expression blocked G-CSF-mediated STAT3 phosphorylation and survivin expression in TCC-SUP-GR cells. Furthermore, 5637-GR cells produced a significantly larger tumor in the subcutaneous nude mice xenograft model.
Conclusions: The G-CSF/G-CSFR autocrine/paracrine signaling loop significantly promotes survival and growth of bladder cancer cells.