Expression of
granulocyte-macrophage colony-stimulating factor (
GM-CSF) by metastatic
Lewis lung carcinoma cells (LLC-LN7) was previously shown to contribute to the maintenance of phenotypic characteristics associated with an increased capacity to metastasize. In the present study, pre-incubation of LLC-LN7 cells with neutralizing anti-
GM-CSF antibodies diminished the capacity of the
tumor cells to form experimental
metastases after i.v. inoculation, while pre-incubation with recombinant
GM-CSF (rGM-CSF) increased formation of
metastases. In the presence of rGM-CSF, the LLC-LN7 cells exhibited an increased capacity to migrate, invade through a reconstituted basement membrane, and adhere to lung tissue. Studies to identify the signal transduction pathway through which
GM-CSF enhanced the in vitro metastatic properties of the LLC-LN7
tumor cells implicated
protein kinase A (PKA). Signaling through PKA was suggested by the demonstration that the stimulation of
tumor-cell motility by
GM-CSF was blocked in the presence of the
adenylate cyclase inhibitor
nicotinic acid, or the PKA inhibitors A3 or
KT5720. In addition, the role of PKA as a signaling mechanism for
GM-CSF was assessed by using REV-LN7 cells, which are LLC-LN7 cells that have been stably transfected with an expression vector encoding a mutant PKA RI alpha subunit and which, in turn, express a cAMP-resistant PKA. Adherence and invasion by the PKA-defective REV-LN7 cells were not stimulated by rGM-CSF, contrasting with the stimulation observed for wild-type LLC-LN7 cells. These data suggest that rGM-CSF can further enhance the in vitro metastatic characteristics of LLC-LN7
tumor cells and that this is dependent on signal transduction through PKA.