Independent regulation of invasion and anchorage-independent growth by different autophosphorylation sites of the macrophage colony-stimulating factor 1 receptor.

Invasion of tissue by macrophages and implantation into the uterine wall by placental trophoblasts are known to be regulated by the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R, the product of the c-fms proto-oncogene). Recently, the clinical importance of CSF-1 and CSF-1R in invasive breast carcinoma has been recognized, but the significance of coexpression of CSF-1 and CSF-1R in mammary epithelial cell invasion has not been explored. In the present study, we investigated the invasive potential of a noninvasive, CSF-1R-negative, mouse mammary epithelial cell line (HC11) expressing a high level of CSF-1, which was stably transfected with the mouse wild-type CSF-1R. Compared with parental cells, transfected cells expressing a wild-type CSF-1R invaded 100-fold more efficiently through a barrier of reconstituted basement membrane (Matrigel) and formed colonies in soft agar, whereas the cellular growth rate was only slightly increased. Analysis of cell-conditioned medium by zymography and quantitative enzyme activity assays showed that clones transfected with a wild-type CSF-1R expressed significantly higher levels of urokinase-type plasminogen activator than did untransfected clones. Furthermore, after injection into the tail veins of BALB/c mice, CSF-1R-expressing clones also produced a 10-fold higher incidence of lung tumors than the parental cell line. We also analyzed HC11 clones transfected with CSF-1R mutated at two major autophosphorylation sites (Tyr-->Phe807 and Tyr-->Phe721). Mutation at Tyr807 eradicated the stimulatory effect of Fms expression on the invasive ability of HC11 cells and substantially reduced the metastatic potential of the transfected clones but did not alter the Fms-induced anchorage-independent growth in soft agar. In contrast, mutation at Tyr721 of Fms had no effect on invasion as measured in the in vitro assay but markedly abolished Fms-induced colony formation in soft agar and eradicated the metastatic potential of the transfected clones. Our results suggest that expression of CSF-1R can facilitate cellular invasion and anchorage-independent growth in mammary epithelial cells, and these two processes are independently regulated by separate phosphotyrosine sites of CSF-1R.