The differential influence of EGF, IGF-I, and TGF-β on the invasiveness of human breast cancer cells

Dear Editor: The metastatic spread of tumor cells involves a complex cascade which includes cell proliferation, invasion, motility, release of proteases, and establishment of a new microenvironment at the end organ. It appears that peptide growth factors are associated with the metastatic process and have a profound effect on cell motility (Gherardi, 1991). However, the biochemical mechanisms are not well defined. In the present study, epidermal growth factor (EGF), imoulin-like growth factor (IGF-I), and transforming growth factor (TGF-~) were selected for study, since these growth factors, and their respective receptors, are known to be associated with cancer progression. Specifically, EGF and EGF-receptor (EGFR) are associated with highly malignant and invasive breast cancer (Klijn et al., 1992). Further, TGF-~ is an established chemoattractant produced by breast cancer ceils (Arteaga et al., 1993). Thus, EGF and TGF-~ may be autocrine regulators of breast cancer cell motility. On the other hand, IGF-I is secreted by the breast stromal cells, stimulates breast cancer cell proliferation (Cullen et al., 1992; Chen et al., 1993); and thus, may act in a paracrine manner as a chemoattractant for breast cancer cells. The present study was designed to compare the activity of EGF, IGF-I, and TGF-~ on the invasiveness of ER-positive MCF-7 and ER-negative MDA-MB-231 (MDA) breast cancer ceils. The cancer ceils were obtained from American Type Culture Collection and grown as monolayer cultures in Roswell Park Memorial Institute 1640 media as previously described (Tong et al., 1999). The chemoattractant activity of the growth factors was assayed in vitro using Matrigel-coated (100 p~g/well) transwell chambers (Costar, Coming, NY) as previously described (Rajah et al., 1999). Growth factors, tested over a concentration range of 10-14-10 -8 M, were placed on the lower well of the transwell chambers. In experiments with MCF-7 cells, 3 x 105 cells were placed in the top of the Matrigel-coated membrane (1.31 e m 2, 12-t~m pore sized) and incubated for 48 h. Because the MDA ceils are much more invasive, 6.5 X 104 ceils were placed on the membrane (0.31 cm 2, 8-t~m pore sized) and incubated for 24 h. Cell movement through the Matrigelcoated membranes was quantified by counting the number of cells on the lower side of the membrane at the end of the incubation period. The cells were dyed with DiffQuick (Baxter Healthcare Corp, Miami, FL), rinsed, and counted. Invasion data is reported as the percentage of cells that migrated through the membrane, relative to the control cell group. Following incubation, supernatants were collected from upper wells and frozen for collagenase activity assays. The supernatants were assayed for collagenase activity using a radiometric assay in 96-well plates as previously described (Abidi et al., 1997). All experiments were performed in triplicate and the data was expressed as the percentage of collagenase activity, relative to the control group. Our results demonstrated that EGF was the most potent chemoattractant in the ER-positive MCF-7 cells. TGF-~ produced a small chemoattractant effect and IGF-1 displayed no activity (Fig. 1). None of the growth factors altered collagenase release from the MCF-7 cells (data not shown). EGF may increase the invasive motility of MCF-7 cells through alterations in the cytoskeletal signaling pathways and cellular adhesion. Cell membrane ruffling is known to be associated with an increased accumulation of organized actin fibers, and the EGF receptor signaling cascade interacts with actin nucleation (Therlot et al., 1991). EGF-induced enhancement of MCF-7 cell adherence to basement membrane components is another possible mechanism. For example, EGF-induced motility of an immortalized human mammary epithelial cell line was reported to be abolished by incubation with specific integrin antibodies (Matthay et al., 1993). In the ER-negative, MDA cells, TGF-~ caused a significant increase in the number of ceils that migrated through the transwell membranes, while EGF and IGF-I produced no chemoattractant effect (Fig. 2). None of the growth factors altered collagenase release from the MDA cells (data not shown). TGF-~ is reported to enhance breast cancer cell invasion and metastasis in vivo (Welch et al., 1990) and is a potent chemoattractant in various other tumor cell lines (Lyons et al., 1990). There is evidence that TGF-~ may enhance the invasive potential of cancer ceils by increasing the transcription of several cell adhesion receptors (Welch et al., 1990). Additionally, the TGF-[3-mediated inhibition of MDA cell proliferation would allow the cells to direct more metabolic resources to movement and invasion (Zugmaier et al., 1989). In conclusion, this study demonstrates a differential influence of