Emergence of MCF-7 cells overexpressing a transfected epidermal growth factor receptor (EGFR) under estrogen-depleted conditions: Evidence for a role of EGFR in breast cancer growth and progression

Abstrad Overexpression of epidermal growth factor receptor (EGFR) is correlated with loss of estrogen receptor and poor prognosis in breast cancer. To investigate this phenomenon, we transfeded a cytomegalovirus expression vedor direding the expression of EGFR into estrogen receptor-positive MCF-7 breast cancer cells and into a clone of MCF-7 cells previously transfected with transforming growth fador a. Cells arising from single clones or pooled polyclonal populations maintained in charcoal-stripped calf serum, a medium devoid of estrogen, overexpressed EGFR. Switching these cells to a medium containing fetal calf serum or charcoal-stripped calf serum plus 1 713-estradiol resulted in the emergence of a population expressing low EGFR levels. Loss of expression was not a consequence of nonspecific repression of the cytomegalovirus promoter, because expression of the fibroblast growth fador (FGF)-4 complementary DNA in a similar vedor was not lost in fetal calf serum. While loss of EGFR overexpression in fetal calf serum was seen at both the protein and mRNA levels, Southern blotting shows that this was not due to loss of the transfeded gene. Subclones of a cell population with low EGFR expression were capable of increasing expression upon estrogen withdrawal, demonstrating that the changes in EGFR expression were reversible and suggesting a growth advantage conferred by EGFR overexpression under these restridive growth conditions. Overexpression of EGFR did not result in loss of ER expression. These results suggest a role for overexpression of EGFR in the growth of estrogen receptor-positive breast cancer cells in the absence of estrogen. lntrodudion The presence or absence of functional receptors for the steroid hormone estrogen is a valuable prognostic indicator in human breast carcinoma. The responsiveness of ER+3 cells to estrogen is mediated through the ER and can be exploited by treatment with antiestrogenic drugs such as tamoxifen (1 ). However, not all patients with ER+ tumors respond to endocrine therapy, and an almost invariable occurrence in patients that do respond initially is the eventual outgrowth of cell populations where growth is no longer inhibited by antiestrogen treatment (2). Autocrine stimulation of growth by a cell expressing both a growth factor and the cognate growth factor receptor has been postulated as a means by which a cell could overcome estrogen dependence (3). Constitutive overexpression of genes for growth factors or growth factor receptors in an ER+ cell could alleviate the need for estrogen stimulation of growth. One such estrogen-induced gene codes for TGF-a, a ligand for the EGFR (4). The role of TGF-a in the acquisition of an estrogen-independent phenotype has been investigated by transfection of TGF-a into the ER+ MCF-7 breast cancer cell line. Cells that constitutively overexpressed this protein remained estrogen responsive in vitro and did not form tumors in ovariectomized nude mice (5). Similarly, transfection of TGF-a into rodent fibroblast cell lines has been reported to result in a more transformed phenotype (6, 7), but increased tumorigenicity requires overexpression of both ligand and EGFR (8). Conversely, overexpression of EGFR in NIH 3T3 cells has been shown to be transforming only in the presence of EGF (9-1 1). There are several lines of evidence indicating that overexpression of EGFR might be an important event in the progression of breast cancer to an estrogen-independent phenotype. Clinical studies have noted that overexpression of EGFR in breast cancer is correlated with absence of a functional ER (1 2), poor response to tamoxifen treatment (1 3, 1 4), and poor prognosis (1 5-1 7). Furthermore, an inverse correlation between ER and EGFR levels has been reported (1 8, 1 9). These data suggest that, in some breast tumors, overexpression of EGFR may represent a later stage of tumor progression in which stimulation via the ER is no longer required for cell growth. These observations suggest a role for overexpression of EGFR in the proliferation of