Epidermal Growth Factor-dependent Cell Cycle Progression Is Altered in Mammary Epithelial Cells That Overexpress 1

Amplification and overexpression of the c-myc gene are common in primary human breast cancers and have been correlated with highly proliferative tumors. Components of the epidermal growth factor (EGF) receptor signaling pathway are also often overexpressed and/or activated in human breast tumors, and transgenic mouse models have demonstrated that c-myc and transforming growth factor a (a member of the EGF family) strongly synergize to induce mammary tumors. These bitransgenic mammary tumors exhibit a higher proliferation rate than do tumors arising in single transgenics. We, therefore, chose to investigate EGFdependent cell cycle progression in mouse and human mammary epithehial cells with constitutive c-myc expression. In both species, c-myc overexpression decreased the doubling time of mammary epithelial cells by -6 h, compared to parental lines. The faster growth rate was not due to increased sensitivity to EGF but rather to a shortening of the G1 phase of the cell cycle following EGF-induced proliferation. In cells with exogenous c-myc expression, retinoblastoma (Rb) was constitutively hyperphosphorylated, regardless of whether the cells were growth-arrested by EGF withdrawal or were traversing the cell cycle following EGF stimulation. In contrast, the parental cells exhibited a typical Rb phosphorylation shift during G, progression in response to EGF. The abnormal phosphorylation status of Rb in c-myc-overexpressing cells was associated with premature activation of cdk2 kinase activity, reduced p27 expression, and early onset of cychin E expression. These results provide one explanation for the strong tumorigenic synergism between deregulated c-myc expression and EGF receptor Received 1/6/98; revised 4/17/98; accepted 4/29/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Department of Defense Grant DAMD1794-J-4257. S. I. N. was supported by Department of Defense Fellowship DAMD17-94-J-405 1. 2 Present address: Johns Hopkins Oncology Center, Baltimore, MD. 3 To whom requests for reprints should be addressed, at Lombardi Cancer Research Center, Room W41 7B, TRB, Georgetown University, 3970 Reservoir Road NW, Washington, DC 20007. Phone: (202) 6873770; Fax: (202) 687-7505; E-mail: DicksonR@GUNET.Georgetown. edu. signal transduction in the mammary tissue of transgenic mice. In addition, they suggest a possible tumorigenic mechanism for c-myc deregulation in human breast cancer. INTRODUCTION The proto-oncogene c-myc encodes a highly conserved nuclear phosphoprotein with domains that are common to many transcription factors (1-6). When bound to its heterodimeric partner Max, Myc protein binds specifically to DNA and can activate transcription. However, the physiologically relevant targets of myc regulation are not well defined, and thus, its mode of action is not fully understood, despite intense investigation. Myc has been implicated in the regulation of cell proliferation, differentiation, and death by apoptosis (reviewed in Refs. 1-6). Because aberration of any of those normal processes can contribute to tumorigenesis, it is not surprising that deregulated expression of the c-myc gene is often associated with neoplasia. in vitro, c-myc overexpression can cooperate with other oncogenes such as Ras to transform cells, and in vito, the ability of inappropriately expressed Myc to promote tumorigenesis has been clearly demonstrated by transgenic mouse models (7). Classified as an immediate early gene, c-myc expression is tightly regulated and correlated with the proliferative state of the cell (8). In normal quiescent cells, Myc protein levels are very low, and its expression is strongly induced following mitogen stimulation. Similarly, its expression decreases as cells become growth-arrested or undergo differentiation. A reduction in c-mvc levels due to disruption of one copy of the gene results in a lengthened G1 cell cycle phase (9), whereas inhibition of c-myc expression blocks cell cycle progression and leads to G1 arrest (10, 11). Conversely, when c-myc expression is deregulated, cells may grow at a faster rate and are often unable to withdraw from the cell cycle when signaled to do so (12-14). On the basis of the above observations, c-myc has long been thought to control key aspects of the proliferative response. Because passage through the cell cycle is orchestrated by the cychins and their associated cdks4 (reviewed in Ref. 15), these regulatory proteins would be logical targets for such a proposed action of Myc. Normally, expression of the various cyclins is tightly regulated and is characteristic of specific stages of the cell cycle. Several studies in fibroblasts and hematopoietic cells, in fact, suggest that expression or activity of some cychins and cdks may be altered when c-myc expression is deregulated (9, 16-22). 4 The abbreviations used are: cdk, cyclin-dependent kinase; TGF, transforming growth factor; EGF, epidermal growth factor; EGFR, EGF receptor; MEC, mammary epithelial cell; FACS, fluorescence-activated cell sorting; Rb, retinoblastoma; CAK, cdk-activating kinase. Research. on October 16, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 1814 Myc and the Cell Cycle in Breast Cancer Although some mechanistic details of the action of Myc have been studied in rodent fibroblasts, there is considerable interest in further elucidating the mechanisms(s) of malignant transformation by Myc in human epithelial malignancies, in which the oncoprotein has a clear pathophysiological function. Overexpression of c-myc is thought to play a role in the development of breast cancer because it is commonly amplified and/or overexpressed in human breast tumors (reviewed in Ref. 23). Amplification of the c-myc gene is often associated with highly proliferative tumors and poor prognosis. In addition, Myc confers tumorigenicity when it is overexpressed in the mammary gland of transgenic mice. Recent results from our laboratory (24) and others (25) showed that overexpression of TGF-a (which is also common in primary human breast tumors; Refs. 26 and 27) can strongly synergize with c-myc in transgenic mice to promote mammary tumor development in vivo, confirming previous in vitro observations that Myc can cooperate with growth factors such as TGF-a or EGF to transform MECs (28, 29). The contribution of TGF-a may be partly due to the suppression of Myc-induced apoptosis via increased expression of Bdl-xL (30, 3 1). However, tumors and cell lines derived from the double transgenic mice also showed an accelerated growth rate compared to those from single transgenic mice (24, 30). Those results suggest that c-myc may also cooperate with the EGFR signaling pathway to promote aberrant cell cycle progression in MECs. Although a variety of changes in the expression of cell cycle regulators have been identified in human breast cancer cell lines and primary tumors (reviewed in Ref. 32), little is known about the causes or consequences of cell cycle deregulation in breast cancer. Thus, the purpose of this study was to identify changes in cell cycle regulation during EGF-dependent growth of MECs that overexpress c-myc. MATERIALS AND METHODS Cell Lines. A pair of human MEC lines (l84AlN4 and 184A1N4-myc) were used to study the effects of c-myc overexpression on cell cycle regulation. The parental cell line, A 1N4, was derived from normal mammary tissue obtained by reduction mammoplasty and was immortalized with benzo(a)pyrene (33). The A1N4-myc line (29) was established via retroviral infection of A1N4 cells with a construct containing mouse c-myc under the control of the Moloney mouse leukemia virus long terminal repeat. Retention and expression of the c-myc transgene were confirmed by Southern and Western analysis, respectively (data not shown). Both cells lines were maintamed in Iscove’s MEM (Life Technologies, Inc., Gaithersburg, MD) containing 0.5% FCS, 0.5 p.g/ml hydrocortisone, 5 p.g/ml insulin (Biofluids, Rockville, MD), and 10 ng/ml EGF (Upstate Biotechnology Inc., Lake Placid, NY). The cells arrest in G1 in the absence of EGF (34). Two pairs of mouse mammary cell lines (HC14 and HC14myc and MMEC and MMEC-myc) were also used in prehiminary experiments. The HC14 line was established from a midpregnant mammary gland, whereas the MMEC line was derived from an 8-week-old virgin mammary gland. Both cell lines were transfected with a c-myc expression construct driven by the Moloney mouse leukemia virus long terminal repeat (28, 35). Growth Assays. Cells were plated in 96-well plates (Costar, Cambridge, MA) at a density of 1000-2000 cells/well. At various time points, plates were stained as described previously (36) with crystal violet (0.5% in 30% methanol; Sigma Chemical Co., St. Louis, MO), rinsed with water, and dried. At the end of the experiment, the dye was redissolved in 0. 1 M sodium citrate in 50% EtOH, and A540 was measured with an MR700 plate reader (Dynatech Laboratories Inc.). Doubling times were calculated from the slope of the line generated by plotting log(A) versus time. FACS Analysis. Cells were plated (5 X l0 cells/plate) in 10-cm dishes (Falcon 3003; VWR Scientific, Philadelphia, PA) in normal growth medium containing EGF. The next day, the cells were changed to EGF-free medium to arrest them in G1 . After 48 h, the cells were restimulated with EGF ( 10 ng/ml), and cells were harvested at 3-h intervals. Nuclei were isolated and stained with propidium iodide for cell cycle analysis according to the method of Vindelov et a!. (37). Western Analysis. Cells were plated, arrested, and restimulated with EGF as described for FACS analysis. At 1.5or 3-h intervals following EGF stimulation, total cell lysates were prepared. Cells were washed with cold PBS and then s