Acquisition of Hormone-independent Growth in MCF-7 Cells Is Accompanied by Increased Expression of Estrogen-regulated Genes but Without Detectable DNA Amplifications

A hormone-independent but hormone-responsive subpopulation (MCF7/ Mil l ) of the hormone-dependent MCF-7 human breast cancer cell line (R. Clarke et al., Proc. Natl. Acad. Sci. USA 86: 3649-3653, 1989) was further passaged in ovariectomized nude mice and re-established in vitro as the continuous cell line MCF7/LCCI. The lag time to the appearance of proliferating tumors in ovariectomized animals is significantly reduced in MCF7/LCCI when compared with MCF7/MIII cells. In gel denaturation/ renaturation analysis of tumor, genomic DNA does not reveal significant differences in the pattern of detectable DNA amplifications between parent MCF-7 cells and MCF7/LCCI cells. In the absence of estrogen, steadystate levels of phosphoinositol turnover are similar in both MCF-7 and MCF7/LCCI cells, but turnover is increased by estrogen only in MCF-7 cells. MCF7/MIII and MCF7/LCCI, but not MCF-7 cells, express a high baseline level of the estrogen-regulated pS2 mRNA. The baseline level of expression of progesterone receptor protein, but not mRNA, is higher in MCF7/LCCI when compared with either MCF-7 or early passage MCF7/ Mi l l cells. However, while the estrogen receptor is also an estrogenregulated gene, MCF7/MIII and MCF7/LCC1 cells retain estrogen receptor levels equivalent to the parental MCF-7 cells. These data indicate that progression to hormone independence can occur without major gene amplifications or a high constitutive induction of phosphoinositide metabolism. Thus, DNA amplifications may be acquired during the early initiation and/or promotional events of carcinogenesis. Significantly, acquisition of a hormone-independent but responsive phenotype in human breast cancer is associated with perturbations in the expression of specific estrogen-regulated genes. I N T R O D U C T I O N Many breast tumors appear to follow a predictable clinical pattern, being apparently confined to local structures, responsive to endocrine manipulation, and/or sensitive to cytotoxic chemotherapy. However, there is an almost inevitable progression to a more malignant phenotype characterized by invasive/metastatic loci that ultimately become resistant to endocrine manipulation and chemotherapeutic intervention. It is the development of a metastatic, hormone-independent, and drug-resistant phenotype that is responsible for the high percentage of treatment failures among breast cancer patients. The ultimate acquisition of this phenotype may be a fundamental biological property of many human breast cancer cells (1, 2). Received 10/3/91; accepted 10/26/92. 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. I This work was supported by USPHS Grants 1-R55-CA51782, 5-R01-CA58022 (R. C.), and 5-U01-CA51908 and Grant 1P30-CA51008 from the National Cancer Institute (M. E. L., R. C.); Research Grant 90BW65 from the American Institute for Cancer Research (R. C.); Fellowships from the Danish Cancer Society and National Cancer Institute/European Organization for Reasearch on Treatment of Cancer Exchange Program (N. B.); and Fondation pour la Recherche Medicale, and L'Association pour la Recherche sur le Cancer (V. B.). 2 Current address: Finsen Laboratory, Strandboulevarden 49, 2100 Copenhagen, Denmark. 3 To whom requests for reprints should be addressed, at Room S128A, Vincent T. Lombardi Cancer Research Center, Georgetown University Medical School, 3800 Reservoir Road NW, Washington, DC 20007. A major obstacle in the study of human breast cancer progression from hormone-dependent growth to hormone-independent growth has been the lack of suitable model systems. There is no direct biological relationship between the established hormone-dependent and hormone-independent breast cancer cell lines, other than their human breast origin. These cell lines were all derived from different women, at different times, and in some cases from different metastatic sites. In the present study, we describe the phenotype of cells obtained from a further selection of a hormone-independent but hormone-responsive subline (MCF7/MIII) of the hormone-dependent MCF-7 cells. MCF7/ MIII cells were originally isolated from an MCF-7 tumor following a physiologically relevant selection in an ovariectomized athymic nude mouse (3). MCF7/MIII cells no longer require hormonal supplementation to form proliferating tumors (4) and are more invasive and metastatic in vivo than the parental MCF-7 cells (5). We hypothesized that an additional selection of MCF7/MIII cells in ovariectomized athymic mice would induce a cellular morphology indicative of further malignant progression. Further selection of MCF7/MIII cells produced a hormone-independent variant (MCF7/LCC 1) that exhibits phenotypic characteristics that imply a more malignant phenotype than that expressed by the parental MCF-7 and MCF7/MIII cells. The molecular events that enable breast cancer cells to acquire a progressed phenotype remain unclear. For example, hormone-resistant cells can exhibit a partial or complete down-regulation of hormone receptors (6, 7), but loss of receptor expression is not essential for the development of resistance. Approximately 40% of human breast tumors expressing ER 4 are insensitive to the inhibitory effects of antiestrogen treatment (8, 9). Increased tumorigenicity (10, 11) and acquired drug resistance (12) are associated with the presence of amplified DNA sequences in some cancers. The amplification and/or overexpression of the cellular protooncogenes e r b B 2 (13, 14), myc (15), hst, and int2 (16-18) are observed in subsets of breast cancer patients. Overexpression of the receptor for epidermal growth factor is also associated with a more malignant phenotype in human breast cancer (19), and with the acquisition of drug resistance (20). Many of these oncogenes and growth factor receptors utilize perturbations in phosphoinositol metabolism as a signal transduction mechanism. Since MCF-7, MCF7/MIII, and MCF7/LCC 1 cells are derived from a common lineage, they provide a novel model to study the process of malignant progression in human breast cancer. Mechanistically, the acquisition of a more malignant hormone-independent phenotype (MCF7/MIII, MCF7/LCC 1) could be associated with perturbations in the regulation of gene expression. Thus, DNA amplifications (11, 21, 22), alterations in phosphoinositol metabolism, and/or perturbations in the expression of specific E2-regulated genes may be associated with the increased malignant potential exhibited by MCF7/MIII and MCF7/LCC 1 cells. We have previously indicated that progression to hormone independence may be associated with the altered expression of specific hormone-regulated genes (23, 24). We now describe the 4 The abbreviations used are: ER, estrogen receptor; PGR, progesterone receptor; EGF-R, epidermal growth factor receptor; TGF, transforming growth factor; SF, serumfree media; IMEM, improved minimal essential medium; CCS-IMEM, improved minimal essential medium supplemented with steroid-stripped serum.