Mutation and Tamoxifen Resistance in Breast Cancer ’

A substantial portion of patients with estrogen receptor-positive breast cancer fail to respond to estrogen depletion or to the antiestrogen tamoxifen. The molecular changes that bead to tamoxifen resistance and estrogen-independent growth are unknown. To test the hypothesis that a p53 mutation could result in tamoxifen resistance and estrogenindependent growth, the MCF-7 cell line was transfected with p53 cDNA which was mutated at codon 179 (histidine to glutamine). MCF-7 is an estrogen receptor-positive, estrogen-dependent, tamoxifen-sensitive cell line with only wild-type p53. The presence of transfected mutant p53 cDNA was verified by the PCR, and overexpression of p53 protein was assessed by Western blotting. Five separate mutant-transfected clones were selected and tested in subsequent growth experiments. In monolayer culture, there was no consistent evidence of estrogen-independent growth or tamoxifen resistance in the mutant transfectants compared with vector-only controls or the parental cell line. In soft agar growth experiments, four of five mutant transfectants remained sensitive to tamoxifen in a dose-dependent manner. In the presence of wild-type p53, mutant 179 p53 protein does not result in estrogen-independent growth or tamoxifen resistance. These results do not exclude the possibility that other p5.3 mutational types could result in tamoxifen resistance, or that loss of the remaining wild-type allele may be necessary to result in this phenotype. INTRODUCTION The growth of some breast cancers has long been known to be regulated by estrogen. Conversely, antiestrogens such as tamoxifen can inhibit breast cancer growth in laboratory models, and treatment with tamoxifen can result in a probonged survival of breast cancer patients in the adjuvant setting (1) or for metastatic disease (2). Unfortunately, however, only a portion of Received 2/2/95; revised 5/15/95; accepted 6/5/95. 1 This work was supported by an American Cancer Society Institutional Research Grant, The San Antonio Cancer Institute, NIH P30 CA54174, and the Specialized Program of Research Excellence, NIH P50 CA58183-02. R. M. E. is the recipient of a Specialized Program of Research Excellence Careen Development Award. 2 To whom requests for reprints should be addressed, at University of Texas Health Science Center, Department of Medicine/Medical Oncology, 7703 Floyd Curl Drive, San Antonio, TX 78284-7884. breast cancers are sensitive to the effects of tamoxifen and estrogen. This unresponsiveness is only partially explained by back of ER,3 since 50% of patients who have ER-positive tumors still do not respond to tamoxifen therapy, and even those who do eventualby become resistant. The cause of this unresponsiveness remains unclear. p53 plays a central role in the negative growth regulation of a wide variety of cells (3, 4). Mutations in the p.53 gene result in the boss of this negative growth regulation, an acquired ability to transform cells (5), and promotion of malignant cell growth (6). p53 alterations are common in breast cancer and are associated with a worse prognosis, which is an indirect manifestation of a more aggressive cell growth (7-9). By a number of speculative mechanisms, p53 status could alter a breast tumor’s responsiveness to hormonal therapy and result in estrogen-independent growth. One mechanism that might explain tamoxifen resistance in p53-mutated breast cancer cells rebates to the ability of tamoxifen to induce apoptosis, as seen in a mouse mammary carcinoma cell line (10). Studies suggest that p53 plays an important role in apoptosis induced by anticancer agents (11). 1fp53 became mutated, it is possible that tamoxifen-induced apoptosis might also become blocked. Also, p53 mutations can alter growth factor interactions potentially important in the therapeutic response to tamoxifen. Evidence suggests that tamoxifen can produce an inhibitory effect on breast cancer by increasing TGF3, a growth factor that slows breast cancer cell growth (12). In human bronchial epithelium, mutant p53 causes loss of the inhibitory response usually seen with TGF3 (13). Thus, breast cancers with a p53 mutation may no longer be responsive to the normal growth-retarding effects of TGF3. Finally, p53 negatively regulates cell proliferation and entry of cells into the S-phase. ER also influences cell proliferation and transit through the cell cycle. In the presence of mutated pS3, the influence of the ER on cell cycle control might be lost, so that the cell would no longer be responsive to tamoxifen on estrogen. By any of these mechanisms, loss of the function of p53 would disrupt the functional integrity of the ER pathway because of a shared on common component. This would in turn bead to loss of response to agents exerting their effect through ER. Using MCF-7, a human breast cancer cell line that is estrogen dependent, tamoxifen sensitive, and ER positive, we tested the hypothesis that a p53 mutation could result in tamoxifen resistance and estrogen-independent growth. MATERIALS AND METHODS Vector. A full-length p53 eDNA was cloned into a 5.5-kb pRC/CMV plasmid (Invitrogen, San Diego, CA). The eDNA was derived from a lung cancer cell line and contained a Research. on April 19, 2017. © 1995 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 1204 p53 and Tamoxifen Resistance 4 G. Casey, personal communication. mutation that resulted in a histidine to glutamine substitution at codon 179 (14). This is a frequent site of mutation in a number of different cancers (15). The construct also contained a CMV promoter for p53 expression and a neomycin gene allowing G418 selection. A pRC/CMV plasmid without p53 eDNA served as a control. Cell Line. MCF-7 is a webb-characterized ER-positive and progesterone receptor-positive, hormone-dependent, tamoxifen-sensitive human breast cancer cell line. It has no detectable mutations in the conserved region (exons 4-9) of the p53 gene (16). Recently, the entire coding region of the gene was sequenced and no mutation was found.4 When used as a control, MCF-7 cells were not cbonalby derived from a single cell. Transfection. Twenty jig CsC1-punified control or mutant-containing plasmid was transfected into MCF-7 cells with 50 jig Lipofectin (GIBCO-BRL, Gaithersburg, MD) according to the manufacturer’s instructions. Cells were incubated overnight, then transferred to MEM (GIBCO, Grand Island, NY) supplemented with 10% FBS and i09 M insulin. The cells were initially grown in 800 jig/mb G418 (GIBCO-BRL) for 3 weeks, after which individual resistant colonies were harvested and screened for the presence of transfected plasmid by amplifying regions unique to the vector on vector-p53 construct using the PCR method. Western Blotting. Cells were harvested at 50-75% confluency, cytosobs were prepared in 5% SDS, and protein determination was made with the bicinchoninic acid method. Samples were run on 8.5% pobyacrylamide gels. The primary antibody for p53 was DOl (Oncogene Science, Uniondabe, NY) at a concentration of 10 jig/mb. The antibody for proliferating cell nuclear antigen was PC1O (DAKO, Carpentenia, CA) at a 1:100 dilution. The secondary antibody was an antimouse IgG antibody linked to horseradish penoxidase, NA931 (Amersham, Arlington Heights, IL). Blots were developed using a ehemiluminescence reagent (Dupont New England Nuclear, Boston, MA) and exposed to film for 1 to 2 mm. Growth Studies. For 17 3-estnadiob-induced monolayer growth experiments, 1 X i04 cells were plated and grown for 24 h in MEM plus 10% FBS plus 10 M insulin plus 25 jig/mb gentamicin. The cells were then washed, and the medium was changed to phenol red-free MEM plus 10% charcoal-stripped FBS plus 10 M insulin and 25 jig/mb gentamicin. For 4-hydroxytamoxifen growth experiments, 2 X i04 cells were plated, and 10 “ M estradiob was added to the phenol red containing 10% MEM and 10% stripped FBS. Cells were grown to approximately 50% confluency oven a period of 5-7 days. All expeniments were done in triplicate. Cells were harvested and counted with a hemocytometer. For anchorage-independent soft agan cloning, a base plate composed of 0.8% aganose in MEM phenol red-free medium, 10% stripped FBS, 10 tO M estradiob, 10 M insulin, and 25 jig/mb gentamicin was used. An upper layer of 0.4% aganose contained the above constituents, 30,000 celbs/ well, and the indicated concentration of 4-hydroxytamoxifen. Colonies were counted at 7 days. All experiments were pen1 2 3 4 5 6 7 8 9 1011