C-myc gene expression alone is sufficient to confer resistance to antiestrogen in human breast cancer cells.

C-myc is implicated in the initiation, progression and estrogen response of breast cancer. To further investigate the role of c-myc in breast cancer, we have developed clonal MCF-7 human breast cancer cell lines harboring a stably-transfected human c-myc gene, whose expression was stringently controlled by the bacterial reverse tetracycline transcription activator protein. The expression of the endogenous genomic c-myc gene in MCF-7 cells was abolished by the potent pure estrogen antagonist, ICI 182,780. Functional c-Myc protein was identified by both Western immunoblotting and by its ability to transactivate a chimeric plasmid consisting of E-box sequences upstream of the luciferase reporter gene. One MCF-7 clone, 35im, was chosen for further characterization. C-myc induction by doxycycline was rapid and dose dependent; c-myc mRNA appeared as early as 30 min after doxycycline addition and stimulation of c-myc expression required as little as 50 ng/ml doxycycline, with c-myc mRNA levels reaching a plateau at 2.5 microg/ml doxycycline. ICI 182,780 or doxycycline (a tetracycline analog) treatment did not alter the mRNA levels of Max, the c-myc binding partner. As in wildtype MCF-7 cells, the growth of clone 35im was inhibited by 1 microM or less of ICI 182,780 and stimulated by 10 nM to 1 microM 17beta-estradiol. When maintained in a complete medium containing 5% normal fetal bovine serum (FBS) and ICI 182,780, doxycycline induced cell growth by 400% in an 8-day assay. A similar level of growth was achieved with doxycycline treatment in cells that were arrested by the use of charcoal-stripped FBS. Doxycycline had no effect on the growth of a control MCF-7 clone (18c). Apoptosis, assessed by caspase-dependent cleavage of poly(ADP-ribose) polymerase, was unchanged in clone 35im cells after treatments with doxycycline or ICI 182,780. The present study demonstrates that c-myc alone is sufficient to confer antiestrogen resistance in human breast cancer. Our novel c-myc-inducible MCF-7 cell model offers a unique opportunity to study the diverse actions of the c-myc proto-oncogene in human breast cancer.