Growth Regulation of Estrogen Receptor-Negative Breast Cancer Cells Transfected With Complementary DNAs for Estrogen Receptor

Abstract

BACKGROUND: The growth of estrogen receptor (ER)-positive breast cancer cells is hormonally regulated, but the majority of breast cancers are ER negative and unresponsive to hormonal therapy. PURPOSE AND METHODS: To test whether hormonal control over replication can be re-established in ER-negative cells, we transfected ER-negative MDA-MB-231 (clone 10A) cells with sense and antisense constitutive ER expression vectors containing the gene for either wild-type or mutant ER linked to the gene for neomycin resistance aminoglycoside phosphotransferase (neo). A Northern blot analysis was done on total RNA from eight of the 10 transfectant clones produced to detect messenger RNA coding for ER and neo, and a Western blot analysis was done on protein extracted from the cells of one mutant and two wild-type ER sense transfectant clones to determine the molecular weight of the ER in transfectants. Levels of ER in transfectants were measured both by enzyme immunoassay and by ligand-binding methods. To ascertain whether the ER in wild-type and mutant sense transfectants was functional, we tested the effects of 17 beta-estradiol (E2) and/or an antiestrogen, ICI 164,384, on 1) ER-activated gene regulation (by transient transfection of these cells a second time with a reporter plasmid containing an estrogen response element linked to the chloramphenicol acetyl transferase [CAT] gene), 2) induction of progesterone receptor, 3) DNA replication, and 4) cell cycle kinetics. RESULTS: Messenger RNA coding for ER and for neo was detectable in both sense and antisense transfectant clones. Sense transfectants (both mutant and wild-type) expressed ER protein with a molecular weight similar to that found in ER-positive control cells. By the ligand-binding method high levels of ER were detected in both wild-type and mutant transfectants, although by the enzyme immunoassay method lower levels were detected in mutant transfectants. ER from both wild-type and mutant sense transfectants appeared functional, since E2 stimulated the expression of reporter-linked CAT and of progesterone receptor in these transfectants. E2 inhibited DNA replication in wild-type sense transfectants at a concentration of 10(-10) M and mutant sense transfectants at a concentration of 10(-8) M, and ICI 164,384 blocked this effect. CONCLUSION: ER-negative breast cancer cells stably transfected with either a mutant or wild-type ER gene regain hormonal responsiveness; however, E2 inhibits rather than stimulates cell growth. IMPLICATION: Reactivation of quiescent ER may provide a novel therapeutic approach for controlling ER-negative breast cancers.