CLASSIFICATION OF BREAST-CANCER CELLS ON THE BASIS OF A FUNCTIONAL ASSAY FOR ESTROGEN-RECEPTOR

Background: The receptor (ER) for estrogen (E2) is routinely assayed a s a marker to determine the feasibility of anti-hormone therapy agains t breast cancer because ER-positive (ER+) tumors are much more likely to respond to anti-hormone therapy than are ER-negative (ER-). However 40% of ER+ breast cancer patients do not respond to anti-hormone ther apy. We suggest that this unpredictability of therapeutic responses li es in the current ER assays, which measure only an initial component o f the E2-responsive pathway, and that the difference depends upon alte red downstream processes. We propose a functional criterion that subcl assifies breast cancers on the basis of specific binding of ER to its cognate DNA sequence, the estrogen response element (ERE). Materials a nd Methods: ER was identified in breast cancer cell lines by immunoflu orescence assay, Western blot analysis, identification of ER-specific mRNA, and by interaction of the ER-ERE complex with three different ER -specific antibodies. ER-ERE complex formation was measured by electro phoretic mobility shift assay (EMSA). Transactivation of the E2-respon sive gene was studied by transfection of cells with fusion gene constr uct with the promoter-containing ERE sequence and assay of reporter ge ne activity in the cell extracts. Results: The growth of ER+ T47D cell s was sensitive to tamoxifen, ICI-182,780, and ethynyl estradiol (EE2) , whereas another ER+ breast cancer cell line, 21PT, was resistant to these compounds. The estrogen receptor (ER) in the nuclear extracts of MCF-7 and T47D demonstrated hormone-dependent interaction with the re sponse element (ERE) and also downstream transactivation of the E2-res ponsive PS2 promoter. But in the 21PT cell line that was designated as ER- on the basis of ligand-binding assay and was found to be ER+ by a ll the other ER assays, ER-ERE interaction and PS2 promoter transactiv ation were independent of hormone. Conclusions: On the basis of the do wnstream functional assay of ER interaction with ERE, ER+ breast tumor cells can be subclassified into two categories. The first is E2-depen dent (ERd+) and these cells should respond to anti-hormone therapy. Th e second type of ER interacts with ERE independent of E2 (ERi+) and co nstitutively transactivates responsive genes. It is predicted that the latter type of breast cancers will not respond to antihormone therapy .