Classification of Breast Cancer Cells on the Basis of a Functional Assay for Estrogen Receptor

Abstract

Background: The receptor (ER) for estrogen (E2) is routinely assayed as a marker to determine the feasibility of anti-hormone therapy against 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 therapy. We suggest that this unpredictability of therapeutic responses lies in the current ER assays, which measure only an initial component of the E2-responsive pathway, and that the difference depends upon altered downstream processes. We propose a functional criterion that subclassifies breast cancers on the basis of specific binding of ER to its cognate DNA sequence, the estrogen response element (ERE). Materials and Methods: ER was identified in breast cancer cell lines by immunofluorescence 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 electrophoretic mobility shift assay (EMSA). Transactivation of the E2-responsive gene was studied by transfection of cells with fusion gene construct with the promoter-containing ERE sequence and assay of reporter gene activity in the cell extracts. Results: The growth of ER+ T47D cells was sensitive to tamoxifen, ICI-182,780, and ethynyl estradiol (EE2), whereas another ER+ breast cancer cell line, 2 1PT, was resistant to these compounds. The estrogen receptor (ER) in the nuclear extracts of MCF-7 and T47D demonstrated hormone-dependent interaction with the response element (ERE) and also downstream transactivation of the E2-responsive 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 all the other ER assays, ER-ERE interaction and PS2 promoter transactivation were independent of hormone. Conclusions: On the basis of the downstream functional assay of ER interaction with ERE, ER+ breast tumor cells can be subclassified into two categories. The first is E2-dependent (ERd+) and these cells should respond to anti-hormone therapy. The second type of ER interacts with ERE independent of E2 (ER'+) and constitutively transactivates responsive genes. It is predicted that the latter type of breast cancers will not respond to antihormone therapy.

Calmodulin Is Essential for Estrogen Receptor Interaction with Its Motif and Activation of Responsive Promoter

“…Cell Lines and Culture-MCF-7 and MDA-MB-231 cell lines were obtained from ATCC and were grown in rich medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2.8 M hydrocortisone, 1 g/ml insulin, and 12.5 ng/ml epidermal growth factor) under standard tissue culture conditions (20,21). For hormone studies Dulbecco's modified Eagle's medium without phenol red (DCC) supplemented with 10% stripped serum (dextran-coated, charcoaltreated fetal bovine serum; HyClone) was used.…”

Epidermal growth factor-induced nuclear factor κB activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

“…The ERϪ MDA-MB-231, MDA-MB435, and BT549, and ERϩ MCF-7 and T47D breast cancer cell lines were obtained from American Type Culture Collection. Growth conditions of these cells have been described previously (38). The rich medium, designated by R, is DMEM supplemented with 10% FBS, and the basal medium, designated by B, is DMEM supplemented with 10% FBS stripped with dextrancoated charcoal (HyClone) (38,39).…”

“…Nuclear extracts from cells were prepared as described by Dignam et al (40). The DNA-binding activity of NF-B was determined by EMSA as described previously (38)(39)41). The DNA-protein complex was detected as a retarded radioactive band by autoradiography of the dried gel (6,7).…”

“…The complementary strands of the oligonucleotide (5Ј-TCGACAGGGACTTTC-CGAGAG-3Ј) containing the NF-B motif (boldface) were custom synthesized by Integrated DNA Technologies (Coralville, IA). These strands were annealed to generate the doublestranded NF-B oligonucleotide, end labeled with 32 P-ATP (NEN) and T4 kinase (New England Biolabs), as described previously, and used for electrophoretic mobility-shift assay (EMSA) (38,39). E2, hydrocortisone, insulin, DTT, DMSO, and phenylmethylsulfonyl fluoride were obtained from Sigma.…”

Human 21T breast epithelial cell lines mimic breast cancer progression in vivo and in vitro and show stage-specific gene expression patterns

“…Although all three cell lines were initially thought to be ER and PR negative, subsequent work showed the 21PT cell line to express a variant ER-receptor, which is not well detected by conventional anti-ER antibodies. 37,38 To further explore the potential of the 21T series human breast cell lines as a functional model of early breast cancer progression, we have investigated their behavior both histologically in vivo and in 3D in vitro cultures. Characterization of these cells at the molecular level, using gene expression profiling of cells grown in 3D culture revealed stage-specific differences in genes involved in certain signaling pathways and functional categories reflective of progression to a more aggressive phenotype.…”