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Tian et al. EMT drives anti-estrogen resistance in breast cancer

promoter of breast cancer growth and metastasis. [1-3] (USA) and cultured as previously described, while
[15]
Although the molecular mechanisms responsible for human luminal B BT474 cells were kindly provided by
eliciting the dichotomous activities of TGF-β remain to Dr. Mark W. Jackson (Case Western Reserve University,
be fully elucidated, accumulating evidence implicates USA) and propagated in DMEM supplemented
canonical Smad2/3-dependent signaling in mediating with 10% fetal bovine serum (FBS; Thermo Fisher
tumor suppression by TGF-β and noncanonical Scientific, USA) and 1% Pen/Strep (Invitrogen, USA).
Smad2/3-independent signaling in mediating its tumor Pharmacological agonists and inhibitors used herein
promoting activities. [1-3] Amongst the best characterized are described in the Supplementary Table 1.
noncanonical signaling pathways operant in
coupling TGF-β to tumorigenesis are: (1) integrins Cell biological assays
and components of focal adhesion complexes; (2) Analyzing the effects of TGF-β and EMT programs
MAP kinase and small GTP-binding protein family on ER-α signaling in MCF-7 and BT474 cells was
members; and (3) PI3K/AKT and NF-κB pathways; determined as follows: (1) cell proliferation assays:
[4]
they also function to drive epithelial-mesenchymal cells were treated in the absence or presence of
transitions (EMT) stimulated by TGF-β, thereby TGF-β1 (5 ng/mL; R&D Systems, USA) for 72 h to
promoting breast cancer dissemination, stemness, induce EMT, at which point they were subcultured in
and chemoresistance. [5] 96-well plates (10,000 cells/well) for 5 days with either
diluent or inhibitors to the TGF-β type I receptor (TβR-I;
Within the mammary gland, signaling by estrogen 100 ng/mL), the epidermal growth factor receptor
receptor (ER-α) plays an essential role not only (EGFR; 1 mol/L), the insulin-like growth factor 1
during glandular development and differentiation, but receptor (IGF1R; 1 mol/L), mitogen-activated protein
also during the initiation and progression of luminal kinase kinase (MEK; 10 mol/L), or ER-α (0.1 mol/L;
breast cancers. [6-8] Indeed, the oncogenic activities Supplementary Table 1). Differences in cell growth
of dysregulated ER-α signaling underlie the clinical and survival were determined by incubating the cells
success of anti-estrogen drugs (e.g. tamoxifen) as with MTS Plus reagent (20 µL; Promega, USA) for 1 h
first-line therapies to treat ER-positive breast cancers. at 37 ˚C, followed by measuring absorbance at 490
However, despite their initial efficacy, anti-estrogen nm on a Promega Modulus II Microplate Multimode
drugs often become ineffective as patient tumors instrument (Promega, USA); (2) 3-dimensional (3D)
develop resistance and undergo disease recurrence. [9,10] growth assays: 3D-cultures were prepared by diluting
At present, the mechanisms resulting in acquired pre- or post-EMT MCF-7 and BT474 cells in complete
anti-estrogen resistance are not fully understood. media supplemented with 5% Cultrex (Trevigen,
However, compelling evidence implicates nongenomic Gaithersburg, USA), which subsequently were seeded
ER-α signaling as a major culprit of resistance to anti- onto solidified Cultrex cushions (500 µL/well) contained
estrogen-based therapies. [9,11-13] Likewise, aberrant in 6-well plates (150,000 cells/well). Afterward, the
expression of a truncated metastasis tumor antigen 1 cells were cultured in the absence or presence of
(MTA1) mutant was found to bind and sequester ER-α TGF-β1 (5 ng/mL), estradiol (1 nmol/L), tamoxifen
in the cytoplasm, thus enhancing the nongenomic (0.1 nmol/L), or fulvestrant (0.1 mol/L; Supplementary
actions of ER-α and disease progression in breast Table 1) for 8 days, during which time they were fed
cancers. [14] every 3 days with full growth media supplemented with
5% Cultrex and pharmacological agents. Differences
Given the pathophysiologic parallels that exist between in organoid growth were calculated using NIH Image
nongenomic ER-α and noncanonical TGF-β signaling J; (3) luciferase reporter gene assays: pre- and
in driving breast cancer progression, we speculated post-EMT MCF-7 and BT474 cells were allowed to
that EMT programs induced by TGF-β may elicit adhere overnight to 24-well plates (40,000 cells/well).
nongenomic ER-α signaling and endocrine resistance The cells were transiently transfected as described
in luminal breast cancers. The aim of this study was previously [16,17] with the following reporter plasmids:
to test this hypothesis and further our understanding (a) pSBE-luciferase, which contains 4 copies of the
of how EMT programs drive disease progression and Smad3/4-binding element (4X-CAGA) and serves
acquired resistance to anti-estrogen-based therapies as a direct measure of canonical TGF-β signaling;
in human breast cancers. (b) p3TP-lux, which contains 3 copies of TPA-
[18]
responsive elements and 96 bp of the PAI-1 promoter
METHODS and responds to both canonical (i.e. Smad3/4) and
noncanonical (i.e. AP-1) TGF-β signaling; (c) pERE-
Cell lines and chemical inhibitors TATA-luciferase, which contains 3 copies of the
[19]
Human luminal A MCF-7 cells were obtained from ATCC estrogen response element (3X-GGTCACAGTGACC)
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ August 21, 2017 151