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

and responds to estrogen; (d) pTopFlash-luciferase, protein loading were monitored by reprobing stripped
[20]
which contains 7 copies of the TCF/LEF-binding sites membranes with antibodies against either β-actin,
(7X-AGATCAAAGGgggta) and responds to β-catenin ERK1/2, or p38 MAPK as indicated.
activation; and (e) pCMV-β-gal, which was used
to control for differences in transfection efficiency. Co-immunoprecipitation assays
Afterward, the cells were incubated in the absence or Clarified cell extracts (1 mg/tube; see above)
presence of TGF-β1 (5 ng/mL), estradiol (0.1 nmol/L), prepared from pre- and post-EMT MCF-7 cells were
4-OHT (0.1 mol/L), or TβRI inhibitor II (100 ng/mL; incubated under continuous rotation with 1 µg of
Supplementary Table 1) as described previously; [16,17] antibodies against either β-catenin, E-cad, ER-α, or
(4) direct and indirect immunofluorescence: pre- and Src overnight at 4 ˚C. The resulting immunocomplexes
post-EMT MCF-7 and BT474 cells were cultured were collected by microcentrifugation, washed, and
overnight in 8-well chamber slides (80,000 cells/well) fractionated through 10% SDS-PAGE gels prior to their
prior to being treated for 96 h in serum-free media immobilization to nitrocellulose membranes, which
supplemented with TGF-β1 (5 ng/mL), estradiol subsequently were probed with antibodies against
(0.1 nmol/L), 4-OHT (0.1 µmol/L), or fulvestrant either phospho-tyrosine, β-catenin, EGFR, IGF1R, or
(0.1 µmol/L; Supplementary Table 1). Afterwards, ER-α [Supplementary Table 3]. Differences in protein
the cells were fixed in 4% paraformaldehyde and loading and/or input were monitored by immunoblotting
processed for direct immunofluorescence with with corresponding antibodies as indicated.
phalloidin (0.25 mol/L; Molecular Probes, USA), or
for indirect immunofluorescence with either anti-E- Statistical analysis
cadherin (E-cad) or anti-β-catetnin antibodies (1:250 Statistical values were defined using an unpaired
dilution; BD Biosciences, San Jose, USA), followed Student’s T-test, where a P value < 0.05 was
by sequential incubation with biotinylated secondary considered significant. P values for all experiments
antibodies (5 g/mL; Jackson ImmunoResearch, USA) analyzed are indicated.
and Alexa-streptavidin (1.2 g/mL; Molecular Probes,
USA). Following extensive washing with PBS, the RESULTS
slides were mounted on glass slides with Prolong
mounting medium (Molecular Probes) as described TGF-β induces EMT in MCF-7 cells and
previously. [16,17] potentiates noncanonical TGF-β signaling
EMT programs stimulated by TGF-β have been
Semi-quantitative real-time PCR analysis associated with the acquisition of motile and metastatic
Total RNA from MCF-7 or BT474 cells were purified phenotypes, and with the generation of cancer stem
using the RNeasy Plus Mini kit (Qiagen, USA) according cells and chemoresistance. [4,22,23] With respect to
to the manufacturer’s instruction. Afterwards, cDNAs cancers of the breast, these features of EMT are more
were synthesized by iScript reverse transcription kit commonly associated with basal-like/TNBC subtypes,
(Bio-Rad, USA), diluted 5-fold in H O, and employed in with little evidence related to how TGF-β and its
2
semi-quantitative real-time PCR reactions (20 µL) using stimulation of EMT programs impact ER-α signaling
SYBR Green system (Bio-Rad, USA) that contained in luminal breast cancers. To address this question,
5 µL of diluted cDNA and 0.1 µmol/L of oligonucleotide we administered TGF-β1 to human MCF-7 cells (i.e.
pairs listed in Supplementary Table 2. Differences in luminal A subtype) to induce an EMT program under
RNA concentration were controlled by normalizing 2D- and 3D-culture conditions. Figure 1A shows that
individual gene signals to their corresponding β-actin MCF-7 cells readily transitioned from epithelial to
or GAPDH as indicated. mesenchymal morphologies when stimulated with
TGF-β1. Moreover, these morphological alterations
Western blotting analyses exhibited by MCF-7 cells coincided with their (1)
Pre- and post-EMT MCF-7 and BT474 cells were downregulated expression of the epithelial marker
lysed in Buffer H/1% Triton X-100 and solubilized for E-cadherin in 3D-cultures [Supplementary Figure 1A],
[21]
60 min on ice. After microcentrifugation, the clarified and (2) upregulated expression of mesenchymal
cell extracts were resolved through 10% SDS-PAGE markers vimentin, N-cadherin, Cox-2, and MMP-9
electrophoresis gels, transferred electrophoretically [Supplementary Figure 1B-D], as well as that of the
to nitrocellulose membranes, and blocked in 5% master EMT transcription factors, Snail, Twist, Zeb1,
milk prior to incubation with primary antibodies and Zeb2 [Figure 1B and C]. Likewise, post-EMT
listed in Supplementary Table 3. The resulting MCF-7 cells showed a striking loss of β-catenin from
immunocomplexes were visualized by enhanced the plasma membrane [Supplementary Figure 1E] that
chemiluminescence reactions, and differences in reflected its enhanced phosphorylation on tyrosine
152 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ August 21, 2017