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

rigid microenvironments potentiate EMT programs 3C). The antagonist activities of TGF-β on ER-α
stimulated by TGF-β. [25-27] signaling are magnified in post-EMT MCF-7 cells as
compared to their pre-EMT counterparts [Figure 2G].
Similar induction of EMT programs was observed in Mechanistically, we observed TGF-β stimulation of
human BT474 cells (i.e. luminal B subtype) stimulated EMT programs to rapidly promote ER-α translocation
with TGF-β. For instance, BT474 cells displayed a from the nucleus to the cytoplasm, resulting in ER-α
more mesenchymal morphology in response to TGF-β being entirely excluded from the nucleus by 72 h
[Supplementary Figure 2A]; they also exhibited robust [Figure 2H]. Collectively, these findings suggest that
cadherin switching as the cells transitioned from TGF-β and its stimulation of EMT programs inhibit the
pre- (e.g. E-cadherin dominant) to post-EMT (e.g. genomic functions of ER-α by inducing its cytoplasmic
N-cadherin dominant) states [Supplementary Figure 2B]. sequestration.
Likewise, BT474 cells undergoing EMT programs
exhibited a time-dependent loss of epithelial markers TGF-β stimulation of EMT promotes the
(e.g. β-catenin, CK19, and ZO-1; Supplementary interaction of ER-α with EGFR, IGF1R and Src
Figure 2C) and gain of mesenchymal markers (e.g. in MCF-7 and BT474 cells
MMP9, Twist, and vimentin; Supplementary Figure Previous studies have shown the ability of cytoplasmic
2D). Collectively, these events culminated in the ER-α to interact physically with EGFR and IGF1R,
redistribution of E-cadherin [Supplementary Figure 2E] leading to the activation of MAP kinases and resistance
and β-catenin [Supplementary Figure 2F] from the to tamoxifen. [9,11-13] Because EMT programs stimulated
plasma membrane to the cytoplasm, consistent with by TGF-β resulted in ER-α exclusion from the nucleus,
the ability of TGF-β to induce EMT programs in luminal we speculated that EMT programs may underlie the
breast cancers. associations of ER-α with receptor tyrosine kinases
(RTKs). To test this hypothesis, we determined the
Induction of EMT programs by TGF-β promotes impact of TGF-β on the expression of EGFR and IGF1R
ER-α accumulation in the cytoplasm of MCF-7 in luminal breast cancer cells. As shown in Figure 3A-C,
cells TGF-β significantly stimulated the synthesis of EGFR
We next investigated the interplay between TGF-β and IGF1R mRNA in MCF-7 cells propagated in either
and ER-α in regulating EMT programs in MCF- 2D- or 3D-culture. Similar increases in EGFR transcript
7 cells, especially in light of the ability of TGF-β to levels were also observed in BT474 cells stimulated
inhibit ER-α signaling and vice versa. In doing so, with TGF-β [Supplementary Figure 4A], while the
[28]
we first determined whether ER-α signaling impacted abnormally high levels of IGF1R mRNA in BT474 cells
the coupling of TGF-β to EMT programs in MCF-7 [Supplementary Figure 4B] masked any apparent
cells. Although estradiol treatment had no obvious effects of TGF-β on IGF1R transcript levels. We also
effect on E-cad localization in MCF-7 cells, hormone observed TGF-β stimulation of MCF-7 and BT474 cells
administration did suppress the ability of TGF-β to to induce the production of EGFR proteins, which were
induce the redistribution of E-cadherin from the plasma readily captured in ER-α immunocomplexes [Figure 3D-
membrane [Figure 2A]. ER-α was the predominant F]. Likewise, IGF1R was also readily captured in ER-α
estrogen receptor expressed in MCF-7 [Figure 2B] and immunocomplexes isolated from MCF-7 and BT474
BT474 [Supplementary Figure 3A], indicating that the cells stimulated with TGF-β [Figure 3F and G]. Finally,
actions of estrogen are mediated entirely by ER-α in Src has been reported to facilitate the extranuclear
these breast cancer cells. As such, we also monitored activities and localization of ER-α in breast cancers.
[29]
the impact of TGF-β on ER-α expression in MCF-7 and Accordingly, we also found Src to associate with ER-α
BT474 cells. Figure 2C shows that TGF-β transiently in a TGF-β-dependent manner in MCF-7 cells [Figure
increased the synthesis of ER-α transcripts when MCF- 3H], suggesting a potential role for Src in facilitating
7 cells were propagated in 2D-cultures, an event that the extranuclear localization and function of ER-α.
also trended to occur in BT474 cells [Supplementary Collectively, these findings suggest that TGF-β and
Figure 3B]. Conversely, ER-α transcript levels were EMT programs result in the upregulation of EGFR and
dramatically repressed when MCF-7 organoids were IGF1R, which form cytoplasmic complexes with ER-α
stimulated with TGF-β in 3D-cultures [Figure 2D]. and Src.
Although TGF-β clearly regulated ER-α transcription,
activation of the TGF-β pathway had little-to-no effect TGF-β stimulation of EMT programs enhances
on ER-α protein expression in both MCF-7 and BT474 EGFR, IGF1R, and MAP kinase signaling in
cells [Figure 2E] despite remaining competent to inhibit MCF-7 and BT474 cells
luciferase expression driven by an estrogen-response The activation of MAP kinases by TGF-β is essential
element (ERE; Figure 2F and Supplementary Figure for its stimulation of EMT programs. [4,30] Interestingly,
154 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ August 21, 2017