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Characterization of a membrane-associated estrogen receptor in breast cancer cells and its contribution to hormone therapy resistance using a novel selective ligand.
The Journal of Steroid Biochemistry and Molecular Biology ( IF 4.1 ) Pub Date : 2020-04-11 , DOI: 10.1016/j.jsbmb.2020.105671
Toshifumi Niwa 1 , Junko Takanobu 1 , Kanae Suzuki 1 , Yuta Sato 1 , Yuri Yamaguchi 2 , Shin-Ichi Hayashi 1
Affiliation  

The estrogen receptor (ER) plays a role in the progression of hormone-dependent breast cancer and is a hormone therapy target. Estrogen acts as a transcription factor (genomic action) and also produces a quick non-genomic reaction through intracellular signaling pathways. The membrane associated ER (mER) may regulate both these signals and hormone therapy resistance. However, the details remain unclear because a reliable method to distinguish the signals induced by the estradiol (E2)-mER and E2-nuclear ER complex has not been established. In the present study, we prepared the novel ligand Qdot-6-E2, selective for mER, by coupling E2 with insoluble quantum dot nano-beads. We investigated the characteristics of mER signaling pathways and its contribution to hormone therapy resistance using different cell lines including estrogen depletion resistant (EDR) cells with different mechanisms. Qdot-6-E2 stimulated proliferation of nuclear ER-positive cells, but nuclear ER-negative cells showed no response. In addition, Qdot-6-E2 indirectly activated nuclear ER and increased mRNA expression of target genes. We confirmed that E2 was not dissociated from Qdot-6-E2 using a mammalian one-hybrid assay. We visually demonstrated that Qdot-6-E2 acts from the outside of cells. The gene expression profile induced by Qdot-6-E2-mER was different from that induced by E2-nuclear ER. The effect of anti-ER antibody, the GFP-ER fusion protein localization, and the effect of palmitoyl acyltransferase inhibitor also indicated the existence of mER. Regarding intracellular phosphorylation signaling pathways, the MAPK (Erk 1/2) and the PI3K/Akt pathways were both activated by Qdot-6-E2. In EDR cells, only nuclear ER-positive cells showed increased cell proliferation with increased localization of ERα to the membrane fraction. These findings suggested that Qdot-6-E2 reacts with ERα surrounding the cell membrane and that mER signals help the cells to survive under estrogen-depleted conditions by re-localizing the ER to use trace amounts of E2 more effectively. We expect that Qdot-6-E2 is a useful tool for studying the mER.

中文翻译:

使用新型选择性配体的乳腺癌细胞中膜相关雌激素受体的表征及其对激素治疗耐药性的贡献。

雌激素受体(ER)在激素依赖性乳腺癌的进展中起作用,并且是激素治疗的目标。雌激素起转录因子(基因组作用)的作用,并通过细胞内信号传导途径产生快速的非基因组反应。与膜相关的ER(mER)可能同时调节这些信号和激素治疗耐药性。但是,由于尚未建立区分雌二醇(E2)-mER和E2核ER复合物诱导信号的可靠方法,因此细节仍不清楚。在本研究中,我们通过将E2与不溶性量子点纳米珠偶联,制备了对mER有选择性的新型配体Qdot-6-E2。我们调查了mER信号通路的特征及其对激素治疗耐药性的贡献,使用了不同的细胞系,包括具有不同机制的雌激素耗竭抵抗性(EDR)细胞。Qdot-6-E2刺激核ER阳性细胞增殖,但核ER阴性细胞无反应。此外,Qdot-6-E2间接激活核内质网并增加靶基因的mRNA表达。我们证实,使用哺乳动物一杂交试验,E2并未从Qdot-6-E2上解离。我们从视觉上证明了Qdot-6-E2从细胞外部起作用。Qdot-6-E2-mER诱导的基因表达谱不同于E2核ER诱导的基因表达谱。抗ER抗体的作用,GFP-ER融合蛋白的定位,棕榈酰基酰基转移酶抑制剂的作用也表明存在mER。关于细胞内磷酸化信号传导途径,MAPK(Erk 1/2)和PI3K / Akt途径均被Qdot-6-E2激活。在EDR细胞中,只有核ER阳性细胞显示细胞增殖增加,而ERα在膜部分的定位增加。这些发现表明Qdot-6-E2与细胞膜周围的ERα反应,mER信号通过使ER重新定位以更有效地使用痕量E2,帮助细胞在雌激素耗尽的条件下存活。我们希望Qdot-6-E2是研究mER的有用工具。仅核ER阳性细胞显示出细胞增殖增加,而ERα定位于膜部分增加。这些发现表明Qdot-6-E2与细胞膜周围的ERα反应,mER信号通过使ER重新定位以更有效地使用痕量E2,帮助细胞在雌激素耗尽的条件下存活。我们希望Qdot-6-E2是研究mER的有用工具。仅核ER阳性细胞显示出细胞增殖增加,而ERα定位于膜部分增加。这些发现表明Qdot-6-E2与细胞膜周围的ERα反应,mER信号通过使ER重新定位以更有效地使用痕量E2,帮助细胞在雌激素耗尽的条件下存活。我们希望Qdot-6-E2是研究mER的有用工具。
更新日期:2020-04-11
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