Department of Molecular and Functional Dynamics, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
Department of Molecular and Functional Dynamics, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
J Steroid Biochem Mol Biol. 2020 Jul;201:105671. doi: 10.1016/j.jsbmb.2020.105671. Epub 2020 Apr 11.
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.
雌激素受体（ER）在激素依赖性乳腺癌的进展中发挥作用，是激素治疗的靶点。雌激素作为转录因子（基因组作用），也通过细胞内信号通路产生快速的非基因组反应。膜相关 ER（mER）可能调节这两种信号和激素治疗抵抗。然而，由于尚未建立区分雌二醇（E2）-mER 和 E2-核 ER 复合物诱导的信号的可靠方法，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 具有选择性的新型配体 Qdot-6-E2。我们使用不同的细胞系，包括具有不同机制的雌激素耗竭抵抗（EDR）细胞，研究了 mER 信号通路的特征及其对激素治疗抵抗的贡献。Qdot-6-E2 刺激核 ER 阳性细胞的增殖，但核 ER 阴性细胞无反应。此外，Qdot-6-E2 间接激活核 ER 并增加靶基因的 mRNA 表达。我们使用哺乳动物单杂交测定法证实 E2 未从 Qdot-6-E2 中解离。我们直观地证明了 Qdot-6-E2 从细胞外部起作用。由 Qdot-6-E2-mER 诱导的基因表达谱与由 E2-核 ER 诱导的基因表达谱不同。抗 ER 抗体、GFP-ER 融合蛋白定位和棕榈酰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通路，MAPK（Erk1/2）和 PI3K/Akt 通路均被 Qdot-6-E2 激活。在 EDR 细胞中，只有核 ER 阳性细胞显示出细胞增殖增加，并且 ERα 向质膜部分的定位增加。这些发现表明，Qdot-6-E2 与围绕细胞膜的 ERα 反应，并且 mER 信号通过将 ER 重新定位到质膜以更有效地利用痕量的 E2 来帮助细胞在雌激素耗尽的条件下存活。我们期望 Qdot-6-E2 是研究 mER 的有用工具。
