Department of Internal Medicine, University of Virginia School of Medicine, 450 Ray Hunt Dr., Charlottesville, VA 22903, USA.
J Steroid Biochem Mol Biol. 2010 Feb 28;118(4-5):219-30. doi: 10.1016/j.jsbmb.2009.09.018. Epub 2009 Oct 6.
As breast cancer cells develop secondary resistance to estrogen deprivation therapy, they increase their utilization of non-genomic signaling pathways. Our prior work demonstrated that estradiol causes an association of ERalpha with Shc, Src and the IGF-1-R. In cells developing resistance to estrogen deprivation (surrogate for aromatase inhibition) and to the anti-estrogens tamoxifen, 4-OH-tamoxifen, and fulvestrant, an increased association of ERalpha with c-Src and the EGF-R occurs. At the same time, there is a translocation of ERalpha out of the nucleus and into the cytoplasm and cell membrane. Blockade of c-Src with the Src kinase inhibitor, PP-2 causes relocation of ERalpha into the nucleus. While these changes are not identical in response to each anti-estrogen, ERalpha binding to the EGF-R is increased in response to 4-OH-tamoxifen when compared with tamoxifen. The changes in EGF-R interactions with ERalpha impart an enhanced sensitivity of tamoxifen-resistant cells to the inhibitory properties of the specific EGF-R tyrosine kinase inhibitor, AG 1478. However, with long term exposure of tamoxifen-resistant cells to AG 1478, the cells begin to re-grow but can now be inhibited by the IGF-R tyrosine kinase inhibitor, AG 1024. These data suggest that the IGF-R system becomes the predominant signaling mechanism as an adaptive response to the EGF-R inhibitor. Taken together, this information suggests that both the EGF-R and IGF-R pathways can mediate ERalpha signaling. To further examine the effects of fulvestrant on ERalpha function, we examined the acute effects of fulvestrant, on non-genomic functionality. Fulvestrant enhanced ERalpha association with the membrane IGF-1-receptor (IGF-1-R). Using siRNA or expression vectors to knock-down or knock-in selective proteins, we further demonstrated that the ERalpha/IGF-1-R association is Src-dependent. Fulvestrant rapidly induced IGF-1-R and MAPK phosphorylation. The Src inhibitor PP2 and IGF-1-R inhibitor AG1024 greatly blocked fulvestrant-induced ERalpha/IGF-1-R interaction leading to a further depletion of total cellular ERalpha induced by fulvestrant and further enhanced fulvestrant-induced cell growth arrest. More dramatic was the translocation of ERalpha to the plasma membrane in combination with the IGF-1-R as shown by confocal microscopy. Taken in aggregate, these studies suggest that secondary resistance to hormonal therapy results in usage of both IGF-R and EGF-R for non-genomic signaling.
当乳腺癌细胞对雌激素剥夺疗法产生继发性耐药时，它们会增加对非基因组信号通路的利用。我们之前的工作表明，雌二醇会导致 ERalpha 与 Shc、Src 和 IGF-1-R 结合。在对雌激素剥夺（芳香化酶抑制剂的替代物）和抗雌激素他莫昔芬、4-OH-他莫昔芬和氟维司群产生耐药的细胞中，ERalpha 与 c-Src 和 EGF-R 的结合增加。与此同时，ERalpha 从核内转移到细胞质和细胞膜中。用Src 激酶抑制剂 PP-2 阻断 c-Src 会导致 ERalpha 重新定位到核内。虽然这些变化在每种抗雌激素的反应中并不完全相同，但与他莫昔芬相比，4-OH-他莫昔芬会增加 ERalpha 与 EGF-R 的结合。EGF-R 与 ERalpha 相互作用的改变赋予了对特定 EGF-R 酪氨酸激酶抑制剂 AG 1478 具有抑制特性的他莫昔芬耐药细胞更高的敏感性。然而，随着他莫昔芬耐药细胞长期暴露于 AG 1478，细胞开始重新生长，但现在可以被 IGF-R 酪氨酸激酶抑制剂 AG 1024 抑制。这些数据表明，IGF-R 系统成为适应 EGF-R 抑制剂的主要信号机制。总之，这些信息表明，EGF-R 和 IGF-R 途径都可以介导 ERalpha 信号。为了进一步研究氟维司群对 ERalpha 功能的影响，我们检查了氟维司群对非基因组功能的急性影响。氟维司群增强了 ERalpha 与膜 IGF-1 受体（IGF-1-R）的结合。使用 siRNA 或表达载体敲低或敲入选择性蛋白，我们进一步证明 ERalpha/IGF-1-R 结合依赖于 Src。氟维司群迅速诱导 IGF-1-R 和 MAPK 磷酸化。Src 抑制剂 PP2 和 IGF-1-R 抑制剂 AG1024 极大地阻断了氟维司群诱导的 ERalpha/IGF-1-R 相互作用，导致氟维司群诱导的总细胞 ERalpha 进一步耗竭，并进一步增强了氟维司群诱导的细胞生长抑制。更引人注目的是，通过共聚焦显微镜观察到 ERalpha 向质膜的易位与 IGF-1-R 结合。总之，这些研究表明，激素治疗的继发性耐药导致非基因组信号使用 IGF-R 和 EGF-R。
