The Vancouver Prostate Centre, Canada; The Department of Urologic Sciences, University of British Columbia, Canada.
The Vancouver Prostate Centre, Canada; Interdisciplinary Oncology, University of British Columbia, Canada.
Mol Cell Endocrinol. 2021 Feb 15;522:111136. doi: 10.1016/j.mce.2020.111136. Epub 2020 Dec 25.
Gli is an oncogenic transcription factor family thought to be involved in breast cancer (BrCa) cell growth. Gli activity is regulated by a post-translational proteolytic process that is suppressed by Hedgehog signaling. In prostate cancer cells, however, Gli activation is mediated by an interaction of active androgen receptor proteins with Gli3 that stabilizes Gli3 in its un-proteolyzed form. Here we show that the estrogen receptor (ER), ERα, also binds Gli3 and activates Gli in BrCa cells. Moreover, we show that ER + BrCa cells are dependent on Gli3 for cancer cell growth.
Transfection with Gli-luciferase reporter was used to report Gli activity in 293FT or BrCa cells (MCF7, T47D, MDA-MB-453) with or without steroid ligands. Co-immunoprecipitation and proximity ligation were used to show association of Gli3 with ERα. Gli3 stability was determined by western blots of BrCa cell extracts. ERα knockdown or destabilization (by fulvestrant) was used to assess how loss of ERα affects estradiol-induced Gli reporter activity, formation of intranuclear ERα-Gli3 complexes and Gli3 stability. Expression of Gli1 and/or other endogenous Gli-target genes in BrCa cells were measured by qPCR in the presence or absence of estradiol. Gli3 knockdown was assessed for effects on BrCa cell growth using the Cyquant assay.
ERα co-transfection increased Gli reporter activity in 293FT cells that was further increased by estradiol. Gli3 co-precipitated in ERα immunoprecipitates. Acute (2 h) estradiol increased Gli reporter activity and the formation of intranuclear ERα-Gli3 complexes in ER + BrCa cells but more chronic estradiol (48 h) reduced ERα-Gli complexes commensurate with reduced ERα levels. Gli3 stability and endogenous activity was only increased by more chronic estradiol treatment. Fulvestrant or ERα knockdown suppressed E2-induction of Gli activity, intranuclear ERα-Gli3 complexes and stabilization of Gli3. Gli3 knockdown significantly reduced the growth of BrCa cells.
ERα interacts with Gli3 in BrCa cells and estradiol treatment leads to Gli3 stabilization and increased expression of Gli-target genes. Furthermore, we found tthat Gli3 is necessary for BrCa cell growth. These results support the idea that the ERα-Gli interaction and Gli3 may be novel targets for effective control of BrCa growth.
Gli 是一种致癌转录因子家族，被认为参与乳腺癌 (BrCa) 细胞的生长。Gli 的活性受翻译后蛋白水解过程的调节，该过程受 Hedgehog 信号的抑制。然而，在前列腺癌细胞中，Gli 的激活是由活性雄激素受体蛋白与 Gli3 的相互作用介导的，该相互作用稳定了未被蛋白水解的 Gli3。在这里，我们表明雌激素受体 (ER)、ERα 也与 Gli3 结合并在 BrCa 细胞中激活 Gli。此外，我们表明 ER+BrCa 细胞依赖 Gli3 进行癌细胞生长。
使用 Gli-荧光素酶报告基因转染报告 293FT 或 BrCa 细胞（MCF7、T47D、MDA-MB-453）中 Gli 的活性，有或没有类固醇配体。共免疫沉淀和邻近连接用于显示 Gli3 与 ERα 的关联。通过 BrCa 细胞提取物的 Western blot 确定 Gli3 的稳定性。使用 ERα 敲低或失稳（氟维司群）来评估 ERα 缺失如何影响雌二醇诱导的 Gli 报告基因活性、核内 ERα-Gli3 复合物的形成和 Gli3 的稳定性。在存在或不存在雌二醇的情况下，通过 qPCR 测量 BrCa 细胞中 Gli1 和/或其他内源性 Gli 靶基因的表达。使用 Cyquant 测定评估 Gli3 敲低对 BrCa 细胞生长的影响。
ERα 共转染增加了 293FT 细胞中的 Gli 报告基因活性，雌二醇进一步增加了该活性。Gli3 在 ERα 免疫沉淀物中共同沉淀。急性（2 小时）雌二醇增加了 ER+BrCa 细胞中的 Gli 报告基因活性和核内 ERα-Gli3 复合物的形成，但更慢性的雌二醇（48 小时）减少了 ERα-Gli 复合物，与 ERα 水平降低一致。只有更慢性的雌二醇处理才会增加 Gli3 的稳定性和内源性活性。氟维司群或 ERα 敲低抑制了 E2 诱导的 Gli 活性、核内 ERα-Gli3 复合物和 Gli3 的稳定。Gli3 敲低显著降低了 BrCa 细胞的生长。
ERα 在 BrCa 细胞中与 Gli3 相互作用，雌二醇处理导致 Gli3 稳定和Gli 靶基因的表达增加。此外，我们发现 Gli3 是 BrCa 细胞生长所必需的。这些结果支持 ERα-Gli 相互作用和 Gli3 可能是有效控制 BrCa 生长的新靶标的观点。
