Park Miso, Lee Seung Hyun, Bui Quyen Thu, Kim Young-Mi, Kang Keon Wook
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea.
Department of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, South Korea.
Front Pharmacol. 2022 Dec 2;13:1057276. doi: 10.3389/fphar.2022.1057276. eCollection 2022.
Most breast cancers are hormone-receptor-positive, and thus the first-line therapy for them is an anti-estrogen medication such as tamoxifen. If metastasis occurs or resistance to tamoxifen develops, the 5-year survival rates for breast cancer patients significantly decrease. Hence, a better understanding of the molecular mechanisms that contribute to breast cancer aggressiveness is of great importance. ERα36 is an estrogen receptor variant that is known to be upregulated in breast cancer patients receiving tamoxifen treatment or in triple-negative breast cancer cells. However, the specific molecular mechanism underlying ERα36-induced tamoxifen-resistance is not yet fully understood. ERα36-overexpressing MCF-7 cells were constructed by either plasmid transfection using ERα36 vector or retroviral infection using ERα36-V5-His vector. Target-gene expression was assessed by Western blot analysis and real-time PCR, and YAP activation was evaluated by luciferase assays and immunofluorescence. Cell proliferation and formation of three-dimensional spheroids were evaluated using the IncuCyte S3 Live Cell Analysis System. We found that the expression patterns of Hippo signaling-related genes were significantly changed in ERα36-overexpressing MCF-7 cells compared to MCF-7 cells, which were also similarly observed in tamoxifen-resistant MCF-7 cells. Specifically, the protein expression level and activity of YAP, the core downstream protein of the Hippo pathway, were significantly increased in ERα36-overexpressing MCF-7 cells compared with MCF-7 cells. The aggressive phenotypes acquired by ERα36 overexpression in MCF-7 cells were destroyed by YAP knockout. On this basis, we propose that ERα36 regulates YAP activity by a new mechanism involving Src kinase. Our results suggest that YAP targeting may be a new therapeutic approach to the treatment of advanced breast cancers overexpressing ERα36.
大多数乳腺癌是激素受体阳性的，因此，针对这些乳腺癌的一线治疗药物是抗雌激素药物，如他莫昔芬。如果发生转移或出现对他莫昔芬的耐药性，乳腺癌患者的5年生存率会显著下降。因此，深入了解导致乳腺癌侵袭性的分子机制非常重要。ERα36是一种雌激素受体变体，已知在接受他莫昔芬治疗的乳腺癌患者或三阴性乳腺癌细胞中上调。然而，ERα36诱导他莫昔芬耐药的具体分子机制尚未完全明确。通过使用ERα36载体进行质粒转染或使用ERα36-V5-His载体进行逆转录病毒感染，构建了过表达ERα36的MCF-7细胞。通过蛋白质免疫印迹分析和实时定量PCR评估靶基因表达，并通过荧光素酶报告基因检测和免疫荧光评估YAP激活。使用IncuCyte S3活细胞分析系统评估细胞增殖和三维球体的形成。我们发现，与MCF-7细胞相比，过表达ERα36的MCF-7细胞中Hippo信号相关基因的表达模式发生了显著变化，在他莫昔芬耐药的MCF-7细胞中也观察到了类似情况。具体而言，与MCF-7细胞相比，过表达ERα36的MCF-7细胞中Hippo通路的核心下游蛋白YAP的蛋白表达水平和活性显著增加。在MCF-7细胞中过表达ERα36所获得的侵袭性表型被YAP基因敲除所破坏。在此基础上，我们提出ERα36通过一种涉及Src激酶的新机制调节YAP活性。我们的结果表明，靶向YAP可能是治疗过表达ERα36的晚期乳腺癌的一种新的治疗方法。
