靶向ER-α36阻断ERK/MMP-9通路抑制三阴性乳腺癌增殖转移的分子机制研究

Triple negative breast cancer is a kind of aggressive subtype of breast cancer， with high-grade malignancy, high recurrence rates，and poor clinical prognosis. Due to the lack of effective therapeutic targets, triple negative breast cancer is insensitive to endocrine therapy and molecular targeted therapy, and poorly responds to traditional chemotherapy. Exploring of potential therapeutic targets for triple negative breast cancer has been an advanced research hotspot in recent years. In our previous study, we found that ER-α36, an isoform of estrogen receptor-α, is widely expressed in tumor tissues of patients with triple negative breast cancer, and high ER-α36 expression is significantly correlated with larger tumor and later TNM stage. Overexpress ER-α36 could promote proliferation and metastasis of triple negative breast cancer; knock-down of ER-α36 could inhibit their proliferation and metastasis. Further study firstly revealed that ER-α36 could phosphorylate ERK by direct binding with it, which then activate the transcription of MMP-9 gene. On the basis of previous research, this project aims to further analyze the mechanism underlying protein-protein interaction between ER-α36 and ERK; demonstrate the mechanism during regulation of MMP-9 expression by ER-α36; and explore the mechanism regulating ER-α36 expression in triple negative breast cancer; so as to provide an experimental basis for identifying ER-α36 as a candidate for therapeutic targeting triple negative breast cancer.

三阴性乳腺癌是一种具有明显侵袭性的特殊亚型乳腺癌，恶性度与复发率高，临床预后差。由于缺乏治疗靶点，三阴性乳腺癌对内分泌治疗及分子靶向治疗不敏感；传统化疗效果亦较差。探索三阴性乳腺癌潜在的治疗靶点是近年来乳腺癌研究的热点。我们在前期研究中发现雌激素受体-α的剪切异构体ER-α36在三阴性乳腺癌患者的肿瘤组织中广泛表达，且高表达的ER-α36与更大的肿瘤体积及更晚的肿瘤分期密切相关。过表达ER-α36能促进三阴性乳腺癌细胞的增殖转移；敲低ER-α36表达能抑制其增殖转移。进一步的研究首次发现ER-α36可通过与ERK直接结合促进其磷酸化、进而激活MMP-9基因的转录。本项目拟在此基础上进一步分析ER-α36与ERK的蛋白相互作用机制；明确ER-α36调控MMP-9的机制；探索三阴性乳腺癌中ER-α36的表达调控机制；从而为鉴定ER-α36作为治疗三阴性乳腺癌的候选靶点提供实验研究基础。

三阴性乳腺癌是一种具有明显侵袭性的特殊亚型乳腺癌，恶性度与复发率高，临床预后差。由于缺乏治疗靶点，三阴性乳腺癌对内分泌治疗及分子靶向治疗不敏感；传统化疗效果亦较差。探索三阴性乳腺癌潜在的治疗靶点是近年来乳腺癌研究的热点。本研究发现雌激素受体-α的剪切异构体ER-α36在三阴性乳腺癌患者的肿瘤组织中广泛表达，且高表达的ER-α36与更大的肿瘤体积及更晚的肿瘤分期密切相关。过表达ER-α36能促进三阴性乳腺癌细胞的增殖转移；敲低ER-α36表达能抑制其增殖转移。本研究利用氨基酸点突变试验成功构建了关键氨基酸位点(L133)突变的ER-α36：ER-α36(L133A)，并合成了含有ER-α36(L133A)的表达质粒，具体分析了ER-α36蛋白与ERK蛋白的相互结合机制，首次发现ER-α36可通过与ERK直接结合促进其磷酸化，ER-α36(L133A)与ERK的结合作用减弱，对其磷酸化的促进作用也明显减弱。进一步的研究表明过表达ER-α36能够激活ERK下游的信号通路，而过表达ERα36(L133A)对ERK下游信号通路的激活作用有所减弱。本研究通过慢病毒系统建立了过表达ER-α36的MDA231/Lenti-ER-α36细胞和过表达ER-α36(L133A)的MDA-231/Lenti-ER-α36(L133A)细胞。结果表明MDA231/Lenti-ER-α36细胞具有增强的克隆形成能力、成瘤能力、成管能力以及迁移、侵袭能力；而MDA-231/Lenti-ER-α36(L133A)细胞的克隆形成能力、成瘤能力、成管能力以及迁移、侵袭能力均弱于MDA231/Lenti-ER-α36细胞。本研究发现过表达ER-α36能够能够激活MDA-231细胞中MMP-9基因的转录，上调MMP-9蛋白和mRNA的表达水平；而过表达ER-α36(L133A)对MDA-231细胞中MMP-9基因的转录激活作用有所减弱。这些结果进一步明确了ER-α36调控三阴性乳腺癌增殖转移的分子机制，以及ER-α36通过ERK通路调控MMP-9表达的分子机制，为鉴定ER-α36作为治疗三阴性乳腺癌的候选靶点提供了实验研究基础。