基于GPR30/HIF-1ɑ信号通路调控有氧糖酵解的黄芩素逆转他莫昔芬耐药的研究

Tamoxifen is the most frequently used endocrine agent for treatment of breast cancer patients, but most patients obtain resistance which is the major obstacle for its clinical use. Understanding the mechanisms of tamoxifen resistance and devising sensitizer to overcome drug resistance are urgent tasks. Accumulated evidences indicated that tamoxifen-resistant cells display increased aerobic glycolysis compared with sensitive cells, and inhibition of aerobic glycolysis restores tamoxifen sensitivity in resistant breast cancer cells. In addition, our preliminary experiment results showed that G protein-coupled receptor 30 (GPR30)/hypoxia-inducible factor-1α (HIF-1ɑ) signaling pathway may regulate aerobic glycolysis of tamoxifen-resistant cells. We previously confirmed that baicalein has potent anti-breast cancer activity, meanwhile it possesses inhibitory effect on GPR30 signaling pathway activation. In this study, we will evaluate the effects of baicalein on increasing tamoxifen sensitivity in vitro and in vivo. Meanwhile, we will detect whether baicalein could take effect on aerobic glycolysis of tamoxifen-resistant cells, and the expression, subcellular distribution, activity of GPR30 and HIF-1ɑ, to explore the mechanism that baicalein reverses tamoxifen resistance via down regulation of aerobic glycolysis by inhibiting GPR30/HIF-1ɑ signaling pathway. In general, this project may provide new evidence for the activity and mechanism of baicalein in breast cancer treatment. Furthermore, it may offer a new target and a strategy for discovering sensitizer to overcome tamoxifen resistance.

他莫昔芬（TAM）是乳腺癌患者应用最广泛的内分泌治疗药物，但大多数患者产生耐药是其临床应用的主要障碍，TAM耐药机制和增敏药物的研究亟待突破。已有的研究表明TAM耐药细胞与敏感细胞相比有氧糖酵解水平升高，抑制有氧糖酵解是逆转TAM耐药的潜在途径。本课题组通过预实验进一步发现，可能存在GPR30/HIF-1ɑ信号通路调控TAM耐药细胞的有氧糖酵解。我们前期研究证实，黄芩素能够通过抑制GPR30信号通路的活化，发挥抗乳腺癌的效应。基于此，本项目将考察黄芩素在体内外逆转TAM耐药的药理学活性，并研究黄芩素对耐药细胞有氧糖酵解，以及对GPR30和HIF-1ɑ表达、分布、活性的影响，阐明黄芩素通过抑制GPR30/HIF-1ɑ信号通路下调有氧糖酵解，逆转TAM耐药的作用机制。本项目延续前期研究，为深入探索黄芩素抗乳腺癌的作用环节和机制提供新依据，也为TAM耐药增敏剂的研究提供新靶点和新思路。

他莫昔芬（TAM）是雌激素受体阳性患者的一线治疗药物，患者产生耐药是限制其临床应用的主要障碍，亟待耐药机制和增敏药物的研究。已有的研究表明TAM耐药细胞与敏感细胞相比有氧糖酵解水平升高，抑制有氧糖酵解是逆转TAM耐药的潜在途径，然而调控TAM耐药细胞有氧糖酵解的机制尚不明确。本项目研究发现GPR30通过上调TAM耐药细胞中UCH-L1的表达，下调PHD2和pVHL的表达，增强HIF-1α/UCH-L1的结合，降低HIF-1α/PHD2-pVHL的结合，减少HIF-1α的泛素化，增加其稳定性和转录活性，从而上调TAM耐药细胞的有氧糖酵解水平，即存在GPR30/HIF-1ɑ信号通路调控TAM耐药细胞的有氧糖酵解。进一步通过体内外实验发现GPR30/HIF-1α信号通路能够调控耐药细胞对TAM的敏感性。临床样本分析证实高水平的GPR30/HIF-1α信号通路可提示乳腺癌患者预后不良，且与患者应用TAM治疗预后不良呈正相关。此外，本项目研究发现黄芩素能增加TAM对耐药细胞的生长抑制、克隆形成抑制以及诱导凋亡，并能够增加TAM抑制耐药细胞在NOD/SCID小鼠体内的生长，具有逆转TAM耐药的活性。黄芩素能下调GPR30的表达，上调HIF-1α与PHD2和pVHL的结合，增加HIF-1α的泛素化水平促进其降解，从而抑制GPR30/HIF-1α通路的活化。由此，黄芩素能够下调耐药细胞的有氧糖酵解水平，解除HIF-1α对线粒体的抑制作用，上调耐药细胞的线粒体数目和产生活性氧的能力，恢复TAM诱导耐药细胞生长抑制和线粒体凋亡。综上，本项目阐明了GPR30/HIF-1ɑ信号通路调控TAM耐药细胞有氧糖酵解的新机制，明确了黄芩素通过抑制该通路下调有氧糖酵解逆转TAM耐药的作用，为深入探索黄芩素抗乳腺癌的作用环节提供新依据，也为TAM耐药增敏剂的研究提供新靶点和新思路。