基于乳腺癌CANP/CST系统失调的黄芩素抑制纤连蛋白诱导上皮间质转化的研究

In the mammary gland, the stromal extracellular matrix undergoes dramatic changes during breast cancer tumorigenesis and development. High level of fibronectin (FN) and its ability to induce epithelial-mesenchymal transition (EMT) of breast epithelium show an active role in breast cancer progression. The deregulation of the calpain/calpastatin system could drive breast cancer development. Our preliminary results showed that the expression of calpain 1 and calpain 2 were up-regulated, yet the expression of calpastatin was down-regulated during FN-induced EMT of mammary epithelial cells, but baicalein suppressed this changes of calpain and calpastatin expression. In this study, we evaluated the effect of baicalein on FN-induced EMT of mammary epithelial cells. In addition, to determine whether baicalein could take effect of calpain/calpastatin system, we will detect the expression, subcellular distribution and activity of calpain and calpastatin, as well as their upstream signaling pathway including extracellular signal-regulated kinase (ERK) and calcium ion signaling. Moreover, the MMTV-PyMT mice model with highly aggressive spontaneous mammary tumors will be used to validate the results of the in vitro studies. In general, our project may provide new evidence for clarifying the mechanism of baicalein in breast cancer prevention and treatment. Furthermore it may offer a new target and an approach for drug discovering of breast cancer.

在乳腺癌的进展过程中乳腺上皮细胞周围基质会发生显著的改变，纤连蛋白（FN）表达上调是其关键变化之一。FN可诱导乳腺上皮细胞发生间质转化（EMT）促进乳腺癌的发展。此外，calpain (CANP)/calpastatin (CST)系统失调在乳腺癌的进展中也起着重要作用。本课题组前期研究发现，FN在诱导乳腺上皮细胞发生EMT的同时，使CANP表达上调，CST表达下调，可能存在该系统的失调，黄芩素能抑制这一变化。在本项目中，以FN诱导乳腺上皮细胞EMT为研究对象，考察黄芩素对这一过程的干预作用，并研究黄芩素对CANP和CST表达、分布、活性以及其上游ERK和钙离子信号通路的影响，阐明黄芩素通过调控CANP/CST系统，抑制FN诱导EMT的机制；并通过自发乳腺癌小鼠模型，在整体水平验证体外实验结果。本项目为深入探索黄芩素防治乳腺癌的机制提供新的依据，也为抗乳腺癌药物的研究提供新靶点和新思路。

在乳腺癌进展过程中微环境的基质成分会发生显著改变，纤连蛋白（fibronectin，FN）表达上调是其关键变化之一。FN可诱导乳腺上皮细胞发生间质转化（epithelial-mesenchymal transition，EMT）促进乳腺癌的发展，但其作用机制尚不清楚。本项目研究发现FN可通过诱导calpain（CANP）/ calpastatin（CST）系统失调促进乳腺上皮细胞发生EMT。即FN诱导乳腺上皮细胞发生EMT时，钙中性蛋白酶CANP2表达和活性上调，其内源性抑制剂钙蛋白酶抑制蛋白CST表达下调，并被剪切成小分子片段，失去对CANP的抑制。而CANP抑制剂ALLN, calpeptin、calpain inhibitor IV等能逆转FN诱导乳腺上皮细胞发生EMT。黄芩素能够通过抑制细胞迁移/侵袭能力增强、细胞骨架改变、上调上皮标志物E-cadherin和ZO-1的表达、下调间质标志物N-cadherin、vimentin和snail的表达，抑制FN诱导乳腺上皮细胞发生EMT。黄芩素还能抑制FN诱导的CANP2表达增加、膜分布及活性上调，抑制小分子CST的形成并上调其原型水平。采用质粒转染过表达CANP2，黄芩素逆转FN诱导乳腺上皮细胞EMT的作用被抑制。此外，黄芩素可通过同时抑制Ca2+和ERK信号，下调FN诱导的CANP2表达和活性上调。MMTV-PyMT小鼠可自发形成乳腺癌，黄芩素能够抑制小鼠乳腺肿瘤的发生、生长及肺转移并延长生存期，且在给药过程中未出现明显的内脏器官毒性。随着乳腺癌的进展，MMTV-PyMT小鼠的乳腺肿瘤组织中FN、CANP2和vimentin表达逐渐增加，E-cadherin表达逐渐下调，而在相同的时间点内，黄芩素可显著抑制小鼠乳腺肿瘤组织中FN、CANP2及vimentin的表达、上调E-cadherin的表达。本研究明确了黄芩素可通过抑制CANP/CST系统失调逆转FN诱导乳腺上皮细胞EMT的作用，为深入探索黄芩素防治乳腺癌的机制提供新依据，也为寻找和发现乳腺癌发展过程中的关键信号分子提供参考。