Caveolin-1介导自噬-溶酶体通路促进乳腺癌转移机制及地榆靶向抑制剂的功效研究

Autophagy is considered as a significant mechanism for cancer cells adapting to environmental stress and inducing subsequent metastasis. Chinese Medicine has unique priority in preventing and treating cancer metastasis. Therefore, identification of autophagy-related therapeutic targets and development of natural inhibitors will shed novel light for preventing breast cancer metastasis. Our preliminary results indicated that the Chinese herb, Sanguisorba officinalis L., could significantly inhibit breast cancer metastasis both in vitro and in vivo. Pharmacological study and network analysis further revealed that autophagy greatly contributes to its pharmacological activities, and caveolin-1 was found to be a core regulator in the whole network. What’s more, mechanism study indicated that caveolin-1 plays a critical role in mediating autophagy-lysosome pathway. Based on our preliminary results, we hypothesized that caveolin-1 targeting strategy or caveolin-1 specific inhibitors derived from Sanguisorba officinalis L. could inhibit breast cancer metastasis through the autophagy-lysosome pathway. Our study will explore the detailed molecular mechanisms involved in caveolin-1-regulated autophagy and metastasis by multiple experiments including clinical sample detection, gene editing, gene-protein interaction and chemical-target fishing chip, etc. Besides, we will also study the critical role of caveolin-1 in mediating the metastasis inhibition effects of Sanguisorba officinalis L. and identify its bioactive substance. Taken together, our study will provide novel insights and tools for molecular mechanisms of breast cancer metastasis and development of a new generation of targeting inhibitors.

自噬激活是晚期肿瘤细胞适应生存压力、进而侵袭转移的重要机制，中医药在防治肿瘤转移领域具有独特的思路和优势，鉴定肿瘤自噬调节关键信号及开发天然靶向抑制剂将对防治乳腺癌转移提供安全有效的崭新手段。我们前期基础研究结合生信分析发现地榆能显著抑制体内外乳腺癌转移，自噬是其抗转移重要机制，caveolin-1是其核心靶标，且其高表达能够促进自噬溶酶体活性，由此设想基于caveolin-1设计的靶向策略或地榆中的caveolin-1先导小分子抑制剂能够通过阻断自噬溶酶体通路从而抑制乳腺癌转移。本项目拟通过临床样本检测、体内外基因重组、基因蛋白互作、分子捕获芯片等技术探讨caveolin-1通过介导自噬溶酶体途径促进转移的分子机制，解析caveolin-1在地榆抑制乳腺癌转移过程中的关键作用，最后筛选鉴定地榆的物质基础。研究的开展将为乳腺癌转移机制及靶向药物的开发提供新思路和新工具。

本研究拟探讨Caveolin-1(Cav-1)介导自噬-溶酶体通路促进乳腺癌转移机制及地榆靶向抑制剂的功效。首先，我们从临床样本结合生物信息学分析、基因重组技术、体内外自噬检测、乳腺癌转基因小鼠模型、Co-IP及SILAC-IP/MS等手段证明了Cav-1可通过上调溶酶体GRP78的表达介导自噬流，从而促进乳腺癌体内外转移，其高表达与晚期乳腺癌不良预后显著相关。接着，我们建立了地榆-靶点-乳腺癌疾病网络，预测并验证了该中药能够剂量依赖性抑制Hif-1α/Cav-1通路，通过阻断自噬-溶酶体途径阻断缺氧诱导的自噬，从而显著抑制体内外乳腺癌转移。由于地榆可能通过抑制Hif-1α从而降低Cav-1 mRNA的表达水平，我们继续开展了ChIP-qPCR实验，发现地榆干预后CAV-1基因的启动子区与HIF-1a蛋白的结合能力明显削弱。此外，我们进一步发现了地榆对Hif-1α的抑制作用与VHL介导的泛素蛋白酶体途径密切相关。最后，采用分子动力学芯片结合地榆抗肿瘤活性成分筛选出了地榆中Cav-1天然抑制剂没食子酸(gallic acid, GA)，其生物活性验证发现GA可通过介导自噬-溶酶体途径显著抑制体内外乳腺癌转移，临床前药代动力学及肿瘤靶向性研究进一步提示GA在动物模型体内肝、心、脾、肺、肾和乳腺中均有分布，肺部分布最多，也证实了其抗乳腺癌肺转移的潜在能力。通过该项目的实施，共发表高质量中英文论文11篇；进行国际国内交流4次，海报展示并发表SCI收录会议论文3篇，口头汇报1次；以副主编出版专著1部；参与申请相关专利4项，其中授权1项；参与获得相关省部级奖励2项；培养广州中医药大学青年英才1名，培养硕士毕业1名。研究的开展以期为Caveolin-1在乳腺癌晚期阶段发挥促癌基因功能提供理论基础，同时为中医药靶向药物的开发提供新思路和新工具。