面向Hedgehog信号通路中Gli转录因子的小分子筛选和调控机制研究

The Hedgehog pathway is central in embryonic development, certain types of adult stem cells, and cancer stem cells. It is also an important therapeutic target for regenerative medicine and cancer. The current generation of Hedgehog cancer drugs all physically engage a membrane protein Smoothened. However, resistance has quickly emerged as a major problem for multiple drugs. Amplification of Gli2, a major transcription activator in the pathway, was discovered as the major mechanism. In addition, Gli2 is overexpressed in several types of cancer stem cells. Current study aims to tackle this problem by obtaining a systematic and mechanistic understanding of Gli2 pharmacological modulation, and discovering small molecules with promising activities. A high throughput/content screening system probing Gli2 regulation was developed. High throughput screen of biologically active small molecules were conducted. Drug targets and their mechanisms of action will be investigated through functional genomics approaches. Promising compounds will be identified, and their anti-tumor activity and animal safety will be examined using a mouse model that develops drug resistance against Hedgehog cancer drugs. This project will lay a solid ground for future development towards next generation cancer drugs targeting the Hedgehog pathway.

Hedgehog (Hh)信号通路是胚胎发育、某些组织成体干细胞和癌症干细胞中的核心信号通路之一。它对于再生医学和癌症都是重要的药物靶点。当代的Hedgehog临床靶向药物都作用于膜蛋白Smoothened。但是一些药物很快出现了耐受问题，这主要是由于信号通路中主要转录激活因子Gli2的扩增而导致的。另外，Gli2也是在多种癌症干细胞中过表达的重要转录因子之一。本项目通过筛选Gli2的调控小分子，系统地研究Gli2的药物调控机制。我们开发了一个针对Gli2的高通量/高内涵小分子筛选平台；开展了对生物活性小分子库的筛选；通过对活性分子的分析并结合功能基因组学等手段研究其调控靶点、机理、信号网络；并通过应用一个Hedgehog靶向抗癌药的药物耐受模型检测有潜力的活性分子的抗癌活性和动物安全性。通过本课题，我们将为进一步开发下一代Hedgehog靶向抗癌药打下坚实基础。

Hedgehog信号通路的异常激活可以引起许多种类的癌症。目前两种Hedgehog临床靶向抗癌药Vismodegib和Sonidegib都出现了耐药反应，主要是这些临床靶向抗癌药的靶点膜蛋白SMO容易发生耐药性的点突变以及最下游的Gli2转录因子容易发生基因扩增。抑制GLI2的活性将能够从根本上解决临床药物的耐药性问题。因此，我们构建了一个可以观察GLI2在初级纤毛中定位的高内涵筛选系统。利用这一系统发现了一些前列腺素类的化合物可以抑制GLI2在初级纤毛中的聚集。值得注意的是，其中一个已经被FDA批准的药物PGE1可以克服SMO突变体和Gli2基因扩增造成的耐药性，是一个可以强劲抑制GLI2活性的抑制剂。机制研究表明，PGE1结合在定位于初级纤毛的EP4受体上，激活cAMP-PKA信号通路的活性，促进GLI2的磷酸化，并促进GLI2通过泛素化途径发生降解。我们还证实了PGE1可以有效地抑制耐药的人髓母细胞瘤的生长。我们的发现将为克服目前Hedgehog临床靶向抗癌药的耐药性打下坚实的基础。

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