Notch1的SUMO化与去乙酰化Cross-talk在三阴乳腺癌侵袭转移中的作用机制

The high motality of triple negative breast cancer is characterized with high invasion and metastasis. The molecular mechanisms of invasion and metastasis are still complicated and unclear. Our results and literatures had revealed that activated Notch1 signaling promoted invasion and metastasis of triple negative breast cancer. The expression level of Notch1 is regulated by some post translational modifications. We had found that the expression of Notch1 protein is inhibited by SIRT1-mediated deacetylation, besides, the SUMOylation also regulates the expression and location of Notch1 in preliminary publications. Since the Lysine on Notch1 gene is the mutual site of SUMOylation and deacetylation, we hypothesize that, there may be a cross-talk of the two post translational modifications on Notch1, which will regulate Notch1 signal and affect invasion and metastasis of triple negative breast cancer. To verify our hypothsis, on the basis of partial results in our research, in vitro simulation SUMO environment, co-immunoprecipitation, qChIP, mutation and animal RNAi will be used in different metastatic ability cancer cell lines, triple negative breast cancer tissue and breast cancer mice model to figure out the significance of the Notch1 SUMOylation and deacetylation to Notch1 signal and mechanism of the two translational modifications to invasion and metastasis of triple negative breast cancer.

三阴乳腺癌高侵袭转移特性是其高死亡率主要原因，机制复杂而不详。项目组前期实验和文献显示Notch1通路促进三阴乳腺癌侵袭转移。Notch1表达受多种翻译后修饰调控，前期实验发现Notch1被SIRT1靶向去乙酰化修饰而降低表达，文献报道Notch1的SUMO化修饰对其表达和定位有重要调节作用，Notch1去乙酰化和SUMO化修饰均发生在赖氨酸上，我们推测两者可能有相关性，提出假说：Notch1的SUMO化和去乙酰化修饰可能存在Cross-talk从而调控Notch1通路进而影响三阴乳腺癌侵袭转移。我们在前期研究基础上，模拟体外SUMO环境,运用免疫共沉淀、qChIP、定点突变、动物水平RNAi等技术，以三阴乳腺癌细胞和乳腺癌组织及裸鼠乳腺癌模型为研究对象，分析Notch1的SUMO化和去乙酰化发生的机制及意义，重点明确两者共同作用位点及Cross-talk在三阴乳腺癌侵袭转移中的作用机制。

三阴乳腺癌高侵袭转移特性是其高死亡率主要原因。研究发现Notch1通路分子mRNA和蛋白高表达，发生EMT变化，促进三阴乳腺癌发生发展。Notch1蛋白功能受多种翻译后修饰调控。乙酰化是一种翻译后修饰，我们发现去乙酰化酶SIRT1与Notch1表达负相关并负性调节Notch1表达，免疫沉淀和特异性抗乙酰化赖氨酸抗体显示SIRT1和Notch1相互结合， SIRT1通过去乙酰化功能导致Notch1乙酰化水平降低，抑制了三阴乳腺癌侵袭转移。SUMO化是另一种翻译后修饰，CO-IP和抗SUMO化抗体发现Notch1与SUMO1相互作用并发生Notch1的SUMO化修饰，生物信息学软件预测Notch1的SUMO化位点，根据置信度高、位于Notch1胞内区域、结合文献筛选9个SUMO1结合位点中3个K2049、K2150、K2252进行验证，设计K突变成R定点突变引物，PCR扩增和测序验证，免疫沉淀显示定点突变的Notch1的SUMO化水平降低，抑制三阴乳腺癌侵袭迁移能力。乙酰化和SUMO化修饰均发生在赖氨酸残基上，这两个修饰有无Cross-talk？过表达SIRT1后Notch1的SUMO化水平升高（p=0.0032），沉默SIRT1后Notch1的SUMO化水平降低（p=0.0005）；反之，定点突变Notch1的SUMO化位点，而乙酰化水平无明显变化。进一步沉默SIRT1后转染SUMO1定点突变载体，Notch1的SUMO水平降低（p=0.0003）；过表达SIRT1的细胞体外SUMO化环境培养，Notch1的SUMO水平升高（p=0.001），说明Notch1乙酰化对SUMO化修饰的拮抗作用。我们从人组织水平观察-体外细胞水平检测--裸鼠体内肿瘤水平验证三个层次探讨Notch1的SUMO化与去乙酰化Cross-talk在三阴乳腺癌侵袭转移中的作用机制，为后续治疗提供一些参考。