Snail/p53结合抑制剂SN004的抗肿瘤作用及分子机制研究

Snail/p53 signal axis plays a critical role in in regulating the tumor progression. Compounds that interfere with Snail/p53 interactions is an extremely promising antitumor therapy. We previously screened the compound (SN004) with novel structure compound based on the combining strategy of computer virtual screening and in vitro competition experiments. Further investigation found that SN004 effectively block the binding of Snail and p53, thereby stabilizing the activity of p53, and has excellent anti-tumor effect in vitro. In current project, we aim to elucidate the detailed molecular mechanisms of Snail/p53 regulation by SN004 via implying methods such as immunofluorescence, GST pull-down, gene transfection, dominate point mutation, etc. We will further assess the anti-tumor activity of SN004 in vivo using MMTV-PyMT-induced mammary tumours mice, PyMT or HCT-116 tumor-bearing nude mice model. Finally, these studies will facilitate us to deeply understand the molecular mechanisms and anti-tumor activity of SN004 in vivo, which has great importance to its clinical application in treatment for Snail-related in tandem with p53 wild-type tumor.

Snail的过度表达在野生型p53肿瘤的发生发展中发挥关键的促进作用,干预Snail/p53相互作用是极具应用前景的抗肿瘤治疗手段,具有重要的临床研究价值。我们前期利用虚拟筛选技术和竞争结合实验,发现具有全新结构的化合物SN004可以与Snail高度亲和,有效阻断Snail与p53的结合,进而稳定p53的活性,展现出优异的体外抗肿瘤效果,但其体内增殖抑制效果尚未明确,作用机制有待完善。本项目拟采用免疫共沉淀、基因定点突变、GST pull-down等技术深入探索SN004调控Snail/p53信号轴的的作用机理。应用MMTV-PyMT自发性乳腺肿瘤转基因小鼠模型,乳腺癌PyMT细胞和结肠癌HCT-116细胞裸鼠移植瘤模型全面评价SN004的体内增殖抑制活性,明确SN004的抗肿瘤效果,为Snail相关的p53野生型肿瘤治疗提供新的策略。

锌指转录因子Snail蛋白能在肿瘤的发生、复发过程中扮演重要角色，往往代表着不良预后。靶向Snail相关信号通路，有望抑制肿瘤的恶化进程。然而，直接靶向Snail蛋白的小分子抑制剂尚未见报道。Snail蛋白晶体结构的成功解析，使基于Snail分子结构的特异性筛选抗肿瘤药物成为可能。在本项目中，我们发现化合物SN004与Snail蛋白高度亲和（IC50=180nM），强烈抑制肿瘤细胞增殖，并且化合物对肿瘤细胞的敏感性与p53的表型相关。随后，我们通过流式细胞术和WB检测肿瘤细胞凋亡情况，进一步证实化合物对肿瘤细胞的凋亡依赖于p53诱导的线粒体途径。分子模拟及氨基酸定点突变实验发现SN004与Snail的174位精氨酸结合。SN004在50nM浓度下，可以降低多种肿瘤细胞以及人乳腺癌原代细胞内的Snail蛋白表达水平，并且不影响Snail mRNA变化，提示SN004可能是通过影响Snail蛋白翻译后修饰水平干扰Snail蛋白表达。质粒过表达和免疫共沉淀实验发现化合物可以降低Snail的乙酰化水平，增加Snail的泛素化水平。体外GST-pull down实验，发现化合物可以干扰Snail与CBP的相互作用，进一步证实化合物降低Snail蛋白稳定性的分子机制。SN004（30mg/kg），能够明显抑制PyMT转基因小鼠和HCT-116异种移植瘤裸鼠肿瘤的生长情况。HE结果染色显示化合物对重要器官无明显毒副作用，提示化合物在体内具有较高的耐受剂量。SN004（30mg/kg）能够明显降低HCT-116细胞脾注射的裸鼠肝转移模型中肝结节数目，有效抑制肿瘤肝转移的发生。. SN004是一个毒性较低、结构新颖的靶向Snail的小分子化合物。SN004的发现，为Snail抑制剂的开发奠定了重要的物质基础，拓展了以转录因子为靶点进行抗肿瘤药物治疗的的研究思路。