Snail介导的TFEB-溶酶体抑制在乳腺癌转移中的作用及分子机制

Metastasis cause most of breast cancer patients to death. Recent studies showed that lysosomes played an important role in breast cancer metastasis. Snail is one of the master regulators of EMT. In our work, we found over expression of Snail in immortalized normal breast epithelial cell and breast cancer cells, reduced the expression of TFEB, which suppressed the synthesis of lysosome, and promoted epithelial-mesenchymal transition. From these we inferred that Snail promoted tumor metastasis by inhibiting lysosomal synthesis. Based on this findings, we will further detect the lysosome function inhibition by examined the degradation rate of amino acid by mass spectrometry; construction of tumor metastasis mice model and the clinical pathological samples were analyzed to further elaborate the function of Snail negative regulation of lysosome in tumor metastasis; and through Luciferase activity assay and combined with CHIP-seq to analyze mechanism of Snail transcriptional regulation of TFEB and lysosome. This project will expand the new understanding of Snail regulation in tumor metastasis, and provide a solid theoretical basis for TFEB transformed into a new target treatment of breast cancer.

转移是乳腺癌患者死亡的首要原因。新近研究发现，溶酶体在乳腺癌转移中发挥重要功能。Snail是诱导上皮间质转化（EMT）最重要的转录因子之一，我们前期发现，Snail过表达的永生化正常乳腺上皮细胞和乳腺癌细胞中，溶酶体合成关键调控者--转录因子EB(TFEB)表达下调，细胞溶酶体合成显著抑制，细胞发生明显EMT，提示Snail通过TFEB抑制溶酶体合成，进而促进EMT和肿瘤转移。本项目拟在此基础上，通过质谱法检测溶酶体降解氨基酸的速率以分析溶酶体功能改变；运用Luciferase和CHIP实验进一步解析Snail调控TFEB以及溶酶体合成的分子机制；利用小鼠异体移植乳腺癌细胞构建“原位-肺转移”模型及对乳腺癌病理样本进行分析，在动物和人体层面阐述Snail与溶酶体负向调控关系对肿瘤转移的作用。本项目将拓展对Snail调控肿瘤转移的新认识，为TFEB转化为治疗乳腺癌的新靶标提供坚实的理论依据。

Snail是诱导肿瘤细胞发生EMT的最重要的转录因子，主要通过调控E-cadherin的转录水平，从而发挥促进肿瘤侵袭转移的过程。本研究在构建Snail过表达的细胞模型中，发现Snail的表达可以显著抑制溶酶体的合成，并且影响了一系列溶酶体相关的功能，包括大自噬以及分子伴侣介导的自噬（CMA）。对溶酶体的促进剂和抑制剂干扰溶酶体功能之后，可以改变细胞的EMT表型。进一步的分析和探索，我们用luciferase的方法发现Snail抑制溶酶体相关的功能主要是通过抑制溶酶体合成相关的重要转录因子TFEB的表达，进一步的CHIP实验我们证实了Snail结合TFEB启动子区的具体位点。初步的人体实验中我们发现，在Snail高表达的样本中，TFEB表达减弱，于此对应，在Snail低表达的样本中，TFEB表达稍增强。近期有文章表明p53野生型的病人样本中，Snail高表达与乳腺癌预后差相关，我们的研究发现TFEB在乳腺癌中表达低于正常组织，且TFEB的高表达的乳腺癌却有较好的预后。总之，我们的研究表明，Snail通过抑制TFEB的表达促进肿瘤的发展。