锌指转录因子Snail在恶性乳腺肿瘤发生及发展中的作用及其分子机制的研究

The zinc-finger transcription factor Snail is inappropriately expressed in various types of malignant tumors. As a key molecule that regulates cell epithelial-mesenchymal transition (EMT), Snail is involved in EMT-mediated regulation of cancer stem cell generation and tumor cell dissemination and metastasis. Currently, it is still unclear whether endogenously-derived Snail expression plays a functional role in regulating tumor initiation and progression. As such, we have generated Snail floxed/floxed mice and crossed these transgenic lines into the MMTV-PyMT mouse model of breast cancer. Preliminary studies demonstrate that tissue specific deletion of Snail markedly represses tumor initiation, progression and metastasis. To further investigate the fundamental role of Snail in controlling tumor initiation and progression as well as the potential molecular mechanisms, we propose the following studies: 1) the effect of Snail conditional deletion on expansion and activity of cancer stem cells (i.e. tumor initiating cells) in preneoplastic tissue and established tumors; 2) the impact of Snail on tumor cell proliferation, apoptosis, differentiation, EMT, collective invasion, extravasation and distal metastasis; 3) the regulatory effect of Snail on “housekeeping” tumor suppressor gene p53 and the functional role of Snail/p53 signaling axis in controlling tumor initiation and progression. These studies should provide an important theoretical basis for diagnosis of breast cancer as well as Snail-related cancer therapeutic strategy.

锌指转录因子Snail在多数恶性肿瘤组织中过表达。作为调控细胞上皮-间质转化（EMT）的关键因子，Snail参与了EMT介导的肿瘤干细胞生成、肿瘤细胞播散及转移的调节。迄今，内源性Snail在恶性肿瘤发生发展中的作用仍不清楚。为此，我们构建了自发性乳腺肿瘤、Snail条件性敲除转基因小鼠。初步的研究表明，组织特异性Snail敲除显著抑制了乳腺肿瘤的发生、发展及转移。为深入探讨Snail在恶性乳腺肿瘤发生发展中的作用及其分子机制，本项目拟开展如下研究：1）Snail敲除对癌前及肿瘤组织中肿瘤干细胞（也称起动细胞）扩增及活性的影响；2）Snail基因对肿瘤细胞增殖、凋亡、分化、EMT、集体侵润、血管外渗及转移等的调控；3）Snail对“看家”抑癌基因p53的调控及Snail/p53信号轴在肿瘤发生发展中的作用。本研究将为乳腺肿瘤病理诊断及Snail相关的上皮性肿瘤治疗策略提供重要的理论基础。

公共数据库的Kaplan-Meier生存分析结果表明，Snail1表达水平与p53野生型乳腺癌患者的无复发生存时间、无转移生存时间及总生存时间呈显著负相关；相反，Snail1表达水平与p53突变型乳腺癌患者的预后无显著相关性。进一步的分析结果表明，Snail1表达水平与携带野生型p53管腔型乳腺癌患者的无复发生存时间呈显著负相关，而与携带突变型p53管腔型乳腺癌患者的无复发生存时间无显著相关性。同样地，Snail1表达水平与携带突变型p53基底样型和HER2过表达型乳腺癌患者的无复发生存时间无显著相关性。这些数据表明，Snail1表达水平与携带野生型p53乳腺癌患者的预后呈负相关性，而与携带突变型p53乳腺癌患者的预后无显著相关性。在乳腺癌发生过程中，Snail1基因敲除后，癌前增生的数量显著减少，癌前增生细胞增殖减少、凋亡增加，肿瘤起始细胞的百分比显著减少。同时，Snail1基因敲除小鼠的肿瘤组织呈现出高分化表型，肿瘤细胞增殖减少、凋亡增加、侵袭能力减弱。我们发现Snail1条件性敲除小鼠原位乳腺肿瘤和原位移植瘤中，p53蛋白表达水平显著升高。Western blots实验结果也显示，体外敲除原代乳腺癌前上皮细胞及乳腺癌细胞Snail1基因，显著上调p53及其下游靶基因的蛋白表达水平；RT-qPCR实验结果显示，体外敲除Snail1基因显著上调p53下游靶基因的mRNA表达水平，而不影响p53基因mRNA表达水平。分子生物学研究发现Snail1作为一个桥梁分子，将野生型p53和HDAC1募集到一起，下调p53乙酰化水平，促进其降解，进而降低p53蛋白表达水平。综上所述，本论文阐明了锌指转录因子Snail1与乳腺癌患者的不良预后息息相关。应用转基因动物模型，我们进一步证明了Snail1通过调控肿瘤起始细胞的扩增和活性，进而促进恶性乳腺肿瘤的发生、发展及转移。乳腺癌前增生上皮细胞及乳腺癌细胞的生长和增殖受到Snail1/HDAC1/p53复合物的调控。