Notch3通过上调GSK3β介导Snail降解抑制乳腺癌EMT分子机制及其增强化疗敏感性的研究

The latest studies have indicated that epithelial mesenchymal transition (EMT) is closely related to the malignant proliferation and chemotherapy resistance of cancer cells. Unfortunately, the molecular mechanism of EMT in breast cancer remains unclear completely. The pioneer experiments showed that unlike the other molecules of the Notch family, Notch 3 overexpression could significantly inhibit the EMT and proliferation of breast cancer cells. Through performing comparative analysis of transcriptome between control breast cancer cell line (MDA-MB-231) and MDA-MB-231 cell strain for stable and high expression of recombinant Notch3 intercellular domain (N3ICD), we found Notch 3 overexpression could upregulated the expression levels of GSK3β kinase, tight junction, adhesion junction and epithelial apical-basal polarity complexes. These data suggested that the function of Snail/Slug family of transcriptional repressors, which are considered to regulate the expressions of specific molecules of epithelial cells, may be inhibited. Therefore, basis on our pioneer work we suppose Notch 3 may up-regulate the expression of GSK3β. Furthermore, Snail may be phosphorylated by the upregulted GSK3β and degradated through ubiquitin proteasome pathway. We will prove the hypothesis and reveal the molecular mechanism of Notch 3 inhibiting EMT of breast cancer cells by using cell biology, RT-PCR, Western blot and immunofluorescence staining, animal experiment, confocal laser scanning technology and so on. We also will evaluate the capacity of Notch 3 overexpressing combined chemotherapeutic drugs to kill the breast cancer cells, enhance chemosensitivity in this project. The study will provide theoretical and experimental basis for curing breast cancer and getting new targets and anti breast cancer drugs.

上皮间质转换（EMT）与肿瘤细胞的恶性增殖、化疗耐药密切相关，但其发生的分子机制尚未完全明确。我们前期发现：Notch3（N3）有别于同家族其它分子，其过表达明显抑制乳腺癌细胞EMT。通过研究N3过表达组和对照组的差异表达基因，发现N3过表达组的GSK3β、上皮细胞腔底轴极性分子、紧密连接和粘着连接等分子的表达显著上调，提示：对这些分子具有负调控作用的Snail在N3过表达时可能受抑。已有的研究显示：Snail是GSK3β激酶的底物。本项目在前期基础上，拟采用细胞生物学、RT-PCR、Western blot、免疫荧光染色、动物实验、共聚焦激光扫描等技术，以N3上调GSK3β、后者进一步促进Snail磷酸化并经泛素-蛋白酶途径降解为切入点，揭示N3抑制乳腺癌细胞EMT的机制；也将对N3过表达联合化疗药物杀伤癌细胞、抑制化疗耐药性作进一步的研究。为寻求乳腺癌治疗的新靶点提供理论和实验依据。

在本项目经费的支撑，我们在克服乳腺癌耐药、抑制EMT、浸润与转移方面取得了突破性的进展。首先，我们发现靶向沉默Cohesin复合物核心元件及Mediator亚单位12抑制乳腺癌的耐药性，具体为，SMC1A促进乳腺癌的发展并与三阴性乳腺癌的耐药性有关；STAG2促进乳腺癌的增殖和分化；RAD21促进乳腺癌的增殖分化并可能影响乳腺癌的内分泌治疗；MED12可能促进乳腺癌的增殖。其次，现已明确Hippo/YAP通路是调控乳腺癌上皮细胞EMT的重要分子途径，然而Hippo分子的表达明显地受表观遗传学的影响，主要体现在组蛋白甲基化酶（EZH2）和DNA甲基转移酶（DNMT1）共同调节wwc1基因（Hippo分子）的表达。第三，众所周知，基因组DNA中第五位胞嘧啶（5mC）的甲基化是调控基因表达的重要因素之一， 5mC 最初被发现位于CpG岛内，CpG岛是富含CpG二核苷酸的基因启动子区域常见的一段DNA片段。人类基因组含有大约1% 的甲基化胞嘧啶，是最丰富、最广泛的DNA修饰，该修饰被通常认为是基因表达的一种稳定的抑制性调控因子。而我们发现在不同分子亚型的乳腺癌上皮细胞，Notch3和HIF-1α基因启动子中均含有不同程度的Non-CpG甲基化并发现在乳腺癌上皮细胞中，Notch3基因的启动子区域的Non-CpG甲基化是导致其沉默表达的主要因素。第四，脂肪组织在乳腺癌的增值和迁移方面起到了关键的作用。通过高通量的质谱测序方法筛选到3T3-L1细胞可能分泌的能够影响乳腺癌上皮细胞迁移和增值的细胞因子。质谱结果显示Enpp2、Mif、Postn三个基因的蛋白在细胞上清中的表达在Notch3过表达时降低，在Notch3干扰时升高，可做为我们后续的研究方向。最后，我们发现Notch3跨膜区在修正紧密连接分子、极性分子的定位，促使其发挥正常的功能、抑制乳腺癌上皮细胞EMT过程中具有重要作用。