STAT3在细胞有丝分裂及肺上皮细胞癌变中的作用实验研究

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor which is encoded by the STAT3 gene in human. STAT3 can promote oncogenesis by being constitutively activated through various pathways. However, the role of STAT3 in mitosis is largely unknown. In our previous study, we found that a sesquiterpene lactone, 6-O-angeloylplenolin (6-OAP), caused mitotic arrest in lung cancer and multiple myeloma cells through activation of spindle assembly checkpoint (SAC) and blockage of ubiquitiniation and subsequent proteasomal degradation of cyclin B1. By using biotin labeling of 6-OAP and protein chip assay techniques, we found that STAT3 is a direct target of 6-OAP. We also found CDC2 is one of the proteins which can bind STAT3 by using Co-Immunoprecipitation. These results suggest that STAT3 may have a critical role in the regulation of mitosis and SAC, and its abnormal activation may confer proliferation advantage to cancer cells. In this project, we will therefore investigate the role for STAT3 to play in cell division cycle and SAC by transfection of this factor into normal lung epithelial cells and silencing STAT3 in lung cancer cells. We will explore the effects of STAT3 on protein components of SAC, anaphase promoting complex (APC)/CDC20 and cyclin B1.We will dissect STAT3's role in lung epithelial cell transformation. These efforts may shed new lights on mitosis regulation and lung cancer pathogenesis, and provide novel therapeutic strategy for lung cancer. We will demonstrate the molecules mechanism of STAT3 involved in mitotic spindle assembly and find effective therapeutic targets.

STAT3 是信号传导与转录激活因子家族的重要成员。近年来多项研究结果显示, STAT3 在多种肿瘤中组成性激活, 与肿瘤的增殖分化密切相关。但STAT3 在细胞周期尤其是有丝分裂期中的作用鲜有报道。我们实验室前期从鹅不食草中分离出一种高效低毒的小分子化合物6-OAP，发现6-OAP能抑制纺锤体与染色体的结合，导致有丝分裂检验点的持续活化, 引起cyclin B1 在细胞内积累，使肿瘤细胞周期阻滞在有丝分裂期。我们进一步对6-OAP生物素标记,通过蛋白芯片发现与6-OAP与STAT3 相互作用，通过免疫共沉淀发现CDC2可以和STAT3结合。提示我们STAT3可能激活有丝分裂检验点,导致有丝分裂异常，促进肿瘤细胞过度增殖。本课题在此基础上，拟通过过表达/干扰等方法明确STAT3 参与有丝分裂纺锤体组装的过程及分子机制，为探索有丝分裂期异常在肿瘤发生中的作用及寻找癌症治疗的新靶点奠定基础。

在国家自然科学基金的资助下，我们在3年的时间（2013.1-2015.12）顺利完成了预期任务，主要研究了天然小分子化合物6-OAP通过作用于靶蛋白STAT3，将肺癌细胞阻滞在细胞周期G2/M期的作用机理与抗癌效果，并研究STAT3在细胞有丝分裂期及肺上皮细胞癌变中的调控作用。首先，我们证实了6-OAP可以结合STAT3的SH2结构域的Arg609, Ser611, Ser613位点，且通过结合STAT3呈时间、剂量依赖性下调STAT3酪氨酸705位的磷酸化。同时发现6-OAP通过下调p-STAT3，从而抑制STAT3 与Skp2启动子区的结合，并影响Skp2转录与表达，引起E-cadherin累积，抑制肺癌细胞侵袭转移。此外， 6-OAP还可以通过抑制STAT3的转录活性，显著降低其下游抗凋亡基因BCL-2和MCL-1的表达，从而诱导肺癌细胞凋亡。进一步，我们通过siRNA干扰下调 STAT3的表达，同样发现下调STAT3可显著抑制肺癌细胞增殖及克隆形成能力，还可通过引起E-cadherin累积，下调MMPs（Matrix metalloproteinases，MMPs）的表达，进而抑制肺癌细胞的侵袭转移。不仅证实了6-OAP是通过抑制STAT3 与Skp2启动子区相结合，影响Skp2转录与表达，进而抑制肺癌细胞侵袭转移及诱导肺癌细胞凋亡；同时也证明STAT3是肺癌治疗的一个理想靶点，在肺上皮细胞癌变中发挥重要作用。最后，我们还建立了H1975皮下移植瘤模型及luciferase-A549原位移植瘤模型。在这两种移植瘤模型中，6-OAP 均能够通过下调STAT3的表达，引起E-cadherin累积，从而显著抑制肿瘤的生长及侵袭，发挥其抗肿瘤效果。本研究采用化学生物学的方法，通过蛋白芯片与生物素标记技术发现药物作用靶点STAT3，揭示了STAT3在肺癌发生中的重要作用，STAT3蛋白可以作为潜在的肺癌预后标志物或治疗靶点， 为开展以小分子抗肿瘤药物为探针的基于化学生物学发现肿瘤治疗新靶点的研究提供了重要依据。