GSK3β-FoxO1-C/EBP-δ轴在食管癌细胞中的核心调控作用

Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role in the regulation of chronic inflammation and has been reported to affect the invasion and metastasis of tumor cells. However, the relationship between the activation of GSK3 and the progression of esophageal cancer (EC), especially the related intracellular mechanisms remain less understood. Our preliminary data showed that (i) the expression of GSK3β increases, whereas the phosphorylation of GSK3β decreases in EC tissue as compared with the para-cancerous tissues; (ii) Inhibition of GSK3β by pharmacological inhibitors including Lithium Chloride (LiCl) and SB216763 suppresses the proliferation and metastasis of EC cell lines; (iii) the prediction of phosphorylation sites by the software (NetworKIN; NetworKIN; MetaCor; Scansite) indicates that FoxO1, a known caner-suppressor protein, is the optimum substrate of GSK3β and could directly phosphorylated by GSK3β; and (iv) FoxO1-mediated phosphorylation of C/EBP-δ has been shown with suppressive effects on multiple tumors. Based on the evidence, we hypothesize that GSK3β exacerbates the invasion and metastasis of EC through promoting the emigration of tumor suppressor protein, FoxO1, from nucleus to cytoplasm, and then diminishing the expression of the other anti-tumorigenic protein C/EBP-δ. In this study, we will utilize multiple EC cell lines, biopsy samples from EC patients, tumor-bearing SCID mice, and employ several techniques including gene modification, immunohistochemistry, immunoprecipitation, Immunoblots, and confocal imaging to (i) determine the expression, sub-cellular localization of GSK3β, FoxO1, C/EBP-δ along with the emigration of FoxO1 from nucleus to cytoplasm; (ii) assess the influence of GSK3β-FoxO1-C/EBP-δ signaling axis on the invasion, proliferation, and metastasis of EC; and (iii) elucidate the molecular mechanisms of this signaling axis in the development of EC, and thus identify novel therapeutic targets for the control of EC progression, which will also pave the way for the development of novel and innovative immunoregulatory therapeutics to control the development of other cancers well beyond EC.

糖原合成酶激酶3β(GSK3β)是慢性炎症调控的关键靶点，其对肿瘤细胞侵袭转移能力的影响已有报道。但GSK3β表达及活性与肿瘤的关系，特别是分子机制尚不清楚。预试验发现食管癌组织中GSK3β表达水平升高、磷酸化水平降低；在裸鼠荷瘤模型中GSK3β抑制剂对食管癌细胞成瘤具有抑制作用；软件预测提示抑癌蛋白FoxO1是GSK3β下游靶蛋白之一，且在细胞系中证实前者可直接被后者磷酸化；鉴于FoxO1调控的C/EBP-δ被证实具有抑癌作用。因此设想GSK3β通过促进FoxO1核-浆转位，调控C/EBP-δ从而影响食管癌侵袭和转移。本研究拟在食管癌细胞系、组织样本及裸鼠模型中通过免疫组化、免疫印迹、基因修饰和共聚焦显微镜等观察GSK3β、FoxO1和C/EBP-δ的表达及FoxO1核-浆转位，探明GSK3β-FoxO1-C/EBP-δ轴对细胞活力、动力、侵袭力影响并探讨相关分子机制，为治疗提供新靶点。

通过本课题的研究，分析食管鳞癌（Esophageal squamous cell carcinoma, ESCC）中GSK3β、FoxO1和C/EBP-δ表达特征，确定其异常表达与临床病理特征及预后的相关性；检测GSK3β及FoxO1基因沉默后对ESCC细胞增殖、迁移及裸鼠皮下成瘤能力的影响，分析GSK-3β、FoxO1、C/EBP-δ三者之间的潜在调控关系，探讨其在ESCC发生发展的作用及机制。研究结果表明，GSK-3β、FoxO1和C/EBP-δ 异常表达发生于食管鳞癌，可能调控了食管鳞癌发生发展的过程；GSK-3β、FoxO1和C/EBP-δ与食管鳞癌患者生存具有相关性，是食管鳞癌患者生存预后的潜在分子标志物；GSK-3β表达升高促进了食管鳞癌发展，可能成为食管鳞癌治疗的新靶点。