ILK调控Wnt/β-catenin信号通路影响食管鳞癌放化疗敏感性的机制研究

Esophageal cancer is one of the most serious malignant tumors that has been threatening public health, with high morbidity and mortality. It has been listed the high-grade malignant tumor in Xinjiang, there is obvious ethnic differences(among Han、Uigur and Kazakh nationality) . We found that there were different efficacy and prognosis among different ethnic groups; Proteomic technique was used to screen out potential proteins related to therapeutic survival among different ethnic groups. High frequency of ILK(Integrin-linked kinase) was found in peripheral blood in patients with esophageal carcinoma,which was significantly correlated with curative chemoradiotherapy efficacy and prognosis. ILK gene silencing inhibited the progression of esophageal squamous cancer cells. In view of our previous study, this project will focus on the scientific issue of " The molecular mechanisms of ILK regulating Wnt/β-catenin pathway to influence the sensitivity of Chemoradiotherapy in Esophageal Squamous Cell Carcinoma", using these techniques as lentiviral vector construction、 RT-PCR、 animal tumorgenesis and so on , verify the predictive value of ILK abnormal expression on the diagnosis and prognosis in esophageal squamous cell carcinoma in multiple levels including molecular、cell, tissue specimens and organics; Definite the effect of ILK on chemoradiation sensitivity of esophageal squamous cell carcinoma; Reveal that ILK plays a key role in regulating the mechanism of chemoradiotherapy sensitivity by the Wnt/β-catenin pathway; Explore a new mechanism of chemoradiotherapy resistance of esophageal cancer and provide a theoretical basis and new strategy for the accurate treatment .

食管癌是严重威胁人民健康的恶性肿瘤之一，发病率和死亡率高。在新疆已列为特高发肿瘤，发病有明显的民族（汉族、哈萨克族、维吾尔族）差异。我们发现不同民族间疗效及预后不同；应用蛋白组学技术筛选出影响不同民族放化疗疗效的差异蛋白；其中ILK高频率出现，与疗效预后显著相关；且从细胞水平证实ILK基因沉默抑制食管癌细胞进展。鉴于我们前期的研究结果，本项目拟围绕“ILK基因调控Wnt/β-catenin信号通路影响食管鳞癌放化疗敏感性的分子机制”这一科学问题，通过慢病毒载体构建、RT-PCR、动物成瘤等技术，在分子、细胞、组织标本、机体等多水平、多层次验证ILK异常表达对食管鳞癌诊断及疗效预后的预测价值；明确ILK对食管鳞癌放化疗敏感性的影响；揭示ILK通过Wnt/β-catenin信号通路发挥作用，以调控放化疗敏感性的分子机制，探索食管癌放化疗抵抗的新机制，为食管癌的精准治疗提供新策略。

食管癌是最常见的恶性肿瘤之一，发病率和死亡率高，在新疆已列为特高发肿瘤。我们通过蛋白组学技术发现食管鳞癌（ESCC）患者血清中ILK高表达，且与治疗欠佳、预后差相关，已在ESCC细胞中证实沉默ILK可明显抑制细胞的恶性表型。鉴于前期研究结果，本项目完成了ILK对ESCC细胞顺铂化疗敏感性的作用及机制研究的探索，同时基于ILK在ESCC中的生物功能，利用分子对接技术筛选出靶向ILK的抑制剂Niltotinib，并对其抗ESCC的作用及机制研究进一步探索。研究发现ILK基因过表达可显著增强ESCC细胞增殖、迁移以及侵袭的能力，减少细胞凋亡，沉默ILK则具有相反的作用；沉默ILK会降低胞内GS3Kβ的磷酸化水平，抑制β-catenin和Wnt信号通路活性及耐药相关蛋白MDR1的表达，增强ESCC细胞CDDP敏感性，过表达ILK则会降低ESCC细胞CDDP敏感性，其机制可能是ILK通过磷酸化GS3Kβ，影响β-catenin的表达水平进而影响Wnt信号通路活性引起的；裸鼠成瘤实验结果显示，ILK过表达可削弱体内ESCC细胞对CDDP敏感性，沉默ILK作用相反。我们采用药物再利用策略用分子对接技术筛选出ILK新型抑制剂Nilotinib，其可显著抑制ESCC细胞恶性生物学行为，过表达ILK可逆转Nilotinib对ESCC细胞的毒性，同时Nilotinib可影响ESCC细胞线粒体功能，增加ESCC细胞对X射线的敏感性、ESCC细胞照射后的凋亡水平及G2/M期的阻滞；细胞转录组测序发现Nilotinib可引起AIRE下调，在ESCC敲减AIRE可显著抑制ESCC细胞的增殖、侵袭迁移能力，促进细胞凋亡， Nilotinib处理和沉默ILK均可引起AIRE表达下调，通过CHX和MG132处理后发现Nilotinib影响AIRE蛋白稳定性继而导致表达下调；荷瘤裸鼠模型实验显示Nilotinib可显著抑制ESCC生长及AIRE的表达。本研究揭示ILK通过Wnt/β-catenin信号通路继而调控ESCC化疗敏感性，探索出了食管癌化疗抵抗的新机制，为食管癌的精准治疗提供新策略；Nilotinib在体内外实验证明其靶向ILK影响AIRE蛋白稳定性发挥抗食管鳞癌的作用，为靶向ILK治疗食管鳞癌提供研究价值，同时为开拓Nilotinib新靶点及新应用范围奠定临床试验前基础。