Gli2/DEC1/ABCC4通路诱导胃癌干细胞化疗耐药的机制研究

Gastric cancer is a leading cause of cancer-related deaths. In addition to surgery, the postoperative chemotherapy is the primary mean of treatment. However， nearly all patients develop chemotherapy resistance. The cancer stem cells (CSCs) theory postulates that cancers harbor a subset of cells that share characteristics of stem cells, with a capacity for self-renewal and an ability to differentiate into many cell types. Numerous studies have demonstrated that putative CSCs are more resistant to chemotherapy than non-CSCs. The Hedgehog (Hh) signaling pathway has been regarded as a key regulator of CSCs maintenance and drug resistance. However, the role of Hh pathway in gastric CSCs remains largely unknown. In our previous studies, we identified putative CSCs from human gastric cancer cell lines through tumor sphere culture. Gastric cancer cells of BGC823 and MGC803 could grow as tumor sphere in serum free conditioned medium. Compared with monolayer cells, both sphere cells highly expressed stemness genes Sox2，Oct4 and Bmi1. More importantly, sphere cells were more resistant to chemo-drugs 5-Fu，CDDP and ADM compared to monolayer cells. We found a series of genes differentially expressed between BGC823-sphere and BGC823-monolayer by gene array analysis. Gli2, the key regulatory factor of Hh signaling pathway was found highly expressed in sphere. Furthermore, DEC1, a Gli potential target gene, and ABCC4 are also increased in sphere. ABCC4 are known to regulate CSCs stemness and drug resistance. More importantly, inhibition expression of Gli2 decreased the cisplatin sensitivity, which is associated with down- regulation of DEC1 and ABCC4, Therefore, we propose that Gli2 is essential for maintenance of CSCs drug resistance in gastric cancer, through regulate DEC1 and ABCC4 expression. To investigate this hypothesis, we will isolate two gastric cancer sphere cells. The chemo-drug IC50 and the expression of Gli2, DEC1 and ABCC4 will be detected and compared between the sphere cells and the monolayer cells. Luciferase reporter gene systems, chromatin immunoprecipitation assay (ChIP) will be used to clarify the possible mechanism by which Gli2 regulates DEC1. We will knockdown Gli2 、DEC1 and ABCC4 expression using CRISPR, and determine the chemo-drug IC50 in sphere cells and tumorigenic capacity in nude mice. We will also determin the expression levels of Gli2, DEC1 and ABCC4 by immunohistochemistry, qPCR and Western Blot in clinical patient samples. In the clinical cohort level, we will analyze the correlation between them, revealing the clinical significance how “Gli2/DEC1/ABCC4” signaling pathway acts in the development and prognosis of gastric cancer.We hope that some of our studies may help us understand the new regulation mechanism of Hh signaling pathway, and have significant clinical implications in gastric cancer management.

胃癌化疗效果不佳且预后差，耐药是根本原因。肿瘤干细胞(CSCs)被认为是肿瘤耐药的根源，Hedgehog(Hh)信号通路与CSCs化疗耐药密切相关，但其在CSCs中激活的分子靶点尚不明确。我们前期建立了富集胃癌CSCs的sphere，sphere对化疗药物的IC50是贴壁细胞的3倍以上；芯片检测发现sphere中Hh核心转录分子Gli2，及其潜在的下游分子DEC1、维持CSCs干性及耐药的分子ABCC4表达上调；生物信息学分析显示DEC1启动子区存在Gli2结合位点；贴壁细胞中发现三者表达具有相关性，抑制Gli2表达，顺铂敏感性增高。我们提出“Gli2/ DEC1/ ABCC4通路诱导胃癌CSCs化疗耐药”的假说。拟采用CRISPR抑制该通路，从CSCs、动物、临床标本三个水平明确Gli2/DEC1/ABCC4通路对胃癌CSCs化疗耐药的调控机制，以期探寻Hh通路调控胃癌化疗耐药的新靶点。

胃癌化疗效果不佳且预后差，耐药是根本原因。肿瘤干细胞(CSCs)被认为是肿瘤耐药的根源，Hedgehog(Hh)信号通路与CSCs化疗耐药密切相关，但其在CSCs中激活的分子靶点尚不明确。我们通过建立胃癌CSCs 研究细胞模型sphere，并证实sphere对化疗药物的IC50是贴壁细胞的3倍以上；qPCR证实sphere 中Gli2、DEC1、 ABCC4 表达明显高于贴壁培养的细胞；过表达Gli2， sphere形成率高于于对照组细胞，同时胃癌细胞对5-Fu、CDDP的敏感性降低，且凋亡率降低；过表达Gli2，DEC1、 ABCC4表达明显上调；采用CRISPR抑制DEC1表达， sphere形成率低于对照组细胞，并增强了sphere对化疗药物的敏感性；过表达ABCC4， 胃癌细胞对5-Fu、CDDP的敏感性降低，且凋亡率降低；在CRISPR-DEC1细胞中过表达Gli2， 胃癌细胞IC50高于单独CRISPR-DEC1组， DEC1、ABCC4的表达水平增高；裸鼠成瘤结果表明，胃癌MKN45-sphere皮下成瘤明显大于贴壁细胞，且对化疗药物5-fu的敏感性明显低于贴壁细胞；抑制胃癌细胞DEC1表达后，成瘤体积及肿瘤生长明显减慢。抑制胃癌细胞DEC1表达后，成瘤体积及肿瘤生长明显减慢。我们的研究表明Gli2/DEC1/ABCC4通路诱导了胃癌CSCs化疗耐药。本研究丰富了Hh 信号通路，为后续研究及靶向药物的研发奠定理论和实验基础。