应用人胃类器官研究SMAD4突变调节自噬在胃癌发生中的作用和机制

Organoids are 3D self-organizing structures grown from stem cells in vitro, which can recapitulate the architecture, multi-lineage differentiation, genetic signature and functionality of the original tissues in vivo. As a novel preclinical model, organoid is one of the great technological breakthroughs of the last decade. Recent and our previous studies highlighting that human organoids can be precision models to investigate the functional roles of driver gene mutations in carcinogenesis, based on clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) genome engineering technologies. The analysis of TCGA data revealed that SMAD4 mutation was one of the key driving mutations in gastric cancer, which was associated with low expression of autophagy-related genes (ATGs). We hypothesize that SMAD4 mutation could promote gastric carcinogenesis by inhibition of autophagy in CRISPR/Cas–engineered organoids. Based on the human stomach biopsy normal tissues derived organoids that we have already cultured, organoids with SMAD4 mutation will be obtained by CRISPR/Cas9 genome editing. Phenotypic comparative analysis will be used to clarify the SMAD4 mutation-mediated phenotypic neoplastic transformation of gastric organoids. Organoids culture, time-lapse live-cell imaging and rescue experiments will be used to analyse the changes of autophagy and tumor phenotypes in organoids, to reveal the novel mechanism of SMAD4 mutation in gastric carcinogenesis through regulating autophagy. These studies will highlight the validity of the CRISPR/Cas–engineered human organoids as disease modeling, and shed light on deeper understanding the roles and mechanisms of SMAD4 mutation in the pathogenesis of gastric cancer, providing novel strategies for targeted therapy of gastric cancer.

类器官是干细胞在体外培养形成的三维结构，其多种细胞成分、稳定的遗传和表型特征均与来源组织类似，是能代表体内条件的个体化理想模型。新近研究及我们前期发现，CRISPR/Cas编辑的类器官是研究肿瘤驱动基因突变作用的精准体外平台。TCGA分析发现SMAD4突变是胃癌发生的关键驱动突变之一，与自噬相关基因（ATGs）低表达相关。我们推测：CRISPR/Cas9编辑的人胃类器官SMAD4突变可通过抑制自噬促进胃癌发生。我们前期已建立人胃活检正常组织来源类器官，拟通过CRISPR/Cas9基因编辑构建SMAD4突变胃类器官，通过表型对比分析，阐明SMAD4突变与胃上皮表型肿瘤性转化之间的关系；通过类器官培养、活细胞时差显微摄像、分子细胞生物学及挽救实验等方法，分析类器官自噬及肿瘤表型的变化，从调节自噬这一新角度阐明SMAD4突变在胃癌发生中的机制。为研究胃癌体外模型、发生机制及药物研发提供科学依据。

胃癌高居我国恶性肿瘤发病率第二，死亡率第三，仍缺乏有效的治疗方案。而胃癌研究使用的传统体外模型，如永生化的细胞系等，无法真实反映患者来源组织细胞的遗传特性。类器官是干细胞在体外培养形成的三维结构，其多种细胞成分、稳定的遗传和表型特征均与来源组织类似，是能代表体内条件的个体化理想模型。自噬在肿瘤的发生发展中发挥重要作用。本项目对TCGA数据库及临床样本分析发现，SMAD4基因作为胃癌的关键性驱动基因之一，在胃癌组织中的突变率为7.1%，且与胃癌患者预后不良及自噬相关基因低表达相关。通过体外三维培养建立人源性胃类器官，CRISPR/Cas9基因编辑和筛选，构建SMAD4突变型单克隆胃类器官。与野生型相比，SMAD4突变类器官显示肿瘤性转化，自噬相关基因表达降低、自噬受到抑制。而加入自噬激活剂雷帕霉素后，SMAD4突变类器官的的自噬相关基因表达回升，肿瘤性转化受到抑制。本项目以CRISPR/Cas9编辑人源性胃类器官作为核心研究平台，在类似人体内的、无种属及其他遗传变异干扰的条件下，证实SMAD4突变促进人源性胃类器官的肿瘤性转化，激活自噬则有利于逆转SMAD4突变类器官的肿瘤性转化过程。研究为探索胃癌研究新模型、发生机制和药物研发奠定实验基础。