多基因协同诱导的神经内分泌肺癌小鼠遗传模型建立

We found that the negative feedback loop in the regulatory network between the tumor suppression gene MEN1 and protooncogene K-ras is essential for the onset of pulmonary neuroendocrine tumor (P-NET). The activation of K-ras drives the pathogenic progression of P-NET, while MEN1-mediated epigenetic regulatory mechanisms determine the final fate of lung adenocarcinoma cells with neuroendocrine differentiation. MEN1 knockout mice bearing K-ras mutations develop P-NET. However, to what extent that this mouse model could represent human P-NET remains to be further explored. The biological characteristics and histological type of these murine tumors need to be further identified. Here we focus on the establishment and characterization of P-NET animal model by utilizing lung specific promoter and optimize genetic regulation strategies. A series of experiments will be carried out, including the optimization and standardization of P-NET mouse model, the establishment of relevant analytical methods and the optimization of the therapeutic treatment etc. The aim of this study is to identify the following factors co-shared between the mouse model and human P-NET, including the molecular bio-markers, histological type, disease progression and so on. We will build up the preclinical assessment database for P-NET and elucidate the underlying epigenetic mechanism of P-NET. Our work will potentially provide a new animal disease model with defined genotype and phenotype, which may benefit the molecular diagnostics, disease risk assessment and drug screening of P-NET.

我们发现，MEN1基因与Kras之间的负反馈调控网络是神经内分泌型肺癌（P-NET）发病的关键节点，Kras突变是P-NET发病的驱动因素，而MEN1调控的染色质重塑等表观遗传调控机制则决定肺癌细胞的神经内分泌分化命运。MEN1基因敲除联合Kras突变可诱导P-NET动物模型，然而该模型在多大程度上模拟人类P-NET有待进一步探讨，肿瘤生物学特性、病理分型等需要深入鉴定。本研究围绕P-NET动物模型建立这一科学问题，通过肺特异性启动子导入及遗传调控方式优化等策略，开展小鼠P-NET遗传模型的优化及标准化、模型分析方法建立、治疗方案优化等研究；鉴定以上模型与人类同类型肺癌在分子标志物、病理组织学分型、疾病发病进程等方面的相似度，建立这一类肺癌评估临床前数据库；深入阐明P-NET发病的表观遗传学机制。有望为P-NET的分子诊断、疾病风险预警及药物筛选提供基因型及表型明确的新型动物疾病模型。

神经内分泌（Neuroendocrine，NE）定向分化是肺癌细胞逃避应激反应、获得治疗耐受性的重要适应性防御机制之一。目前临床上对这一类肺癌尚缺乏有效的治疗模式或特异性治疗措施，主要原因仍是对神经内分泌型肺癌（P-NET）发病的分子机制缺乏深入了解。建立一种能够从整体水平模拟导致人类P-NET发病的天然突变的动物疾病模型，将有助于深入揭示P-NET发病的分子机制，对设计和检验靶向药物的有效性及副作用提供科学的研究平台与实验工具。本项目基于前期研究提出科学假设，抑癌基因MEN1与原癌基因Kras之间的负反馈调控网络是P-NET发病的关键节点，即Kras突变是P-NET发病的驱动因素，而MEN1基因调控的染色质重塑等表观遗传调控机制则决定肺癌细胞的NE分化命运。通过本项目研究，解决了如下关键科学问题：（1）通过肺上皮细胞特异性启动子导入、基因敲除/转基因遗传调控方式优化等策略，建立了上皮细胞特异性KrasG12D;MEN1f/f;CreER诱导的P-NET小鼠模型，较好地模拟了人类同类型疾病自然发病进程及天然遗传学突变；（2）开展了基于动物疾病模型的P-NET发病分子机制研究，阐明了依赖P53/Rb通路的MEN1调控上皮细胞NE定向分化的分子机制，鉴定了menin调控P53通路稳定性的新型表观遗传学机制；（3）鉴定了menin维持基因组稳定性及调控细胞衰老的关键生物学功能，证实了MEN1缺失通过积累基因组DNA损伤、逃避细胞衰老程序促进肺泡上皮细胞向NE方向定向分化的细胞学机制；（4）从表观遗传靶向小分子化合物库中筛选出LSD1的靶向抑制剂SP2509为潜在的P-NET治疗药物，开展了基于动物疾病模型的P-NET靶向小分子药物的药效学鉴定及治疗方案优化，为NE型肿瘤的靶向治疗提供了新的方向。综上所述，本研究结果为P-NET发病机制研究、风险预警、分子诊断、治疗策略及高通量药物筛选提供了理想的研究工具与平台，开展了小鼠P-NET疾病遗传模型的优化及标准化、模型分析方法建立、治疗方案优化等研究；并利用该动物疾病模型深入阐明了肺癌发病的多基因协同作用机制及靶向治疗研究。