用转座子介导的体细胞突变小鼠模型研究肿瘤克隆进化

Darwin''s Theory of Evolution is a leap in the understanding of the nature. After one and a half centuries of development, especially the establishment of Mendelian Inheritance and the decipherment of the DNA double helix, the application of the theory of evolution has gone far beyond the traditional concept of species formation, and it is also applicable to micro-evolutionary processes, such as the differences between individuals within a species, the somatic cell heterogeneity existed in one organism. Furthermore, the process of tumorigenesis and metastasis is understood as a process of Darwinian evolution, called somatic or clonal evolution. Recently, large-scale sequencing analysis has revealed extensive heterogeneity between individual tumors, and intratumor heterogeneity has also been elucidated which may be responsible for the failure of cancer treatments. Intratumor heterogeneity is the consequence of somatic evolution and provides us a clue to explore the details of tumorigenesis and to understand evolutionary events at the cellular level, like the discovery of the “driver” mutation and unveiling of the tree of somatic evolution. So we will focus on the discovery of the different mutations in the multi-region of the individual tumors including the related metastasis and recurrences, and the profile of evolutionary relationships of these mutations.. To achieve this objective, we have developed the tissue-specific piggyBac transposon-mediated somatic insertional mutagenesis system in mice, which will accelerate the formation of specific tumors [Hepatocellular carcinoma (HCC) and colorectal cancer (CRC) in this research]. Since piggyBac is capable of genome-wide mutagenesis, the normal expression of genes including the potential tumor suppressor genes (TSGs) will be disrupted randomly. And then the insertional sites will be cloned and sequenced respectively in the multi-regional samples from individual tumors, including varied sections in primary lesions, recurrences and metastasis. The mass data are analyzed by Gaussian Kernel Convolution from which the common integration sites (CISs) will be picked out and recognized as the TSG candidates. By comparison among these CISs from different regions within individual tumors, we will figure out the relationships between these mutations and those between the mutations and the phenotypes, like metastasis and recurrences. Eventually, we are able to reveal the tree of clonal evolution and demonstrate the mechanisms underlying these changes which may contribute to personalized therapeutic approaches on cancer treatment.

今天，达尔文进化论早已突破了物种形成这一范畴，也适用于个体间的差异、体细胞的异质性等微进化过程。肿瘤细胞具有异质性，它的发生就是一个基于克隆增殖、突变积累、自然选择的体细胞“进化”过程。这种异质性为我们以肿瘤发生为模型研究体细胞进化提供了很好的思路。我们拟利用组织特异性的PiggyBac转座子介导的体细胞突变系统，失活潜在的肿瘤抑制基因(TSG)，在小鼠模型中诱发肝细胞癌及结直肠癌；分别克隆出肿瘤原发灶不同部位、转移灶及复发灶中的突变位点并测序，通过海量数据分析出整合热点（CIS），发现新的TSG及其作用机制；比较不同部位肿瘤组织之间突变的差异，分析这些突变之间的进化关系，并研究它们相互之间的作用关系；将在临床标本中验证新的发现，为这两类肿瘤的治疗提供新的靶点；用微进化的思想理解肿瘤的耐药、复发、转移等过程，为临床肿瘤治疗的“个体化”提供科学依据。

肿瘤细胞具有异质性，它的发生就是一个基于克隆增殖、突变积累、自然选择的体细胞“进化”过程。这种异质性为我们以肿瘤发生为模型研究体细胞进化提供了很好的思路。我们拟利用组织特异性的 PiggyBac 转座子介导的体细胞突变系统，失活潜在的肿瘤抑制基因(TSG)，在小鼠模型中诱发肿瘤形成；分别克隆出肿瘤原发灶不同部位、转移灶及复发灶中的突变位点并测序，通过海量数据分析出整合热点（CIS），发现新的 TSG及其作用机制；比较不同部位肿瘤组织之间突变的差异，分析这些突变之间的进化关系，并研究它们相互之间的作用关系；将在临床标本中验证新的发现，为肿瘤的治疗提供新的靶点；用微进化的思想理解肿瘤的耐药、复发、转移等过程，为临床肿瘤治疗的“个体化”提供科学依据。. 我们已经分别建立了含有高、中、低拷贝数转座子的转基因小鼠tg(p53RNAi/PBGT)六个株系,并用FISH技术确定了各株系中转座子的原始整合位点；用qPCR和Southern Blot明确了各株系小鼠中转座子的拷贝数。我们发现高拷贝的转座会引起小鼠胚胎致死（E7株系）；中拷贝转座的小鼠在出生后两周体型较小（B6、D3株系）。其中四株用于后续肿瘤形成的观察和研究。对成年后的小鼠进行生存状态监测中发现：与对照小鼠相比，转座导致小鼠生存时间明显缩短，有明显增高的肿瘤发生率。目前，我们已经收集了十多个的肿瘤样品以及相对照的非肿瘤样本，分别利用Splinkerette PCR联合传统测序和二代测序进行了转座子整合位点的分析。初步发现：转座子整合位点在肿瘤和非肿瘤样本中有明显不同的富集；肿瘤基因组中部分整合位点高度富集，而非肿瘤样本中整合位点分布则相对平均。 这些结果符合我们的预期，较好地验证了肿瘤的细胞克隆进化学说，也将有力的帮助我们明确单个肿瘤之中潜在的肿瘤驱动基因。下一步将完成所有样本的检测并利用生物信息学的方法分析大量数据，确定潜在的肿瘤驱动基因并在细胞水平加以验证。