中国科学院昆明动物研究所机构知识库

胰腺癌是一种恶性程度很高，预后差的消化道肿瘤，5年生存率仅7%左右,在美国癌症死亡原因中排第四位，预期将很快发展为第二大原因，在中国目前排第六位，但在癌症中的比例逐渐升高。有很多关于胰腺癌基因组的研究，发现一些在胰腺癌中发挥重要作用的基因，如Kras，Trp53，Cdkn2a及Smad4等，这些基因也是人类胰腺癌中变异率最高的基因。在胚胎时期就开始表达突变的KRASG12D可以诱导小鼠形成胰腺癌，Trp53，Cdkn2a和Smad4等基因的灭活可以加速这一过程。但是随着小鼠年龄增加KRASG12D诱导肿瘤形成的能力逐渐减弱，在成年小鼠（P≥60）中完全消失，即使再加上Trp53，Cdkn2a基因的灭活也不能诱导胰腺癌。因此在成体动物中诱导胰腺癌所需的驱动突变仍是未知。在本研究中，我们发现Cdkn2b的灭活对于胰腺癌的诱导是必需的，在表达KRASG12D及灭活Trp53，Cdkn2a的基础上，加上Cdkn2b的灭活就能很好的在成体小鼠中诱导胰腺癌形成。该小鼠胰腺癌具有人类胰腺导管腺癌的主要病理及分子特征，起源于导管细胞的恶性转化，癌组织中差异表达的基因可以富集到人胰腺癌中变异最多的12条信号通路，说明小鼠胰腺癌在一定程度上可以模拟人胰腺癌的分子机制。在机制方面，我们发现KRASG12D在导管细胞中的表达激活MEK-ERK-EGR1信号级联，上调转化生长因子TGF-β的表达，并通过Smad4上调CDKN2B的表达，CDKN2B和CDKN2A一起抑制Rb1的磷酸化，阻滞细胞周期，使细胞走向衰老，从而抵抗KRASG12D介导的细胞转化；同时灭活CDKN2B和CDKN2A就可以促进Rb1的磷酸化，使得Rb1和E2F分离，促进E2F介导的转录，促进细胞周期，最终促进KRASG12D介导的细胞转化及肿瘤的形成。上述结果证明了CDKN2B的灭活在胰腺癌的形成中起着关键作用，为胰腺癌的研究提供一个很好的成体小鼠胰腺癌模型。动物肿瘤模型为探索人类疾病的病因、机制及治疗手段等做出杰出贡献，与人类亲缘关系越近的动物模型更有助于人类疾病的研究。树鼩被认为是最接近灵长类的动物，在进化地位、免疫系统和基因同源性等方面都比小鼠更接近人类。近年来越来越多的人利用树鼩取代灵长类进行生物医学研究，陆续在树鼩中建立多种肿瘤模型，包括肝癌、乳腺癌和脑胶质瘤等。在本研究中，我们尝试用在小鼠模型中鉴定的胰腺癌驱动突变，即过表达KRASG12D及灭活Tp53和Cdkn2a/b在成体树鼩中诱导胰腺癌，发现注射病毒后3-7周就能诱导树鼩胰腺癌，发病率达到100%，该模型起源于腺泡细胞的恶性转化，也具有人类胰腺癌的主要病理特征，并表达人胰腺癌的分子标记。为了分析树鼩模型的优势，我们比较了人、小鼠及树鼩三者间与胰腺癌相关基因的蛋白同源性及修饰位点，发现建模时所用的TP53、CDKN2A等基因的蛋白，树鼩与人同源性远高于小鼠与人同源性，而且许多重要的修饰位点在树鼩中也是保守的，树鼩胰腺癌基因表达谱也比小鼠胰腺癌更类似于人，说明树鼩胰腺癌模型在分子机制上比小鼠更接近于人类疾病。上述结果证明了树鼩胰腺癌模型可能为胰腺癌的基础研究及抗肿瘤药物的探索提供更好的选择。胰腺癌的细胞起源一直是一个有很大争议的问题，主要构成胰腺的腺泡、导管及胰岛细胞均被认为是胰腺癌的起源。最早导管细胞起源观点被广泛接受，可最近研究证据更支持腺泡细胞起源。在本研究中，我们发现在成体小鼠中，慢病毒主要感染导管细胞，且小鼠胰腺癌起源于导管细胞恶性转化；有趣的是，在成体树鼩中，慢病毒主要感染腺泡细胞，树鼩胰腺癌起源于腺泡细胞的恶性转化。而且导管细胞和腺泡细胞恶性转化所需的驱动突变一致，即KRASG12D的过表达及Tp53和Cdkn2a/b的灭活，说明导管细胞和腺泡细胞对人类胰腺癌中突变率最高的基因介导的转化的敏感程度是一致的。我们的模型能为不同细胞起源的胰腺癌的研究提供不同的选择，将有助于胰腺癌的基础研究和治疗探索。未来我们将通过模拟人类胰腺癌突变率最高的遗传改变，在非人灵长类中建立胰腺癌模型，非人灵长类是目前公认的与人类最接近的实验动物，如果该模型构建成功，将更进一步增强我们对人类胰腺癌的认识。 

Pancreatic cancer is a highly malignant gastrointestinal cancer with poor prognosis and five-year survival rate is less than 7%, which is the fourth leading cause of cancer-related deaths in the U.S and is projected to be the second one by 2030. It ranks sixth in China but the rate to all cances keeps increasing every year. Despite extensive pancreatic cancer genome researches were carryed out and found some important drivers of pancreatic cancer, such as Kras, Tp53, Cdkn2a and Smad4 which are the most frequent genetic alterations in human PDAC. The expression of oncogenic KRASG12D at embryonic stages can induce pancreatic cancer in mice, which is accelerated by inactivation of Trp53, Cdkn2a or Smad4. However, the capacity of KRAS to induce PanIN in mouse pancreatic acinar cells decreases with age and is completely abolished in adult mice (P≥60). Moreover, KRASG12D expression in mature acinar cells did not induce obvious lesions even in combination with Tp53 or Cdkn2a deficiency. So the genetic events that lead to pancreatic carcinogenesis in adults remain unclear. In the present study, we demonstrate that inactivation of Cdkn2b (p15ink4b) is necessary for induction of pancreatic cancer with full penetrance by oncogenic KRASG12D expression and inactivation of Tp53 and Cdkn2a in adult mouse (P60 or older). Mouse pancreatic cancers possess the main histological and molecular features of human PDAC and originated from malignant transformation of ductal cells. Specifically, genes that were differentially expressed between mouse pancreatic cancers and normal pancreatic tissue from 9-week old mice enriched in Ras, P53, and Rb as well as other 12 core pathways that contribute to human pancreatic cancer development. KRASG12D overexpression in ductal cells activated the MEK-ERK-EGR1 cascade and the transforming growth factor-b signaling and upregulated expression of CDKN2B which, along with CDKN2A, inhibited Retinoblastoma phosphorylation, leading to cellular senescence and protection of cells from KRAS-mediated transformation. Finally, inactivation of both Cdkn2b and Cdkn2a was necessary for Rb phosphorylation and transcription mediated by E2F to encompass oncogene-induced cellular senescence and promote carcinogenesis. These results demonstrate a critical role for CDKN2B inactivation in pancreatic carcinogenesis, and provide a useful adult animal model for investigating the contribution of genetic alterations to this process.Animal cancer models have made outstanding contribution for understanding the pathogenesis, mechanism and treatment of human diseases. Animals are more close to human, the model may be more useful. The tree shrew (Tupaia belangeri chinensis) is a primate-like animal that is more similar to humans at phylogenetically, immune system and whole-genome level than rodents. Currently, there are more and more attempts to employ tree shrew to replace primate to create animal models of human disease. Tree shrew also has been used to establish several cancer models, including hepatocellular carcinoma, breast cancer and glioblastoma. In this study, the driver mutations identified in mouse model were used to induce pancreatic cancer in adult tree shrew, lentivirus expressing KRASG12D and inactivating Tp53 and Cdkn2a/b could induce pancreatic cancer in tree shrew with full penetrance 3-7 weeks after injection. The tree shrew pancreatic cancer originated from acinar cells and possessed the main histological and molecular features of human PDAC. To analyze the advantages of tree shrew model, we compared protein homology and modification sites of genes in KEGG pancreatic cancer pathway between human and mouse or tree shrew. Several genes in KEGG pancreatic cancer pathways are more similar to their human homologs in terms of protein sequence and post-translational modification sites, including TP53, p16Ink4a and p14arf that are the most frequently mutated in human PDAC and were applied to induce pancreatic cancer in this study. Moreover, the gene expression profile of tree shrew PDAC is closer than that of mouse to the human counterpart. These results suggest that tree shrew pancreatic cancer may be more similar to human disease in molecular mechanisms and It can provides alternative choice of pancreatic cancer by recapitulating genetic mutations in human disease and may be potentially beneficial to the basic pancreatic cancer research and anti-tumor drug discovery.The cell-of-origin of PDAC has been a disputed question. Pancreas was mainly composed by acinar, ductal and islet cells, all of which have been proposed as the cell-of-origin of pancreatic cancer. The concept of ductal origin was widely accepted for long, however, recent studies have suggested that acinar cells are the more possible cell of origin. In this study, we found lentivirus mainly infected the ductal cells in adult mice and the mouse pancreatic cancer was proved to be originated from malignant transformation of ductal cells. Interestingly, the lentivirus same as used in mouse mainly infected acinar cells in adult tree shrew. And the tree shrew pancreatic cancer was proved to be originated from malignant transformation of acinar cells. Our results imply that pancreatic ductal and acinar cells are equally susceptible to malignant transformation by KRAS expression and Tp53/Cdkn2a/b inactivation, all of which are the most frequently mutated in human PDAC. Our results provide alternative choice of pancreatic cancer model derived from different cell-of-origin by aberrant expression of genes frequently and concurrently mutated in human pancreatic cancer. It may be potentially benefit the basic pancreatic cancer research and anti-tumor drug discovery.In future work, we will induce pancreatic cancer in non-human primate by recapitulating the most frequent genetic alterations in human PDAC. This model will promote our understanding of human PDAC extremely as non-human primate was proposed as the most close to human.