胰腺癌前病变PanIN细胞恶性转化中端粒盖帽蛋白TRF2过表达的作用及机制研究

Our genetically engineered mouse models have showed that endogenous Kras hot-spot mutation G12D initiated precancerous lesion pancreatic intraepithelial neoplasia (PanIN), and the inactivated tumor suppressive genes, including Tp53, p16, Smad4 or BRCA2 genes, promoted KrasG12D-driven carcinogenesis, respectively. The distinct phenotypic routes of these murine models recapitulated human invasive pancreatic cancer. It did not, nevertheless, share the same overall mutational spectra occurring in human pancreatic ductal adenocarcinomas. Another TRF2 gene was found with over expression in human pancreatic ductal adenocarcinomas, which was considered as an oncogene. It is unclear whether the TRF2 overexpression is sufficient to promote PanIN with oncogenic kras G12D to develop pancreatic cancer? We speculate that overexpression of TRF2 may be an important precancerous molecular genetic event occurring in middle/late stage of PanIN, and may act as a common initiate step for pancreatic carcinogenesis. Based on the PanIN cell line isolated from the Pdx1-Cre; LSL-KRASG12D compound mutant mouse we established previously, and a found which overexpression of TRF2 promoted rapid colony formation of PanIN cells in soft agar assay, the current study will be further carried out. To define the role of TRF2 overexpression in pancreatic carcinogenesis, we will construct lentiviral vector, in which lentivirus-mediated TRF2 expression will be employed to induce the endogenous expression of TRF2 in the PanIN cell line. Furthermore, cells transformation, caused by overexpression of TRF2 in PanIN cells, will be determined and the biological behavior of cells will be observed. To determine the molecular mechanisms by which TRF2 overexpression regulates cellular functions, moreover, we will analyze gene expression profiling by microarray and bioinformatics studies, and validate the microarray results to find a novel potential gene involved in pancreatic carcinogenesis. The study will provide new mechanistic insights of pathogenesis of pancreatic cancer, and will also have a clear implication that such study may also provide a more efficient strategy to identify relevant genes involved in pancreatic carcinogenesis, and overexpression of TRF2 should be further investigated as candidate targeted therapies for pancreatic cancer.

包括我们的小鼠基因打靶模型证实：Kras突变启动了胰腺上皮内瘤变(PanIN)，Tp53等四抑癌基因失活可从四途径上分别促进PanIN发展为胰腺癌，但模型尚存缺陷。近年发现人胰腺癌TRF2过表达，具癌基因作用。端粒TRF2过表达对PanIN作用如何以及是否可作为共同起始途径并单独促进PanIN细胞恶性转化为胰腺癌仍不清楚？在我们首次成功建立Kras突变启动的PanIN细胞株和TRF2过表达可使PanIN细胞在软琼脂中形成集落的研究基础上，本项目拟进一步采用高效慢病毒介导PanIN细胞TRF2过表达，通过分析肿瘤生物学行为、染色体不稳定性等，探索PanIN细胞在TRF2过表达后的恶性转化作用；应用Microarray技术等，鉴定TRF2过表达后新的相关下游靶基因，并研究其在恶性转化中的作用。因此，本项目研究有助于阐明PanIN细胞恶性转化新途径及机制，为胰腺癌TRF2靶向治疗提供新理论依据。

近来研究发现人胰腺癌TRF2过表达，可能具癌基因作用。TRF2过表达如何促使正常胰腺上皮细胞经上皮内瘤变最终发展为胰腺癌的机制目前仍不清楚。在我们首次成功建立Kras突变启动的PanIN细胞株的基础上将慢病毒介导的TRF2过表达载体侵染小鼠PanIN细胞，筛选得到稳定TRF2高表达PanIN细胞；通过体内、外生物学实验证实TRF2过表达促进PanIN细胞恶性转化；分析TRF2过表达后PanIN细胞中与胰腺癌相关的主要癌基因、抑癌基因表达情况，发现TRF2与以癌基因Tp53表达密切相关；应用Microarray技术，筛选并验证TRF2过表达后差异表达的相关的新下游基因，从中挑选出与Tp53突变密切相关的明显高表达变化的候选基因WISP1及PPP3CA；免疫组化验证发现WISP1及PPP3CA在人胰腺癌组织中的表达显著高于癌旁组织，且生物学行为实验发现WISP1过表达促进PanIN细胞恶性转化，提示PPP3CA及WISP1在胰腺癌发生发展过程中起到癌基因作用；进一步研究发现Tp53突变促使人正常胰腺导管上皮细胞株HPDE6-C7（Tp53野生型）中PPP3CA高表达，而外源性导入野生型Tp53载体抑制人胰腺癌细胞株Panc-1（Tp53突变型）中PPP3CA的表达，研究提示Tp53突变与否与PPP3CA表达密切相关；此外，PPP3CA可通过调节亚基PPP3R1与β-catenin结合，且WISP1过表达可促使PanIN中β-catenin入核，提示PPP3CA及WISP1可能与β-catenin通路存在交叉对话。因此，本项目研究有助于阐明PanIN细胞恶性转化新途径及机制，为胰腺癌TRF2及其下游基因PPP3CA以及WISP1靶向治疗提供新理论依据。