Dysregulation of TGF Beta Action Pancreatic Cancer

描述(申请人提供)：胰腺导管腺癌(PDAC)过度表达所有三种转化生长因子β(TGF-2)亚型和抑制性Smad7，并经常含有Kras突变。PDAC中高水平的转化生长因子-2水平与疾病进展有关，但Smad7和致癌基因Kras通常阻断转化生长因子-2介导的Smad2和Smad3核转位，从而导致转化生长因子-2信号在PDAC中的失调。在这项建议中，我们将检验这样一种假设，即Smad7过表达通过调节癌细胞中的转化生长因子-2的作用来促进癌细胞的生长、存活和化疗耐药，以及Smad7与突变的和成分活性的Kras协同促进胰腺癌的进展和转移。将使用三种方法来检验这一假设。首先，将在体外从生物学作用和信号转导方面确定Smad7在三个PDAC细胞系中过表达的后果，并从肿瘤形成、生长、侵袭、转移、血管生成和凋亡方面确定在裸鼠中过表达的后果。其次，在表达巢蛋白的细胞中表达转录沉默(loxP-Stop-loxP)Smad7的小鼠将被培育成在Pdx1启动子(Pdx1-Cre)控制下表达Cre重组酶的小鼠，以评估Smad7单独在胰腺癌发生发展中的作用。同时携带Nestin-LSL-Smad7和LSL-KrasG12D等位基因的动物将被培育成Pdx1-Cre小鼠，以寻找Smad7和突变的Kras之间的体内协同作用。只有三重复合突变体将激活Kras在整个胰腺中的表达，而Smad7的表达将仅与表达Nestin的亚群重叠。第三，由于高水平的Smad7会导致pRb的功能失活，因此，如果在外显子19两侧的内含子两侧(LSL-RB)表达Rb的小鼠携带LSL-KrasG12D等位基因和Pdx1-Cre，以确定突变的Rb是否模仿Smad7的体内作用。AIMS II和AIMS III中的对照和突变小鼠的胰腺将在指定的时间点进行组织学分析，并将确定对胰腺上皮内瘤变(Panin)、癌症形成和改变的转化生长因子-2信号的影响。项目简介：胰腺导管腺癌(PDAC)是美国成年人癌症死亡的第四大原因，死亡率几乎等同于发病率。这种疾病在未来可能是一个更严重的问题，因为它的发病率在50岁以后增加，而且美国的普通人口正在老龄化。这项建议旨在更好地了解导致这种疾病侵袭性的生物学原因，最终目的是制定更好的诊断和治疗策略。

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinomas (PDACs) overexpress all three transforming growth factor beta (TGF-2) isoforms and inhibitory Smad7, and frequently harbor Kras mutations. High TGF-2 levels in PDAC correlate with disease progression, yet Smad7 and oncogenic Kras generally block TGF-2-mediated Smad2 and Smad3 nuclear translocation, implicating dysregulated TGF-2 signaling in PDAC. In this proposal we will test the hypothesis that Smad7 overexpression contributes to pancreatic cancer progression and metastasis by modulating TGF-2 actions in cancer cells in a manner that promotes their growth, survival and chemoresistance, and that Smad7 synergizes with mutated and constitutively active Kras to promote pancreatic cancer progression and metastasis. Three approaches will be used to test this hypothesis. First, the consequences of Smad7 overexpression in three PDAC cell lines will be determined in vitro with respect to biological actions and signaling, and in athymic mice with respect to tumor formation, growth, invasion, metastasis, angiogenesis and apoptosis. Second, mice that express transcriptionally silenced (LoxP-Stop-LoxP) Smad7 in Nestin-expressing cells will be generated for breeding with mice expressing Cre recombinase under control of the Pdx1 promoter (Pdx1-Cre), in order to assess the role of Smad7 alone in the initiation and progression of pancreatic cancer. Animals carrying both Nestin-LSL-Smad7 and LSL- KrasG12D alleles will then be bred to the Pdx1-Cre mice in order to look for in vivo synergy between Smad7 and mutated Kras. Only the triple compound mutants will have activated Kras expressed in the whole pancreas while Smad7 expression will overlap only with the Nestin-expressing subpopulation. Third, since high Smad7 levels lead to the functional inactivation of pRb, mice that express Rb with a floxed Stop cassette in the introns flanking exon 19 (LSL-Rb) will be bred with mice carrying LSL-KrasG12D alleles and Pdx1- Cre, in order to determine whether mutated Rb mimics the in vivo actions of Smad7. Pancreata from control and mutant mice in aims II and III will be analyzed histologically at defined time points, and the consequences on pancreatic intraepithelial neoplasia (PanIN), cancer formation and altered TGF-2 signaling will be determined. Project Narrative: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in adults in the United States, with mortality virtually equaling incidence. This disease may be an even more serious problem in the future since its incidence increases after age 50 and the general population in the United States is aging. This proposal aims to provide an improved understanding of the biological causes that contribute to this disease aggressiveness, with the ultimate goal of devising better diagnostic and therapeutic strategies.