Dissecting Hedgehog, TGF beta and BMP Signaling During Pancreatic Tumorigenesis

描述(申请人提供)：胰腺癌是美国癌症相关死亡的主要原因，2009年估计有3.5万人死于这种疾病。KRAS2癌基因的激活突变发生在90%的胰腺导管腺癌(PDAC)中，这是最常见的胰腺癌类型，被认为是PDAC的起始病变。在大多数PDAC标本和细胞系中发现Hedgehog途径成分水平升高。大约一半的PDAC病例会发生Smad4转录调控因子的丢失，而作用于Smad4上游的转化生长因子受体RII、BMPRII和ALK4受体激酶在一小部分PDAC病例中会发生失活。然而，Hedgehog通路和由转化生长因子家族配体激活的信号通路在PDAC中的确切作用尚不清楚。因此，本申请建议使用一种新的小鼠建模系统和补充细胞培养和体外研究来系统地研究这些途径在胰腺癌发生中的作用。 以前的研究提供了相互矛盾的数据，关于刺猬配体在胰腺肿瘤发展过程中是以自分泌和旁分泌的方式起作用，还是只以旁分泌的方式起作用。因此，Aim 1的研究将使用新的小鼠模型来直接比较Sonic Hedgehog(Shh)及其下游转录因子Gli1和Gli2在体内胰腺肿瘤形成过程中与激活的Kras协同作用的能力。为了达到这个目的，我们将对肿瘤样本和肿瘤细胞系进行分析，以确定Shh是同时在癌细胞和反应性间质中刺激信号，还是仅在反应性间质中刺激信号。最后，使用一种新的显性抑制基因Gli3等位基因，将确定在Kras诱导的胰腺肿瘤发生中对Gli转录活性的需求。 AIM 2中的研究将确定转化生长因子和骨形态发生蛋白信号缺陷在胰腺肿瘤发生中的相对作用。靶向Smad4、转化生长因子RII或BMPRII的shRNA将在体内被特异性地递送到胰腺上皮细胞，并确定它们与激活的Kras的合作能力。将在诱导肿瘤来源的癌细胞系中进行互补实验，以确定转化生长因子-β或骨形态发生蛋白信号缺陷是否会改变胰腺癌细胞的表型，以及转化生长因子-β家族配体如何影响胰腺癌细胞的行为。 Aim 3中的研究将确定胰腺癌细胞中Hedgehog和转化生长因子-B信号级联信号之间是否存在串扰，以及这种串扰如何影响这些细胞的表型。最后，基因表达谱将被用来确定Hedgehog和转化生长因子-B信号级联下游的重要基因。通过对AIMS 1和AIMS 2中产生的癌细胞株的研究，以及这些基因在体内胰腺肿瘤形成过程中与激活的Kras协同作用的能力的研究，将验证这些基因在胰腺肿瘤发生中的生物学重要性。

DESCRIPTION (provided by applicant): Pancreatic cancer is a leading cause of cancer-related mortality in the United States, with an estimated 35,000 people dying from this disease in 2009. Activating mutations in the KRAS2 oncogene occur in >90% of pancreatic ductal adenocarcinomas (PDAC), the most common type of pancreatic cancer, and are believed to be initiating lesions in PDAC. Elevated levels of hedgehog pathway components are found in a majority of PDAC specimens and cell lines. Loss of the Smad4 transcriptional regulator occurs in approximately half of PDAC cases, and inactivation of the TGFßRII, BMPRII, and ALK4 receptor kinases that act upstream of Smad4 occurs in a smaller subset of PDAC cases. Yet, the precise roles of the hedgehog pathway and the signaling pathways activated by TGF-ß family ligands in PDAC remain unclear. This application therefore proposes to use a novel mouse modeling system and complementary cell culture and in vitro studies to systematically investigate the roles of these pathways in pancreatic carcinogenesis. Prior studies have provided conflicting data on whether hedgehog ligands act in both autocrine and paracrine fashion, or only a paracrine manner, during pancreatic tumor development. Therefore, studies in Aim 1 will use novel mouse models to directly compare the ability of sonic hedgehog (Shh) and its downstream transcription factors Gli1 and Gli2 to cooperate with activated Kras during pancreatic tumorigenesis in vivo. Analyses of tumor samples and cell lines generated from the tumors induced in this Aim will be performed to determine whether Shh stimulates signaling in both the cancer cells and the reactive stroma, or only within the reactive stroma. Finally, using a novel dominant-repressor Gli3 allele, the requirement for Gli transcription activity in Kras-induced pancreatic tumorigenesis will be determined. The studies in Aim 2 will identify the relative contributions of defects in TGF-ß and BMP signaling to pancreatic tumorigenesis. ShRNAs targeting Smad4, TGFßRII, or BMPRII will be delivered specifically to the pancreatic epithelium in vivo, and their ability to cooperate with activated Kras determined. Complementary experiments in cancer cell lines derived from the induced tumors will be performed to determine whether defects in TGF-ß or BMP signaling alter the phenotypes of pancreatic cancer cells, and how TGF-ß family ligands influence pancreatic cancer cell behavior. The studies in Aim 3 will determine whether there is cross-talk between the hedgehog and TGF-ß signaling cascades in pancreatic cancer cells, and how this cross-talk influences the phenotypes of these cells. Finally, gene expression profiling will be used to identify important genes downstream of the hedgehog and TGF-ß signaling cascades. The biological importance of these genes during pancreatic tumorigenesis will be validated using studies of the cancer cell lines generated in Aims 1 and 2, and the ability of these genes to cooperate with activated Kras during pancreatic tumorigenesis in vivo.