The Characterization of the New Tumor Suppressor USP9X in Pancreatic Cancer

 描述(申请人提供)：胰腺导管器官和胰腺癌中肿瘤抑制基因USP9X的特征胰腺导管腺癌(PDAC)是美国第四大癌症死亡原因。PDAC患者的五年生存率是常见癌症中最低的，尽管积极努力改善临床预后，但仍仅为6%。传统的单层培养胰腺癌细胞的方法使我们能够剖析PDAC中的分子途径和药物反应。然而，在体外和体内之间存在着显着的翻译差距。例如，当原位注射时，生长在单层中的PDAC细胞系不会产生间质反应，这与来自基因工程小鼠模型(GEMM)或人类患者的肿瘤不同。此外，利用GEMM以高通量的方式验证基因的功能和药物的响应是一项艰巨的任务。因此，迫切需要弥合体外和体内模型之间的差距。在这里，我建议建立三维培养和移植模型，以研究PDAC的进展和鉴定原代胰腺导管细胞的基因功能。我已经成功地从GEMM(野生型，Kras+/LSL-G12D，Pdx1-Cre；Kras+/LSL-G12D或Pdx1-Cre；Kras+/LSL-G12D；Trp53+/LSL-R172H)中成功地在三维半固体基质(以下称为胰腺类器官)中培养了正常、癌前和恶性小鼠胰腺导管上皮细胞。更重要的是，在原位移植时，植入的器质细胞与2D细胞系不同，它忠实地概括了PDAC的进展。因此，小鼠原位移植器官(OGO)模型将为研究PDAC进展、生物标记物发现和治疗靶点的功能验证提供一个独特的平台。此前，我们已经通过睡美人插入突变筛选确定了去泛素酶USP9X是PDAC中的一种新的肿瘤抑制因子。我们发现，缺乏USP9X的细胞在基质脱离后对凋亡的抵抗力增强，我们发现USP9X的底物E3泛素连接酶Itch参与了这一过程。然而，USP9X和Itch在PDAC中的作用在很大程度上仍不清楚。利用三维胰腺导管细胞器，我将研究USP9X及其底物Itch在PDAC进展中的作用，使我们能够研究USP9X和Itch在正常和癌前胰腺导管细胞中的作用。USP9X和Itch有蛋白质靶点，可以解释它们的肿瘤抑制特性。这些靶蛋白将通过全蛋白质组和泛素-蛋白质组方法进行鉴定。为了确定USP9X和Itch的功能，我们将用Ogo小鼠研究USP9X和Itch的单独底物的作用。新发现的USP9X和Itch底物将阐明PDAC进展中新的肿瘤抑制途径，为新的治疗方法铺平道路。

 DESCRIPTION (provided by applicant): Characterization of the tumor suppressor Usp9x in pancreatic ductal organoids and pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death due to cancer in the United States. The five-year survival rate for PDAC patients is the lowest among common cancers, and remains at only 6% despite intensive efforts to improve clinical prognosis. The conventional tissue culture methods to grow pancreatic cancer cell lines in monolayer allow us to dissect molecular pathways and drug responses in PDAC. However there is a significant translational gap between in vitro and in vivo. For example, when orthotopically injected, PDAC cell lines grown in monolayer do not produce a stromal reaction, unlike tumors from genetically engineered mouse models (GEMMs) or human patients. Moreover it is a daunting task to utilize GEMMs to validate the function of genes and the responses of drugs in a high throughput manner. Therefore, it is urgently required to bridge the gap between in vitro and in vivo models. Here, I propose to establish three dimensional culture and transplantation models to study PDAC progression and characterize gene function in primary pancreatic ductal cells. I have successfully grown murine normal, premalignant and malignant pancreatic ductal epithelial cells in three dimensional semisolid matrices (called pancreatic organoids hereafter) from GEMMs (wild-type, Kras+/LSL-G12D, Pdx1-Cre; Kras+/LSL-G12D or Pdx1-Cre;Kras+/LSL-G12D; Trp53+/LSL-R172H). More importantly, upon orthotopic transplantation, engrafted organoid cells faithfully recapitulate PDAC progression unlike 2D cell lines. Thus, murine orthotopically grafted organoid (OGO) models will provide a unique platform to study PDAC progression, biomarker discovery and functional validation of therapeutic targets. Previously, we have identified the deubiquitinating enzyme, Usp9x as a new tumor suppressor in PDAC using Sleeping Beauty insertional mutagenesis screening. We found that cells lacking Usp9x had an increased resistance to apoptosis following matrix detachment, and we implicated the E3 ubiquitin ligase Itch, a substrate of Usp9x in this process. However, the role of Usp9x and Itch in PDAC remains largely unknown. Using three dimensional pancreatic ductal organoids, I will study the role of Usp9x and its substrate Itch in PDAC progression, allowing us to investigate the role of Usp9x and Itch in normal and premalignant pancreatic ductal cells. Usp9x and Itch have protein targets that can explain their tumor suppressor properties. These target proteins will be identified by whole proteome and Ubiquitin-proteome methods. To characterize the functions of Usp9x and Itch, the role of individual substrates of Usp9x and Itch will be investigated using OGO mice. The newly identified substrates of Usp9x and Itch will elucidate new tumor suppressive pathways in PDAC progression, paving the road towards new therapeutics.