The Characterization of the New Tumor Suppressor USP9X in Pancreatic Cancer

新型肿瘤抑制剂 USP9X 在胰腺癌中的表征

• 批准号: 8835986
• 负责人: Chang-il Hwang
• 金额: $ 5.6万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835986
• 关键词: Adenocarcinoma Cell; Adherent Culture; Apoptosis; Biological Markers; Cancer cell line; Cause of Death; Cell Line; Cell Transplants; Cells; Clinical; Deubiquitinating Enzyme; Disease Progression; Ductal; Ductal Epithelial Cell; Genetic; Genetically Engineered Mouse; Human; In Vitro; Individual; Insertional Mutagenesis; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of pancreas; Methods; Modeling; Modification; Molecular; Mus; Oncogenic; Organoids; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Pharmaceutical Preparations; Phenocopy; Play; Precancerous Conditions; Premalignant; Process; Property; Proteins; Proteome; Proteomics; Reaction; Research; Resistance; Role; Sleeping Beauty; Structure; Survival Rate; System; Testing; Time; Transplantation; Tumor Suppressor Proteins; Ubiquitin; United States; Validation; anticancer research; base; cancer cell; gene function; genome-wide; high throughput analysis; human disease; improved; in vivo; in vivo Model; monolayer; mouse model; novel; novel therapeutics; outcome forecast; pancreatic cancer cells; public health relevance; response; screening; therapeutic target; tissue culture; tumor; tumor progression; ubiquitin-protein ligase

 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.

 描述（由申请人提供）：胰腺导管类器官和胰腺癌中肿瘤抑制因子Usp 9 x的表征胰腺导管腺癌（PDAC）是美国癌症导致的第四大死亡原因。PDAC患者的五年生存率是常见癌症中最低的，尽管努力改善临床预后，但仍仅为6%。传统的组织培养方法，以单层生长胰腺癌细胞系，使我们能够剖析分子途径和药物反应的PDAC。然而，在体外和体内之间存在显著的翻译差距。例如，当原位注射时，以单层生长的PDAC细胞系不产生基质反应，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反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。本研究拟建立胰腺导管原代细胞三维培养和移植模型，研究胰腺导管原代细胞PDAC的进展和基因功能。我已经成功地在来自GEMM（野生型，Kras+/LSL-G12 D，Pdx 1-Cre; Kras +/LSL-G12 D或Pdx 1-Cre; Kras +/LSL-G12 D; Trp 53 +/LSL-R172 H）的三维半固体基质（下文称为胰腺类器官）中培养了鼠正常、癌前和恶性胰腺导管上皮细胞。更重要的是，在原位移植时，与2D细胞系不同，移植的类器官细胞忠实地再现了PDAC进展。因此，小鼠原位移植类器官（OGO）模型将提供一个独特的平台，研究PDAC进展，生物标志物的发现和功能验证的治疗目标。以前，我们已经确定了去泛素化酶，Usp 9 x作为一个新的肿瘤抑制剂在PDAC使用睡美人插入突变筛选。我们发现缺乏Usp 9 x的细胞在基质分离后对凋亡的抵抗力增加，我们暗示E3泛素连接酶Itch，Usp 9 x的底物在这个过程中。然而，Usp 9 x和Itch在PDAC中的作用在很大程度上仍然未知。使用三维胰腺导管类器官，我将研究Usp 9 x及其底物Itch在PDAC进展中的作用，使我们能够研究Usp 9 x和Itch在正常和癌前胰腺导管细胞中的作用。Usp 9 x和Itch具有蛋白质靶点，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。为了表征Usp 9 x和Itch的功能，将使用OGO小鼠研究Usp 9 x和Itch的各个底物的作用。新发现的Usp 9 x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。