Tumor models for the study of inflammation and oncogenesis

当一个准确的临床前模型可用时，人类疾病治疗方法的合理设计将大大便利。小鼠肿瘤模型是最有用的，当它们复制尽可能多的已知分子损伤，以便最准确地描述身体对肿瘤发生的生理反应。有了体细胞工程(睡美人/SB)，我们可以轻松地同时重现多个病变，而且规模是其他方法无法比拟的。到目前为止，我们的动物肿瘤研究主要集中在肾细胞癌(RCC)上，强调肝脏微环境是转移的常见场所。因此，我们选择肾脏和肝脏作为发展肾细胞癌和肝细胞癌肿瘤模型的两个模型器官。我们的第一个目标是利用SB技术优化癌基因在肝脏和肾脏中的传递。为此，我们将使用报告基因，如高斯荧光素酶DNA，它允许对预期器官的基因输送进行定量测量。我们常规使用流体动力DNA传递，并优化了直接肾内注射以摄取Gaussia荧光素酶表达载体。许多与肝细胞癌和肾细胞癌相关的癌基因和通路已经被描述。对于肝细胞癌，除了丙型肝炎病毒(丙型肝炎病毒)诱导的肝细胞癌外，MET、β-连环蛋白和P53经常被认为是主要的标志物。在肾细胞癌中，MET磷酸化、VHL、TRC8和TSC2是透明细胞肾癌的主要标志。这两种癌症还具有相同的致癌信号通路，包括Myc、RAS、TGFpha、IL-6和TNFpha。在我们的第二个目标中，我们将使用SB筛选这些癌基因的组合以了解它们的致瘤潜力。具体来说，对于肝癌，将测试致癌MET、β-连环蛋白、AKT、T121、Myc和P53的组合。对于RCC-shRNA，将分析与透明细胞相关的基因VHL、TSC2和TRC8以及肝癌癌基因集的敲除。在初步试验中，我们已经通过SB-AKT和SB-β-连环蛋白的共同传递复制产生了肝细胞腺瘤(HCA)，并通过SB-MET和SB-β-连环蛋白的共同传递产生了肝癌。这些肿瘤和任何其他由此产生的肿瘤将根据组织病理学进行分类，并在体内原位传代。由于这些癌基因通常不足以导致肿瘤发生，我们认为当这些启动突变与炎症介导的肿瘤促进相结合时，肿瘤可能会发展起来。因此，在本项目的第三个目标中，我们将确定局部亚临床肝肾慢性炎症的细胞因子和其他炎症介质。与肝细胞癌和肾癌相关或在其各自微环境中发现的炎症介质包括：STAT3、NF-kappaB、IL-6、TNFpha、IL-1α、HMGB1、IL-22、IL-23和丙型肝炎病毒基因产物。我们将首先测试结构性激活的STAT3和NF-kappaB，因为它们是不同效应家族致癌炎症信号的汇聚点。我们推测，这些效应分子可能通过创造有利于肿瘤促进和进展的微环境来影响肿瘤的发生。组织病理学将被用来对组织反应进行评分，重点是识别任何增生、肿瘤前转化或肿瘤形成。最后，该项目的第四个目标结合了癌基因(目标2)和炎性介质(目标3)在肿瘤促进和进展中的作用。为此，我们将提供单一的SB癌基因，标志着第一次击中肿瘤启动子和SB效应，以筛选癌基因依赖的炎症介导的肿瘤促进。此外，我们将联合应用SB-AKT/β-连环蛋白与SB-炎症介质的组合，以筛选癌基因依赖的炎症介导的肿瘤进展为癌症。这可能定义了介导腺瘤-癌序列进展的炎性成分，类似于先前在结肠癌中所描述的。类似地，对于肾癌，我们已经成功地产生了代表癌前病变模型的肾移行细胞乳头状瘤，并将使我们能够研究炎症介质在其恶变过程中可能发挥的作用。这个项目中描述的方法将为我们提供准确的肝细胞癌和肾细胞癌的临床前模型，并促进更有效地针对肿瘤发生的治疗方法的发展。

The rational design of therapeutic approaches to human disease is greatly facilitated when an accurate pre-clinical model is available. Mouse tumor models are most useful when they reproduce as many known molecular lesions as possible, so as to most accurately portray the bodys physiological response to tumorigenesis. With somatic cell engineering (Sleeping Beauty/SB), we can recapitulate multiple lesions simultaneously with ease and scale not possible by other means. Our animal tumor studies to date have focused on renal cell carcinoma (RCC) with an emphasis on the liver microenvironment as a frequent depot for metastases. We have therefore chosen the kidney and liver as two model organs for the development of RCC and hepatocellular carcinoma (HCC) tumor models. Our first aim is to optimize hepatic and renal delivery of oncogenes to the liver and kidney, respectively, using SB technology. For this, we will use reporter genes, such as Gaussia luciferase DNA, which allow for a quantitative measure of gene delivery to the intended organ. We routinely use hydrodynamic DNA delivery and have optimized direct intra-renal injection for the uptake of a Gaussia luciferase expression vector. Many oncogenes and pathways associated with HCC and RCC have been described. For HCC, MET, beta-catenin, and p53 are often cited as primary markers in addition to hepatitis C virus (HCV)-induced HCC. In RCC, MET phosphorylation, VHL, TRC8, and TSC2 are primary markers of clear cell RCC. Both of these cancers also share oncogenic signaling pathways including Myc, Ras, TGFalpha, IL-6, and TNFalpha. In our second aim, we will screen combinations of these oncogenes for their tumorigenic potential using SB. Specifically, for HCC, combinations of oncogenic- MET, beta-catenin, AKT, T121, Myc and p53 will be tested. For RCC- shRNA knockdowns of the clear cell-related genes VHL, TSC2, and TRC8 along with the HCC oncogene set will be analyzed. In preliminary trials, we have reproducibly generated hepatocellular adenomas (HCA) by the co-delivery of SB-AKT and SB-beta-catenin, and HCC by the co-delivery of SB-MET and SB- beta-catenin. These and any other resultant tumors will be classified by histopathology and orthotopically passaged in vivo. Since these oncogenes are not usually sufficient to cause tumorigenesis, we consider it likely that tumors develop when these initiating mutations are coupled with inflammatory-mediated tumor promotion. Therefore, in the third aim of this project, we will identify cytokine and other inflammatory mediators of localized subclinical hepatic and renal chronic inflammation. Inflammatory mediators that are associated with HCC and RCC or found in their respective microenvironments include: STAT3, NF-kappaB, IL-6, TNFalpha, IL-1alpha, HMGb1, IL-22, IL-23 and HCV gene products. We will first test constitutively activated STAT3 and NF-kappaB, since they are convergence points of carcinogenic inflammatory signaling by different effector families. We hypothesize that these effector molecules may impact tumorigenesis by creating a microenvironment that is favorable to tumor promotion and progression. Histopathology will be used to score the tissue response with focus on identifying any hyperplasia, pre- neoplastic transformation, or tumorigenesis. Finally, the fourth aim of this project combines the resulting contributions of oncogenes (Aim 2) and inflammatory (Aim 3) mediators in tumor tumor promotion and progression. For this, we will deliver single SB-oncogenes signifying first hit tumor initiators along with SB-effectors to screen for oncogene-dependent inflammatory-mediated tumor promotion. Additionally, we will co-deliver combinations of SB-AKT/beta-catenin with SB-inflammatory mediators to screen for oncogene-dependent inflammatory-mediated tumor progression to carcinoma. This may define inflammatory components that mediate an adenoma-carcinoma sequence progression, similar to that which has been previously described in colon cancer. Analogously, for RCC, we have successfully generated a renal transitional cell papilloma that represents a model of pre-malignant cancer and will allow us to investigate the role inflammatory mediators may play in its progression to malignancy. The approaches described in this project will provide us with accurate pre-clinical models for both HCC and RCC and facilitate the development of therapeutics that more effectively target tumorigenesis.