Tumor models for the study of inflammation and oncogenesis

当一个准确的临床前模型可用时，对人类疾病的治疗方法的合理设计将大大促进。当小鼠肿瘤模型尽可能多地复制已知的分子病变，从而最准确地描绘机体对肿瘤发生的生理反应时，它们是最有用的。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做到的。迄今为止，我们的动物肿瘤研究主要集中在肾细胞癌（RCC）上，重点是肝脏微环境作为转移的一个常见场所。因此，我们选择肾脏和肝脏作为RCC和肝细胞癌（HCC）肿瘤模型的两个模型器官。我们的第一个目标是利用SB技术优化癌基因分别在肝脏和肾脏的肝脏和肾脏传递。为此，我们将使用报告基因，如高斯荧光素酶DNA，它允许定量测量基因传递到预期器官。我们常规使用流体动力DNA递送，并优化了直接肾内注射以摄取高斯荧光素酶表达载体。许多与HCC和RCC相关的癌基因和途径已经被描述。对于HCC，除了丙型肝炎病毒（HCV）诱导的HCC外，MET、β -连环蛋白和p53常被引用为主要标志物。在RCC中，MET磷酸化、VHL、TRC8和TSC2是透明细胞RCC的主要标志物。这两种癌症也共享致癌信号通路，包括Myc、Ras、TGFalpha、IL-6和TNFalpha。在我们的第二个目标中，我们将使用SB筛选这些致癌基因的组合，以确定其致瘤潜力。具体而言，对于HCC，将测试致癌- MET、β -连环蛋白、AKT、T121、Myc和p53的组合。对于RCC- shRNA敲低透明细胞相关基因VHL、TSC2和TRC8以及HCC癌基因集将进行分析。在初步试验中，我们通过共同递送SB- akt和SB- β -catenin可重复地产生肝细胞腺瘤（HCA），以及通过共同递送SB- met和SB- β -catenin产生HCC。这些和任何其他产生的肿瘤将根据组织病理学和原位传代在体内分类。由于这些致癌基因通常不足以导致肿瘤发生，我们认为当这些初始突变与炎症介导的肿瘤促进相结合时，肿瘤可能会发展。因此，在本项目的第三个目标中，我们将确定局部亚临床肝和肾慢性炎症的细胞因子和其他炎症介质。与HCC和RCC相关或在各自微环境中发现的炎症介质包括：STAT3、NF-kappaB、IL-6、TNFalpha、il -1 α、HMGb1、IL-22、IL-23和HCV基因产物。我们将首先测试组成激活的STAT3和NF-kappaB，因为它们是不同效应家族的致癌炎症信号的汇聚点。我们假设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发生。组织病理学将用于组织反应评分，重点是确定任何增生，癌前转化或肿瘤发生。最后，该项目的第四个目标结合了致癌基因（aim 2）和炎症（aim 3）介质在肿瘤促进和进展中的作用。为此，我们将提供单个sb癌基因，标志着首次击中肿瘤的启动子以及sb效应物，以筛选癌基因依赖的炎症介导的肿瘤促进。此外，我们将联合提供SB-AKT/ β -catenin与sb -炎症介质，以筛查癌基因依赖炎症介导的肿瘤进展为癌。这可以定义介导腺瘤-癌序列进展的炎症成分，类似于先前在结肠癌中描述的炎症成分。类似地，对于RCC，我们已经成功地生成了一个肾移行细胞乳头状瘤，它代表了恶性前癌症的一种模型，这将使我们能够研究炎症介质在其向恶性发展过程中可能发挥的作用。本项目中描述的方法将为我们提供准确的HCC和RCC临床前模型，并促进更有效地靶向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.