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 i s 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.
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