Mechanisms of Prostate Tumorigenesis Using Genetically Engineered Mouse Models

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Cell type susceptibility and androgen independence: The pRb tumor suppressor pathway is frequently altered in human prostate cancer. Our previous studies in multiple cell types have demonstrated the importance of pRb in suppressing tumor initiation. We have developed a strategy to dominantly interfere with pRb and potentially compensatory related proteins p107 and p130 by cell specific expression of the SV40 T antigen domain (T121) that binds to and inactivates all three proteins. We utilized keratin regulation to generate transgenic mice with androgen-independent epithelial subtype- targeted expression of T121 in prostate epithelium. In prostate, basal cells express K5 and K14, while luminal cells express K8 and K18. More recently, intermediate cells have been described with co expression of K5/18 and other keratins such as K15, 17 and 19. To explore the susceptible cell target(s), we used compartment-specific keratin gene transcriptional signals to drive expression of a conditional T121 allele composed of a reporter eGFP gene and stop sequences flanked by loxP sites upstream of T121 coding sequences. Specifically, K5, K18, and K19 genes were chosen to target basal cells, luminal cells, and intermediate cells in PE, respectively. To obtain optimal transgene expression, we have chosen to utilize a bacterial artificial chromosome (BAC) harboring the keratin gene to generate transgenic mice. The use of BAC-trangene minimizes position effects associated with standard transgenes and increases the likelihood that all necessary regulatory elements will be present. The eGFP stop cassette and T121 gene will be inserted into the BACs using recombineering technology pioneered by Neal Copeland and colleagues, such that regulators of the keratin gene will also drive the expression of a conditional T121 allele. The subsequent introduction of Cre recombinase (via somatic viral delivery or a tissue-specific transgene) will remove the stop sequences and induce T121 expression. All three strains of KeGT121 transgenic founder mice have been successfully generated (K5eGT121, K18eGT121, and K19eGT121). F1s have been used for mouse line characterization. Mice were also crossed to PbCre4 to activate T121 expression. To determine if tumor phenotype can be accelerated on Pten deficient background, we have crossed the mice on conditional Pten background. Year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. We are in the processing of rederiving all the single strains to helicobacter free facility. These strains are: K5eGT121 (4 lines), K18eGT121 (2 lines), K19eGT121 (2 lines), PbCre4 (1 line), conditional Pten (1 line). All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues were collected in either 10% formalin, OCT, or -80C. We are in the process of histology data collection. Stomal contribution to prostate tumor progression in APT121 mouse model: APT121;p53cf/+;FSPCreER, APT121;p53cf/f;FSPCreER, and relative control (e.g. p53cf/f;FSPCreER, p53cf/+;FSPCreE, and APT121) mice have been successfully generated. To inactivate p53, we i.p. injected the mice with tamoxifen (1mg/mouse/day) or oil as control at 2 months of age for 5 consecutive days. Mice are aged to various stages. Tissues are harvested for histological evaluation and molecular analysis. In year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues were collected in either 10% formalin, OCT, or -80C. We are in the process of aging the mice, and collecting tissue samples.