Development of animal models and molecular tools to investigate the function of prostate specific membrane antigen.

Abstract

Prostate Specific Membrane Antigen (PSMA) is a transmembrane glycoprotein expressed in human prostate luminal epithelial cells. PSMA expression increases in most prostate cancer cases. PSMA overexpression is associated with an unfavorable prognosis, biochemical recurrence, and metastatic disease in patients treated for prostate cancer. PSMA has both folate hydrolase and glutamate carboxypeptidase enzymatic activity. Given its membrane localization and increased expression in prostate cancer, PSMA has received considerable attention as a diagnostic and therapeutic target. Remarkably, the physiological roles of PSMA in the prostate gland remain unknown, and no animal models expressing human PSMA exist. Neither mice nor rats express endogenous PSMA in the prostate, precluding testing of PSMA-targeting agents in wild type Muridae species. The principal goals of this work were to develop a genetically engineered animal model expressing human PSMA in normal and malignant prostates. Multiple attempts to achieve this goal using three distinct molecular strategies in mice were unsuccessful. However, I succeeded in developing a transgenic rat model that conditionally expresses human PSMA in the prostate. By five weeks of age these rats display heterogenous PSMA expression in ventral and lateral prostate lobes. By twenty-five weeks, PSMA expression approaches homogeneity in luminal prostate epithelial cells without apparent pathological effect. Parallel efforts by others to develop a rat model of prostate cancer based on MYC oncogene expression and Pten tumor suppressor loss encountered technical roadblocks and were unsuccessful, which precluded analyses of PSMA function during malignant progression. Overcoming these technical roadblocks necessitated construction of large, complex, multifunctional transgenes that push the boundaries of extant cloning and recombineering technologies. To expedite this process, I optimized a DNA assembly technology and successfully deployed this approach to assemble up to twelve DNA fragments. I also discovered that non-specific DNA can significantly increase the likelihood of achieving success in the generation of complex assemblies. Taken together, these contributions provide a powerful framework to determine the roles of PSMA in normal and diseased prostate glands and provide new molecular tools to support development of next generation of PSMA-expressing animal models. Such models will be instrumental in advancing PSMA-directed diagnostics and therapeutics toward the clinic.