Imaging androgen receptor function during flutamide treatment in the LAPC9 xenograft model Romyla Ilagan, 1 Liquin Joann Zhang, 1 Jill Pottratz, 1 Kim Le, 1 Sussan Salas, 1 Meera Iyer, 3 Lily Wu, 2,3 Sanjiv S. Gambhir, 3 and Michael Carey 1 Departments of 1 Biological Chemistry, 2 Urology, and 3 Molecular and Medical Pharmacology, School of Medicine, University of California at Los Angeles, Los Angeles, California Abstract The current understanding of the response of androgen receptor to pharmacologic inhibitors in prostate cancer is derived primarily from serum prostate-specific antigen (PSA) levels. In this study, we test whether a novel androgen receptor – specific molecular imaging system is able to detect the action of the antiandrogen flutamide on androgen receptor function in xenograft models of prostate cancer. Adenoviruses bearing an optical imaging cassette containing an androgen receptor–responsive two-step transcriptional amplification system were injected into androgen-dependent and hormone-refractory tumors of animals undergoing systemic time-controlled release of the antiandrogen flutamide. Imaging of tumors with a cooled charge-coupled device camera revealed that the response of AdTSTA to flutamide is more sensitive and robust than serum PSA measurements. Flutamide inhibits the androgen signaling pathway in androgen-dependent but not refractory tumors. Analysis of androgen receptor and RNA polymerase II binding to the endogenous PSA gene by chromatin immunoprecipitation revealed that flutamide treatment and androgen withdrawal have different molec- ular mechanisms. The application of imaging technology to study animal models of cancer provides mechanistic insight into antiandrogen targeting of androgen receptor during disease progression. [Mol Cancer Ther 2005;4(11):1662– 9] Introduction Prostate cancer is a disease driven by the androgen receptor (1 – 4). Androgen receptor is a 110-kDa steroid receptor, which is sequestered in the cytoplasm by chaperones in the absence of its ligand. In the presence of dihydrotestoster- one, androgen receptor dimerizes and enters the nucleus, where it binds to androgen response elements and activates transcription of responsive genes. One of the key challenges in prostate cancer research has been determining how androgen receptor functions in recurrent or androgen- independent prostate cancer (also called hormone-refractory prostate cancer; refs. 5–11) and how pharmacologic inhibitors affect function (12). Our groups have been addressing this problem using gene expression – based bioluminescence imaging to evaluate androgen receptor function in xenograft models (13 – 16), which accurately recreate prostate cancer progression from an androgen- dependent to an androgen-independent phase (17). Imaging provides a means to probe the mechanism of cancer in live animals and facilitates the evaluation of pharmacologic effects on specific signaling events. In this study, we address the mechanism of a nonsteroidal antiandrogen called flutamide by molecular imaging of androgen receptor function and compare the results with the effects of androgen deprivation by castration. Fluta- mide is more potent than Casodex in mice (18, 19). We chose this drug to address whether a specialized molecular imaging system could be employed to detect the inhibitory effect of an antiandrogen on androgen receptor function during prostate cancer growth. In gene expression – based bioluminescence imaging, a promoter is placed upstream of a bioluminescence reporter gene (20 – 22). The reporter cassette is introduced into tumor cells in an animal and the promoter activity is imaged after injection of D-luciferin (for firefly luciferase) or coelenterazine (for Renilla luciferase) using a Xenogen in vivo imaging system (Xenogen Corp., Alameda, CA; ref. 23). In vivo imaging system is a cooled charge-coupled device that measures bioluminescent light. A computer interprets the light and superimposes a pseudoimage, representing the quantity of photons emitted by the tissue, over a gray-scale photograph of the animal. A major challenge in bioluminescence imaging is that cellular promoters are typically weak, and detection of optical signals in dense tissues is hampered by light attenuation and scattering (20, 24). We developed an approach to augment cellular promoter activity and light output based on a concept termed two-step transcriptional amplification (TSTA; ref. 16). A cellular promoter expresses a potent chimeric activator, GAL4-VP16. GAL4-VP16 is a fusion of the high-affinity yeast GAL4 DNA-binding domain to the potent herpes simplex virus VP16 activation domain (25, 26). GAL4-VP16 has a unique potency and specificity not naturally found in mammalian cells. GAL4- VP16 binds a GAL4-responsive reporter gene and generates high levels of firefly luciferase. Our prostate cancer – specific Received 6/15/05; revised 8/8/05; accepted 8/30/05. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: S.S. Gambhir is currently at the Molecular Imaging Program at Stanford, Stanford University School of Medicine, James H. Clark Center, E13 318 Campus Drive, Stanford, CA 94305-5472. Requests for reprints: Romyla Ilagan, Department of Biological Chemistry, School of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, CHS 33-142, Los Angeles, CA 90095-1737. Phone: 310-794-9636; Fax: 310-206-5272. E-mail: milagan@ucla.edu Copyright C 2005 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0197 1662 Mol Cancer Ther 2005;4(11). November 2005 on June 11, 2020. © 2005 American Association for Cancer Research. mct.aacrjournals.org Downloaded from