Award Number: W81XWH-04-1-0836 TITLE: Molecular Profiling of Prostate Cancer to Determine Predictive Markers of Response to Radiation and Receptor Tyrosine Kinase Inhibitor Therapy

Purpose: To determine the efficacy of combining radiation (XRT) with a dual EGFR/VEGFR inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. Methods and Materials: Immunoblotting was performed for EGFR, phosphorylated-EGFR (p-EGFR), and p-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3 and HUVEC cells treated with XRT+/-AEE788. Tumor xenografts were established for DU145 and PC-3 on hindlimbs of athymic nude mice assigned to four treatment groups: 1) Control, 2) AEE788, 3) XRT, 4) AEE788+XRT. Tumor blood flow and growth measurements were performed using immunohistochemistry and imaging. Results: AEE788 effectively reduced p-EGFR and p-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with -6AEE788+XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed radiosensitization effect in HUVEC and increased apoptotis susceptibility. Concurrent AEE788+XRT compared to either alone led to significant tumor growth delay in DU145 tumors. In contrast, PC-3 tumors derived no added benefit to combined modality therapy. In the DU145 tumors, significant reduction in tumor blood flow with combination therapy was demonstrated by power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. MS imaging demonstrated that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. Conclusion: AEE788+XRT showed efficacy in vitro/in vivo with DU145-based cell models while PC3-based were adequately treated with radiation alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and demonstrated effects on both tumor cell proliferation and vascular destruction. INTRODUCTION Prostate cancer is the second leading cause of cancer death in men with an estimated 230,090 men diagnosed with prostate cancer in 2005 (1). The first line of therapy includes surgery or radiation therapy (XRT) (2-4), with intermediate to high risk patients often receiving androgen suppression therapy (5). Once the disease progresses to an androgen independent disease status (AID), therapeutic options for prostate cancer patients diminish, with an overall survival of 6-12 months (6). Chemotherapy confers only a small survival benefit in patients with metastatic prostate cancer (7, 8), suggesting a need for investigation of non-hormonal systemic therapy. In AID patients, combining radiation with systemic agents result in 20-30% therapy failure (9), therefore requiring better treatment approaches. Studies have demonstrated that human prostate cancer cells (DU145 and PC-3) derived from androgen independent prostate tumors express epidermal growth factor receptor (EGFR) (10-13). Furthermore, in pre-clinical studies involving various cancer models, including prostate tumors, EGFR overexpression has been linked to proliferation, angiogenesis, and migration (14-16), and -7has an inverse relationship to tumor radiocurability (17, 18). In prostate cancer, EGFR expression correlates with higher Gleason score, higher PSA values, and is an independent prognostic factor negatively impacting disease free survival (19). New small molecule drugs targeting the EGFR receptor have been developed and show promise in the clinical setting. For head and neck cancer, combining ionizing radiation with EGF signal blockade conferred statistically significant survival benefit in a large randomized phase III study (20). In addition to targeting EGFR, the use of vascular endothelial growth factor receptor (VEGFR) inhibitors is actively being investigated for cancer therapy (21). In fact, a number of preclinical studies have demonstrated that molecular compounds targeting VEGFR-2 (such as SU11248) when combined with radiation leads to improved tumor growth delay, partially due to significant tumor vascular destruction (22). Since there is a direct correlation between angiogenesis as well multiple angiogenesis related proteins with tumor grade (high Gleason Score), tumor stage, progression of disease, metastatic potential and survival (23, 24), analysis of angiogenesis-related proteins has been examined. In particular, VEGFR-2 expression has been reported suggesting that prostate tumors are highly vascular and may also benefit from therapy targeting VEGFR-2 (25). Furthermore, a recent study has suggested a significant advantage to combining both EGFR and VEGFR inhibitors in vitro to reduce the activation of Akt in endothelial cells (26). The development of AEE788, a dual tyrosine kinase inhibitor of both EGFR and VEGFR, now provides an avenue to investigate the effect of simultaneous blockade of EGFR and VEGFR (27-35) when combined with conventional cytotoxic agents such as radiation. We hypothesized that dual inhibition of both targets (EGFR and VEGFR) using AEE788 in prostate cancer models should lead to improved tumor control when combined with radiation. METHODS AND MATERIALS Cell Culture, Animals, and Compounds -8DU145 and PC-3 (ATCC, Rockville, MD) human prostate cancer cells and Human umbilical vein endothelial cells (HUVEC) were obtained from Cambrex (East Rutherford, NJ), and cultivated in vitro according to the recommendations of the supplier. Five to six week old male athymic nude mice (nu/nu) were purchased from Harlan Laboratories and maintained in accordance to guidelines approved by the Vanderbilt Institutional Animal Care and Use Committee (IACUC). AEE788 was provided by Novartis Pharma (Basel, Switzerland). For cellular assays, AEE788 was dissolved in DMSO, and for in vivo experiments, AEE788 was dissolved in a suspension on N-methylpyrroline and PEG300 1:9 (v/v).