Cancer Therapeutics, Targets, and Chemical Biology Research Inhibition of Neurotensin Receptor 1 Selectively Sensitizes Prostate Cancer to Ionizing Radiation Nicholas C.K. Valerie1, Eli V. Casarez2, John O. DaSilva2, Marya E. Dunlap-Brown3, Sarah J. Parsons2,4, George P. Amorino1,2, and Jaroslaw Dziegielewski1,4 Abstract Radiotherapy combined with androgen depletion is generally successful for treating locally advanced prostate cancer. However, radioresistance that contributes to recurrence remains a major therapeutic problem in many patients. In this study, we define the high-affinity neurotensin receptor 1 (NTR1) as a tractable new molecular target to radiosensitize prostate cancers. The selective NTR1 antagonist SR48692 sensitized prostate cancer cells in a dose- and time-dependent manner, increasing apoptotic cell death and decreasing clonogenic survival. The observed cancer selectivity for combinations of SR48692 and radiation reflected differential expression of NTR1, which is highly expressed in prostate cancer cells but not in normal prostate epithelial cells. Radiosensitization was not affected by androgen dependence or androgen receptor expression status. NTR1 inhibition in cancer cell–attenuated epidermal growth factor receptor activation and downstream signaling, whether induced by neurotensin or ionizing radiation, establish a molecular mechanism for sensitization. Most notably, SR48692 efficiently radiosensitized PC-3M orthotopic human tumor xenografts in mice, and significantly reduced tumor burden. Taken together, our findings offer preclinical proof of concept for targeting the NTR1 receptor as a strategy to improve efficacy and outcomes of prostate cancer treatments using radiotherapy. Cancer Res; 71(21); 6817–26. Ó2011 AACR. Introduction neuroendocrine cells and their secretions, which can aid cancer cell proliferation and survival. Neuroendocrine cells Prostate cancer is the most common cancer in men and the exist in the normal prostate gland, regulating prostatic second leading cause of cancer deaths in the United States (1, 2). growth, differentiation, and secretion. However, clusters of Radiotherapy is one of the standard treatment modalities for neuroendocrine-like cells are also found in most prostate prostate cancer (3); however, a major obstacle to effective cancer, and the presence of extensive neuroendocrine features radiotherapy is the limited radiation dose that can be safely in tumors is an indication of increased aggressiveness and delivered to the prostate (<85 Gy; refs. 4, 5). Unfortunately, at androgen independence (6–8). These neuroendocrine-like this dose level, a significant proportion of tumors are resistant cells often arise from prostate cancer cells through the process either not responding or recurring after treatment. An alter- of neuroendocrine transdifferentiation (7, 9). The neuroendo- native to radiation dose increase would be to use radiosensitiz- crine-like cells secrete a variety of factors, including parathy- ing agents selectively targeted to prostate cancer cells while roid hormone–related peptides, serotonin, calcitonin, sparing normal tissue, thus minimizing radiation toxicity by bombesin-related peptide, and neurotensin, that enhance lowering effective therapeutic doses. DNA synthesis, proliferation, and migration of prostate cancer Several different factors could participate in prostate cancer cells in vitro. In vivo, neuroendocrine-like cells can promote development, progression, and resistance to antitumor ther- androgen-independent LNCaP xenograft growth in castrated apy. One of such possible mechanisms involves intraprostate mice (10, 11), supporting their role in prostate cancer andro- gen-independent growth. One of the neuropeptides secreted by neuroendocrine-like Authors' Affiliations: Departments of 1Radiation Oncology and 2Micro- prostate cells is neurotensin (12), a 13 amino acid peptide, that biology, 3Molecular Assessment and Preclinical Studies Core, and 4Cancer has numerous physiologic effects (13) mediated predominant- Center, University of Virginia School of Medicine, Charlottesville, Virginia ly through its cognate high-affinity receptor, neurotensin Note: Supplementary data for this article are available at Cancer Research – Online (http://cancerres.aacrjournals.org/). receptor 1 (NTR1). Similar to other G-protein coupled recep- tors, stimulated NTR1 activates multiple pathways, namely, Dedicated to the memory of Dr. George P. Amorino. þ mobilization of intracellular Ca2 , production of cyclic AMP Corresponding Author: Jaroslaw Dziegielewski, Department of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, and GMP, and formation of inositol triphosphate, resulting in Charlottesville, VA 22908. Phone: 434-982-0076; Fax: 434-243-9789; important physiologic responses in both the central nervous E-mail: [email protected] system and periphery. However, it has been shown that doi: 10.1158/0008-5472.CAN-11-1646 neurotensin has significant stimulatory activity in several Ó2011 American Association for Cancer Research. human neoplastic tissues (13–15). For example, NTR1 is www.aacrjournals.org 6817 Valerie et al. expressed in 91% of invasive ductal breast cancer cases (16), Eagle's Media supplemented with 10% FBS (PC-3M), RPMI and its expression is associated with the grade and size of the supplemented with 5% FBS (LNCaP and C4-2B), or serum-free tumor and its invasive potential (17). Similarly, NTR1 is keratinocyte medium supplemented with EGF and bovine expressed in most colon cancer cell lines and primary tumors, pituitary extract (RWPE-1). Phenol red–free and serum-free especially in the highly invasive adenocarcinomas but not in RPMI mediumwas usedin allserum starvationexperiments. All normal adult colon cells (18). In addition, neurotensin and cell culture media and reagents were purchased from Invitro- NTR1 expression has been implicated in the invasiveness of gen. SR48692, a generous gift from Sanofi-Aventis, was dis- head and neck squamous cell carcinomas, and high levels of solved in dimethyl formamide (DMF) as a 2 mmol/L stock and NTR1 expression are a predictive marker for a poor prognosis stored at À80C. Antibodies specific for EGFR, EGFR phospho- (19). We and others have shown that NTR1 is expressed and tyrosine 992 and 845, and Src and Src phospho-tyrosine 416 activated in aggressive prostate cancer cells (15, 20, 21) but not were obtained from Cell Signaling Technology, pan-phospho- in normal prostate epithelial cells. In such prostate cancer tyrosine (Y20) from Abcam, and secondary antibodies conju- cells, stimulation with neurotensin increased mitogen–acti- gated to infrared dyes from LI-COR. SDS-PAGE electrophoresis, vated protein kinase and phosphoinositide-3-kinase activa- agarose gel electrophoresis supplies, and PCR reagents were tion (22) and epidermal growth factor receptor (EGFR), Src, obtained from BioRad. SMARTpool NTR1 and nontargeting and STAT5 phosphorylation (15, 22), resulting in enhanced siRNA were obtained from Dharmacon. Other miscellaneous DNA synthesis, cell proliferation, and survival. reagents were purchased from Sigma Chemicals. NTR1 signaling may not only be responsible for increased proliferation but also for the intrinsic radioresistance of Drug treatment and irradiation prostate cancer cells. Thus, inhibition of the NTR1 receptor Incubation with SR48692 was conducted at 37Cat1mmol/L and its downstream signaling represents a target to enhance and 24 hours before irradiation, except where noted. An the sensitivity of prostate cancer to radiotherapy. NTR1 can be equivalent volume of drug vehicle (DMF) was added to the selectively and efficiently inhibited by the commercially avail- control dishes (final concentration 0.01%). Drug-treated and able small-molecule antagonist, SR48692 (meclinertant; control cells were irradiated using a 220-keV X-ray irradiator Sanofi-Aventis; refs. 23–25). SR48692 is a nonpeptide antag- at a dose rate of 2 to 3 Gy/min and received a dose of 6 Gy, onist that binds preferentially to NTR1 and inhibits down- except where noted. All irradiations were carried out at room stream signaling events such as EGFR and Src activation. temperature, and the samples were returned to 37C (for SR48692 has been shown in vitro to be effective in inhibiting further incubation) or lysed on ice (to assess immediate proproliferative and prosurvival signaling in colon (26), pan- effects). creatic (27, 28), head and neck (19), and prostate cancer cells (15, 29). SR48692 has also shown promising activity in vivo in a Clonogenic survival assay small lung cancer mouse model (30). However, its potential The assays were conducted as described previously (32). In use as a radiosensitizer has never been tested. brief, exponentially growing cells were treated with SR48692 (or In the current study, we tested the hypothesis that inhibi- sham treated with vehicle) and irradiated. Cells were trypsi- tion of NTR1 by SR48692 would radiosensitize prostate cancer nized, rinsed, and counted, and appropriate numbers were cells and tumors. Our results show that, indeed, combined plated for the colony formation assay. After 10 to 14 days of treatment of SR48692 and radiation effectively kills cancer incubation, colonies consisting of more than 50 cells were cells in vitro and in vivo and suggest the need for clinical counted. All data points were determined in triplicate and testing to establish whether anti-NTR1 treatment combined experiments were carried out at least 3 times. The experimental with