Cancer Therapeutics, Targets, and Chemical Biology Research

Inhibition of 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 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 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. 2011 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 , serotonin, , sparing normal tissue, thus minimizing radiation toxicity by -related , 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 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 2011 American Association for Cancer Research. human neoplastic tissues (13–15). For example, NTR1 is

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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 80C. 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 radiotherapy may increase local control of the tumor and results were fitted to standard linear quadratic dose–response decrease its metastatic potential. curves and corrected for effects induced by the drug treatment alone. The dose enhancement ratio (DER) was defined as the Materials and Methods ratio between the radiation dose in the absence of radiosensi- tizer and the dose in the presence of radiosensitizer resulting in Cell lines and reagents the same reduction of cell survival to 37% (DER37). PC-3M-luc-C6 (PC-3M) human prostate carcinoma cell line transfected with the luciferase gene was purchased from Xeno- Growth inhibition assay gen Corporation, which also provided luciferin. RWPE-1 (im- Cells (100,000 cells per well) were plated in 6-wells plates, mortalized normal prostate epithelial cells) cell line was allowed to attach, and treated with SR48692 for indicated obtained from American Type Culture Collection. These 2 cell times. Attached cells were collected at indicated time points lines were authenticated by the suppliers, and fresh-frozen and counted using trypan blue exclusion to discriminate live/ stocks were used for the experiments. The LNCaP (andro- dead cells, or the cell number was estimated by sulforhoda- gen-dependent human prostate carcinoma) and C4-2B cell mine B staining of cellular proteins. lines (androgen-independent human prostate carcinoma) were established and characterized by Dr. L. W. Chung at the Cell lysates, immunoprecipitation, and Western University of Virginia (31) and resuscitated from frozen stocks blotting prior to the experiments. All cell lines were maintained in a Cells were plated in 100-mm dishes (2 106 cells per 37 C/5% CO2 humidified chamber in Dulbecco's Modified dish),allowedtoattachovernight,andtreatedwithSR48692

6818 Cancer Res; 71(21) November 1, 2011 Cancer Research NTR1 Inhibition Sensitizes Prostate Cancer to Radiotherapy

(or sham treated) as indicated in the Results section. In vivo orthotopic human prostate cancer xenografts Following irradiation (6 Gy) and different postincubation Human PC-3M-luc-C6 cells were used in an orthotopic times, cells were washed with ice-cold PBS and harvested xenograft model in male athymic nude mice [NIH Balb/cAn on ice in CHAPS lysis buffer (0.6% CHAPS, 1% Triton X-100, NCr-nu (nu/nu); 5–6 weeks old]. Surgery was conducted to 50 mmol/L Tris, pH ¼ 8.0, 150 mmol/L NaCl, 2 mmol/L expose the prostate, and 20 mL of the tumor cell suspension EDTA) supplemented with protease and phosphatase inhi- (5 105 cells) was injected into the dorsolateral lobe of the bitors (Sigma; ref. 15). Lysates were clarified by centrifuga- prostate gland. Following closure, mice were treated with tion, and protein concentrations were quantified. To assess SR48692 (25 mg/kg in PEG800) or vehicle control for 5 whole-cell protein expression, equal amounts of protein consecutive days, according to the schedule described in were resolved on 4% to 15% gradient gels, transferred to the Results section. Drug was administered orally in 0.2 mL nitrocellulose, and probed with specific antibodies. Infrared volumes 4 hours prior to radiation treatments. Mice were then dye–conjugated secondary antibodies were used for signal anesthetized with a ketamine/xylazine mix, and a dose of 2.5 development, and quantification of signal intensity was Gy X-ray was given to the prostate area while shielding the conducted using a LiCOR Odyssey imager and software. body with lead (34). Animals were imaged on a weekly basis by For immunoprecipitation, 1 mg of precleared protein lysate anesthetizing with isoflurane before and during imaging and was incubated overnight at 4C with EGFR antibody (5 mL, injecting intraperitoneally with luciferin (a substrate for immunoprecipitation-specific mAb; Cell Signaling) or a luciferase) at 150 mg/kg in a volume of 0.1 mL (35). Animals negative control antibody, with constant rocking. Protein were imaged at a peak time of 10 to 20 minutes post-luciferin A/G agarose beads were then added, and immunoprecipi- injection via a Xenogen IVIS instrument, using exposure times tates were washed 3 times with lysis buffer. Finally, samples and sensitivity settings to avoid saturation. Image processing were eluted with 2 sample buffer (100C/5 minutes) and was conducted using Living Image software (Xenogen) by subjected to Western blotting procedure as described region-of-interest analysis of total photons/sec for each above. tumor, with appropriate background subtraction.

RNA isolation and reverse transcription PCR Statistical analysis Total cellular RNA was isolated from exponentially growing All error bars represent the SEM from n ¼ 3 independent cells using the RNeasy Kit (Qiagen). Reverse transcription was experiments, unless indicated otherwise. P values were cal- carried out with 2 mg of total RNA using iScript reagent (Bio- culated using a one-factor ANOVA; values of P < 0.05 were Rad). The PCR amplification was carried out using 25 pmol of considered statistically significant. specific NTR1, NTR2, NTR3, and neurotensin primers as described elsewhere (ref. 33; NTR1, forward 50-TCATCGCC- Results TTTGTGGTCTGCT-30, reverse 50-TGGTTGCTGGACACGCT- GTCG-30; NTR2, forward 50-GTCTCCTCAGCTTCATCGTAT-30, SR48692 sensitizes PC-3M prostate cancer cells to reverse 50-TCCCCAAAGCCTGAAGCTGTA-30; NTR3, for- ionizing radiation ward 50-AGAATGGTCGAGACTATGTTG-30, reverse 50-AAGA- We tested the hypothesis that inhibiting the NTR1 receptor, GCTATTCCAAGAGGTCC-30; and neurotensin, forward 50- and therefore its downstream proproliferation and prosurvival ACTTGGCTTGTTAGAAGGC-30, reverse 50-TGTGGAGCTGG- signals, will enhance cell-killing effects of ionizing radiation in TATATTGTC-30) and JumpStart REDTaq polymerase prostate cancer cells. For initial experiments, we chose an mix (Sigma). The thermal profile consisted of initial denatur- androgen-independent and highly metastatic prostate adeno- ation at 95C for 5 minutes, followed by 30 cycles of dena- carcinoma cell line (PC-3M) and a nontumorigenic prostate turation at 94C for 30 seconds, annealing at 62C for 60 epithelial cell line (RWPE-1). Cells were treated with an NTR1 seconds, extension at 72C for 45 seconds, and by a final antagonist, SR48692, irradiated, and their colony-forming extension at 72C for 7 minutes. PCR products were resolved (clonogenic) ability assessed. At this concentration, SR48692 on a 1% agarose gel, stained with SYBR Safe, and quantified significantly (P < 0.01) enhanced radiation effects in PC-3M using the ImageJ Program (http://rsbweb.nih.gov/ij/). cells (Fig. 1A), resulting in a decrease in surviving fraction at 2 The NTR3-specific product was used to normalize the Gy (SF2) from 0.575 to 0.331, and a clinically relevant dose signals. enhancement ratio at 37% survival (DER37) of 1.77. As shown in Supplementary Fig. S1, SR48692-induced radiosensitization Caspase activation assay is dose and time dependent, reaching a maximum at 1 mmol/L Cells were plated in 96-well plates (10,000 cells per well), and 24 hours of treatment. Importantly, SR48692 pretreatment allowed to attach, treated with SR48692 (1 mmol/L) or sham did not sensitize normal epithelial RWPE-1 cells (Fig. 1B). treated (equal volume of DMF) for 24 hours, and irradiated (6 Gy). Following 24 to 48 hours of incubation, the activation of Prostate cancer cells, but not normal prostate epithelial cellular caspases 3 and 7 was measured by a fluorescence- cells, express NTR1 based assay (Apo-ONE Homogeneous Caspase-3/7 Assay; The results of Fig. 1A and B showed that SR48692-induced Promega). Multiple (6–12) wells per treatment condition were radiosensitization is specific to prostate cancer cells. Here, we used to obtain average fluorescence signals and normalized to confirmed that neurotensin receptors (NTR1, NTR2, and sham-treated controls. NTR3) are differentially expressed in normal and cancer

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A 1 PC-3MB 1 RWPE-1

0.1 0.1

Ctrl Ctrl SR SR Surviving fraction

0.01 0.01 02468 02468 X-ray (Gy) X-ray (Gy)

NTR1 NTR2 CD E NTR3 NT PC-3M RWPE-1 1.0 0.8 (bp) (kDa) NTR1 NTR2 NTR3 NT NTR1 NTR2 NTR3 NT RWPE-1 PC-3M 700 0.6 55 NTR1 500 0.4 55 300 35 Actin 0.2 12 12345678 0.0 mRNA expression (NTR3=1) mRNA expression PC-3M RWPE-1

Figure 1. Blocking NTR1 receptor sensitizes prostate cancer cells to ionizing radiation. A, clonogenic survival of PC-3M cells treated with SR48692 and radiation. Cells were incubated with 1 mmol/L SR48692 (SR) for 24 hours or left untreated (Ctrl), irradiated and replated for colony formation. Results are normalized for effects of the drug alone and fitted to a standard linear quadratic model. Data points are derived from at least 4 independent experiments (SEM). **, P < 0.01 denotes statistical significance compared with radiation only. B, clonogenic survival of RWPE-1 normal prostate epithelial cells treated with SR48692 and radiation. C, expression of NTR1 receptor protein in RWPE-1 and PC-3M cell lines assessed by Western blotting. D, expression of neurotensin receptors (NTR1, NTR2, and NTR3) and neurotensin (NT) mRNA in RWPE-1 and PC-3M cell lines assessed by semiquantitative RT-PCR. E, quantification of RT-PCR products from agarose gel electrophoresis (D). Expression of NTR3 was used to normalize signal intensities.

prostate cell lines. Fig. 1C shows that NTR1 protein, the of NTR1 radiosensitized prostate cancer cells (Supplemen- specific target of SR48692, is expressed in PC-3M cells but tary Fig. S2B) and reduced cell proliferation (Supplementary not in RWPE-1 cells. Surprisingly, the mRNA for NTR1 was Fig. S2A). Importantly, treatment with SR48692 did not present in both cell lines (Fig. 1D), although the level in RWPE- further increase the radiosensitivity of PC-3M cells with 1 was approximately 50% lower than in PC-3M cells (Fig. 1E). depleted NTR1 receptor. Both cell lines contained similar levels of mRNA for NTR3 (Fig. 1D, NTR3 lanes) but none for NTR2 (Fig. 1D, NTR2 lanes). SR48692 alone inhibits neurotensin-induced prostate Protein levels of NTR3 and NTR2 correlated with mRNA levels cancer cell growth (data not shown). NTR1 protein was also expressed in LNCaP, Existing literature suggests that NTR1 stimulation enhances C4-2B, and DU-145 prostate cancer cells at comparable levels prostate cancer cell growth, and inhibiting this pathway with (Supplementary Fig. S3). In addition, PC-3M expressed mRNA SR48692 reduces cell proliferation. Our results (Fig. 1) show that for the neurotensin/neuromedin gene, whereas the neuroten- SR48692 reduces clonogenic survival of cancer cells exposed to sin-specific reverse transcription PCR (RT-PCR) product was ionizing radiation; however, our preliminary experiments sug- absent in RWPE-1 (Fig. 1D, neurotensin lanes). This finding gested that the drug alone did not significantly affect cell was confirmed using an ELISA assay (Supplementary Fig. S4), survival under similar conditions. Thus, we attempted to where we detected significant levels of neurotensin in PC-3M reconcile these observations using PC-3M cells maintained cell culture medium but not in RWPE-1 or LNCaP culture under different conditions: exponentially growing cells in com- media. These observations suggest that autocrine stimulation plete medium (as used in radiosensitization experiments) and of prostate cancer cells occurs via endogenously expressed quiescent cells in serum-free medium (as used in stimulation/ agonist (neurotensin), which stimulates expressed NTR1. proliferation experiments; refs. 15, 22). As shown in Fig. 2A, To validate our hypothesis that the radiosensitizing neurotensin stimulated proliferation of serum-starved PC-3M effects of SR48692 are mediated through its interactions cells, whereas blocking the NTR1 receptor with SR48692 (con- with NTR1, we silenced the receptor mRNA expression in comitant with neurotensin stimulation) completely abrogated PC-3M cells using SmartPool siRNA (Dharmacon). The this response. Moreover, SR48692 pretreatment diminished results in Supplementary Fig. S2 show that knocking down neurotensin-induced EGFR and Src phosphorylation in

6820 Cancer Res; 71(21) November 1, 2011 Cancer Research NTR1 Inhibition Sensitizes Prostate Cancer to Radiotherapy

A B NT (min) SR 15 60 15 60 WB: Ctrl Ctrl 250 EGFR SR 1.50 130 pY845 NT 250 NT+ SR EGFR 1.25 130 pY992

250 EGFR IP: EGFR 1.00 130

0.75 72 Src 55 pY416 Cell number (relative) Cell number 72 0.50 Src PC-3M RWPE-1 55 12345

C SR

Ctrl EGF NT Ctrl EGF NT WB: 250 EGFR 130 pY845 250 EGFR

130 EGFR IP: D 123456 1 E 60

50

40 G Growth (24 h) 30 1 Surviving fraction Survival (14 d) Active S Cell (%) 20 G2/M 0.1 0.01 0.1 1 10 10 SR48692 (μmol/L) 0 Ctrl SR

Figure 2. Differential effects of NTR1 antagonist in serum-free versus serum-containing medium. A, SR48692 inhibits stimulatory effects of neurotensin in prostate cancer cells maintained in serum-free medium. PC-3M and RWPE-1 cells were serum starved for 48 hours, pretreated with SR48692 (SR; 1 mmol/L for 24 hours), stimulated with neurotensin (NT; 100 nmol/L for 24 hours) and proteins quantified by sulforhodamine B staining. Results are normalized to the untreated control, and data points are from 2 to 4 independent experiments (SEM). B, neurotensin-induced EGFR and Src phosphorylation in serum-starved PC-3M cells. Cells were lysed following 15 to 60 minutes of incubation with neurotensin (100 nmol/L) with or without SR48692 (1 mmol/L), cellular EGFR was immunoprecipitated (IP), and EGFR phosphorylation was assessed using phosphospecific antibodies (tyrosine 845, pY845; tyrosine 992, pY992). Src phosphorylation (tyrosine 416, pY416) was assessed using straight Western blotting (WB) of protein lysates. Total EGFR and Src levels were also assessed. C, under similar conditions (serum-free medium), neurotensin (100 nmol/L for 1 hour) does not induce EGFR phosphorylation in RWPE-1 cells lacking NTR1 receptor. EGF stimulation (100 ng/mL for 5 minutes) was used as a positive control. D, NTR1 antagonist has a minimal effect on growth and survival of prostate cancer cells maintained in complete growth medium. PC-3M cells were treated with indicated concentrations of SR48692 for 24 hours and counted (growth) or maintained continuously for 14 days. Established colonies scored (survival). E, SR48692 has no effect on cell-cycle progression in cells maintained in serum-containing medium. Exponentially growing PC-3M cells were treated with SR48692 (SR; 1 mmol/L for 24 hours), and the cell-cycle distribution was assessed by BrdUrd incorporation and DNA staining. *, P < 0.05; **, P < 0.01 denote statistical significance compared to control samples. serum-starved PC-3M cells (Fig. 2B). RWPE-1 normal prostate cell-cycle progression of PC-3M cells growing exponentially epithelial cells did not respond to neurotensin stimulation nor in complete medium (Fig. 2E). On the basis of these results, SR48692 treatment(s) (Fig. 2A and C) most likely because they we can also conclude that observed radiosensitizing activity in do not possess NTR1 receptors (Fig. 1C and D). cells growing in complete medium is not due to effects such as Strikingly, PC-3M cells grown in complete medium were cell-cycle perturbation and/or induction of cell death by the minimally affected by SR48692 treatment. Fig. 2D shows that drug alone. SR48692 minimally affected cell growth, and similar negligible effects were noted in long-term clonogenic survival assays SR48692 radiosensitizes androgen-sensitive cell lines (14 days colony formation). Even at the highest dose tested LNCaP and C4-2B (10 mmol/L), the drug effects did not exceed 30% inhibition. In To determine whether SR48692-radiosensitizing activity is agreement with these findings, SR48692 had no effect on confined only to androgen-insensitive PC-3M cells or is also

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0.481 to 0.336) following combined treatment are statistically AB1 1 significant (P < 0.05), although smaller in magnitude than the decrease in SF2 in PC-3M cells, whereas the DER37 (1.43) calculated for both cell lines is also smaller than for PC-3M cells. On the basis of these observations, we hypothesize that 0.1 0.1 inhibition of NTR1 radiosensitizes prostate cancer cells inde- pendently of their androgen receptor and p53 status. LNCaP C4-2B SR48692 affects EGFR phosphorylation in response to

Surviving fraction radiation or neurotensin 0.01 Ctrl 0.01 Ctrl SR SR It is known that radiation induces EGFR phosphorylation and activates downstream signaling pathways leading to 0246 0246 increased cell survival (36, 37). Therefore, we determined X-rays (Gy) X-rays (Gy) whether SR48692 pretreatment interferes with radiation- and/or neurotensin-induced EGFR phosphorylation. For short-term studies in complete medium, the SR48692-treated Figure 3. Inhibition of NTR1 radiosensitizes androgen receptor– PC-3M cells were irradiated, incubated for 5 minutes at 37C, expressing and androgen-sensitive prostate cancer cells. LNCaP (A) and lysed in CHAPS buffer. Total cellular EGFR was immuno- or C4-2B (B) prostate cancer cells were pretreated with SR48692 (SR; 1 mmol/L for 24 hours) or sham-treated (Ctrl), irradiated, and subjected precipitated, and Western blotting was carried out using to a colony formation assay. Data were gathered from at least 3 antibodies against EGFR phosphotyrosine 992 (pY992) and independent experiments (SEM); *, P < 0.05; **, P < 0.01 denote statistical total phosphotyrosine (pY20). Fig. 4A shows that under these significance compared with radiation only. conditions, radiation induced an increase in EGFR phosphory- lation as determined by both pY20 and pY992 phospho-specific present in androgen-sensitive prostate cancer cells, we tested antibodies. SR48692 alone did not affect EGFR phosphorylation LNCaP and its derivative, C4-2B, in clonogenic survival assays. levels; however, it reduced radiation-induced phosphorylation Both cell lines express androgen receptor and respond to to 83% (pY992) and 67% (pY20) of initial levels. androgen stimulation, unlike PC-3M, which is androgen re- Fig. 4B shows a modest increase in the phosphorylation ceptor negative and androgen insensitive. The difference levels of EGFR (pY845 and pY992) and Src (pY416) in serum- between the two cell lines is that C4-2B can grow in the starved PC-3M cells following an extended time course (15–60 absence of androgen, whereas LNCaP growth is androgen minutes) of irradiation alone (X) and significant increase with dependent. In addition to androgen receptor status, LNCaP combined treatment of radiation plus neurotensin (X þ NT). and C4-2B cell lines also differ significantly from PC-3M in The radiation-induced phosphorylation, observed 5 minutes their p53 status: Both express wild-type protein, whereas PC- postirradiation in Fig. 4A, was reduced to almost basal levels 3M cells are p53 null. As shown in Fig. 3A and B, both cell lines at 15 or 60 minutes following irradiation (Fig. 4B, X). With were sensitized to radiation by pretreatment with SR48692. combined treatment (Fig. 4B, X þ NT), EGFR and Src phos- The decreases in SF2 (LNCaP from 0.599 to 0.419, C4-2B from phorylation reached a maximum at 60 minutes, closely

AB5 min X (min) X+NT (min) SR SR Ctrl 15 60 15 60 15 60 15 60 WB: X+SR SR X Ctrl WB: 250 EGFR 250 130 pY845 pY20 250 130 EGFR Fold change: 1 0.71 5.3 3.5 130 pY992 250 EGFR IP: 250 EGFR EGFR 130 130 pY992 EGFR IP: Fold change: 1 0.98 1.7 1.4 72 Src 250 55 pY416 72 EGFR Src 130 55 1234 123456 7 89

Figure 4. SR48692 affects neurotensin- and radiation-induced EGFR phosphorylation. A, PC-3M prostate cancer cells were treated with SR48692 (SR; 1 mmol/L for 24 hours) and/or irradiation (X; 6 Gy), and cellular lysates were prepared after 5 minutes. Total tyrosine (pY20) and EGFR tyrosine 992–specific (pY992) phosphorylation were assessed in EGFR immunoprecipitates. Fold changes represent the mean of 2 independent experiments, with phosphospecific signal intensities normalized to total EGFR levels. B, serum-starved PC-3M cells were preincubated with SR48692 (SR; 1 mmol/L for 24 hours), stimulated with neurotensin (NT; 100 nmol/L for 1 hour) as indicated, and irradiated (X; 6 Gy). EGFR phosphorylation (Y845 and Y992), Src phosphorylation (Y416), and the expression of total EGFR and Src proteins were analyzed by immunoprecipitation and Western blotting.

6822 Cancer Res; 71(21) November 1, 2011 Cancer Research NTR1 Inhibition Sensitizes Prostate Cancer to Radiotherapy

Combined treatment of SR48692 with radiation elicits A X-ray synergistic antitumor activity against PC-3M tumor Ctrl 24h 48h 72h WB: xenografts in mice SR SR SR SR The orthotopic PC-3M human prostate cancer model was (kDa) DNA-PK used to test the hypothesis that SR48692 can be used as a 250 pS2056 radiosensitizing agent in vivo. The schematic outlines of the in 130 vivo experiments are presented in Fig. 6A and the results in PARP 72 Fig. 6B and C. In the first experiment, animals (8 per treatment 55 group) were randomized on day 5 postinoculation, and drug β-Actin 35 treatment/irradiation was started immediately thereafter. In 12345678 the second experiment, tumor development was monitored by bioluminescence for 14 to 21 days, and then, the animals with B 4 24 h similar tumor burden were randomized into treatment groups 48 h and treated. Treatment regimen was the same in both experi- 3 ments and consisted of 5 daily does of SR48692 (25 mg/kg) followed by 2.5 Gy ionizing radiation on days 3 and 5 (Fig. 6A). 2 Fig. 6B and C shows the tumor burden (measured as bioluminescence from PC-3M cells) in animals in experiments 1 and 2, respectively. In both experiments, SR48692 alone (SR) 1 had no significant effect on tumor progression, whereas radiation alone (X) was only partially efficacious. However,

Relative caspase 3/7 activity Relative þ 0 the combined treatment (X SR) showed the most prominent Ctrl SR X X+SR effect, significantly reducing tumor growth in the treated animals (X vs. X þ SR; P < 0.05). This reduction was especially Figure 5. SR48692 treatment enhances apoptosis in irradiated prostate noticeable when the combined treatment was used on animals cancer cells. A, PC-3M cells were treated with SR48692 (SR; 1 mmol/L for inoculated only 5 days before (Fig. 6B, experiment 1). The 24 hours) and irradiated (X; 6 Gy), and samples, including floating cells, reduced tumor burden was confirmed by visual examination were collected at specified times (24, 48, and 72 hours postirradiation). A of excised urogenital tracts (Fig. 6D, representative samples representative Western blot analysis shows full-size and cleaved PARP from experiment 2). and phosphorylated (pS2056) DNA-dependent protein kinase after indicated treatments. b-Actin was used as a loading control. B, caspase 3/7 activity in SR48692-treated (SR; 1 mmol/L for 24 hours) and -irradiated Discussion (X; 6 Gy) PC-3M cells was measured 24 and 48 hours postirradiation as described in Materials and Methods. Fluorescence signals were On the basis of the incidence of cancer recurrence and normalized to the fluorescence of sham-treated controls (Ctrl). The results were obtained in 2 independent experiments (6–12 intraexperimental radioresistance, improvements in radiation therapy, in gen- replicates). *, P < 0.05. eral, and in prostate tumor treatments, in particular, are urgently needed. In the present study, we show that inhibition following the kinetics of phosphorylation induced by neuro- of NTR1 is a novel method for radiosensitization of prostate tensin alone (Fig. 2B). In both cases, pretreatment with cancer. Our results show that combined treatment of SR48692, SR48692 completely abrogated this long-term EGFR or Src a selective inhibitor of NTR1, and ionizing radiation efficiently phosphorylation (Fig. 4B, SR lanes). kills cancer cells in vitro and significantly lowers tumor burden in vivo. In addition, combination of NTR1 antagonist and Pretreatment with SR48692 enhances radiation- radiotherapy is effective independent of cancer cell p53 and induced apoptosis in PC-3M cells androgen receptor status. Most importantly, the combined Treatment of PC-3M cells with a combination of SR48692 treatment provides selectivity between normal and cancer and radiation resulted in a significant increase in apoptosis cells. within 48 hours, as shown by the enhanced cleavage of PARP The magnitude of sensitization to radiation seen here with – and DNA-dependent protein kinase (DNA-PK) phospho-S2056 SR48692 in cell lines (Figs. 1 and 3, DER37 1.3 1.8) is lower (Fig. 5A, bottom bands). Both proteins are known to be targets than often observed with anticancer drug sensitization. Nev- for caspase 3–mediated degradation during apoptosis (38, 39). ertheless, the differential radiosensitization is significant as Consistent with this observation, there was a statistically compared with, for example, clinically used and experimental significant (P < 0.05) increase in caspase 3 and 7 activity in EGFR targeting sensitizing agents such as erlotinib (40) or cells treated with combined agents (Fig. 5B, X þ SR) when C225 (41, 42). Our in vivo experiments confirm that SR48692 compared with radiation-only treatment (Fig. 5B, X). It is treatment combined with radiation causes significant reduc- worth to note that, at the dose and times assessed, the drug tion in tumor growth (Fig. 6). alone in the presence of serum did not induce cellular We postulate that the differential expression of NTR1, its apoptotic death, in line with our previous results on the high expression in prostate cancer cells but not in normal effects of SR48692 on cell growth and cell cycle (Fig. 2D prostate epithelial cells (Fig. 1), is a major factor in the and E). selectivity of SR48692-induced radiosensitization. However,

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A Experiment 1 (B) Experiment 2 (C) InoculationBioluminescence imaging Inoculation Bioluminescence imaging SR48692 SR48692

0 7 14 21 28 35 0 7 14 21 28 35 Postinoculation (d) Postinoculation (d) Radiation Radiation B C 1E + 10 1E + 10 Ctrl Ctrl SR X 8E + 09 X 8E + 09 X + SR X + SR 6E + 09 6E + 09

4E + 09 4E + 09

2E + 09 2E + 09

Photon flux (photon/s) 0E + 00 Photon flux (photon/s) 0E + 00 0 1428425670 0 1428425670 Time postinoculation (d) Time postinoculation (d) D

Normal Ctrl X X + SR

Figure 6. Effects of NTR1 antagonist combined with radiation on the growth of PC-3M orthotopic xenografts in nude mice. A, schematic description of experiments 1 and 2 as detailed in the Results section. B, experiment 1: 5 days postinoculation mice were randomized, treated with SR48692 (SR; 25 mg/kg) for 5 consecutive days, and irradiated (X; 2.5 Gy delivered to prostate area only) on second and fourth days of treatment. Bioluminescence imaging was conducted on a weekly basis. Data points represent means from 8 animals in the group (SEM). *, P < 0.05 compared with radiation only (one-factor ANOVA). C, experiment 2: the presence and size of tumors were assessed 2 weeks postinoculation, and the mice were randomized into 4 treatment groups. Treatment was conducted during week 4 (as described for A). D, representative images (from C) of the urogenital system in nontreated (normal), PC- 3M inoculated (Ctrl), and treated (X and XþSR) mice. Samples were collected from mice euthanized 50 to 70 days postgrafting.

there are at least 3 different neurotensin receptors (NTR1, cancer selectivity of SR48692 combined with radiation. Our NTR2, and NTR3; ref. 43), therefore, one may ask whether results (Fig. 1D and Supplementary Fig. S4B) show that PC-3M SR48692-induced radiosensitization is related to the inhibition cancer cells express and secrete neurotensin, whereas normal of NTR2 and/or NTR3. In support of our hypothesis, SR48692 prostate cells do not. The autocrine loop in cells expressing has been shown to have a high selectivity for NTR1 and does both the receptor (NTR1) and its agonist (neurotensin) could not interact strongly with NTR2 or NTR3 (44, 45) or other significantly enhance survival after irradiation, and disruption unrelated receptors (44). In addition, the literature (33) and with SR48692 should result in radiosensitivity. It has been our results (Fig. 1D) show that NTR2 is not expressed in most reported that abdominal irradiation increases neurotensin prostate cells (including cell lines used in this study). The case secretion in rat ileum (47). Although we did not observe of NTR3 is more complicated—this receptor is ubiquitously similar effects in cell culture, it is plausible that it can occur expressed throughout the human body and highly expressed in vivo during prostate radiotherapy. in the studied cell lines (Fig. 1D). However, its activation is not Several possible mechanisms of NTR1-dependent radio- likely blocked by SR48692 treatments, as there are 2 orders of sensitization could be operative, including, but not limited K magnitude difference in d for binding to NTR1 and NTR3 (45, to, the disruption of cell-cycle progression, inhibition of DNA 46). NTR3 has been shown to cooperate with NTR1 in trans- damage signaling and/or repair, and enhancement of apopto- ducing the neurotensin signal (33, 45). Therefore, it is not very tic responses. Our results (Fig. 2B) clearly show that SR48692 probable, although still possible, that SR-induced radiosensi- has no effect on cell cycle of cancer cells growing in complete tization is a result of blocking neurotensin binding/stimula- medium, whereas under these conditions, radiosensitization tion of NTR3 receptor. However, the requirement of NTR1 in is observed. This rules out the cell-cycle perturbation as a SR48692-induced radiosensitivity was confirmed using siRNA- major factor in sensitization. Even though SR48692 has only mediated silencing of the receptor (Supplementary Fig. S2). On a minimal effect on PC-3M cell growth in complete medium, it the basis of these considerations, we believe that the observed can completely block neurotensin-induced EGFR and Src radiosensitizing effect is due predominantly to the inhibition phosphorylation (Fig. 4A) and significantly lower radiation- of NTR1. induced EGFR phosphorylation (Fig. 4A). It is known that In addition to the differences in NTR1 expression, the neurotensin functions as a prosurvival and mitogenic stimulus differences in neurotensin secretion could contribute to in cancer cells, operating mostly through EGF receptor

6824 Cancer Res; 71(21) November 1, 2011 Cancer Research NTR1 Inhibition Sensitizes Prostate Cancer to Radiotherapy

transactivation (15, 29). It has also been shown that AKT acts siveness of tumors in humans (13, 14, 16–19, 21, 26–28, 30). downstream of EGFR (48) as an important prosurvival and We hypothesize that the molecular mechanism of cancer- antiapoptotic factor in response to ionizing radiation (49, 50). selective radiosensitization induced by SR48692 could be Reducing the threshold to undergo apoptosis by interference similar in different cell/cancer types and mostly based on with apoptosis resistance pathways would be expected to differential NTR1 expression and its connection to prosur- sensitize tumor cells to ionizing radiation by conditioning vival and antiapoptotic pathways through EGFR and mito- them to induction of cell death. Our results support the gen–activated protein kinase signaling. Therefore, a hypothesis that NTR1 inhibition lowers, or completely abro- combined therapy, as proposed here for prostate tumors, gates, EGFR phosphorylation (Fig. 4), which results in an should be applicable to other cancers that express NTR1 increase in apoptosis following irradiation (Fig. 5). Interest- receptor. ingly, it has been reported that neurotensin acts as an anti- In summary, we have showed that SR48692 radiosensitizes apoptotic factor, protecting serum-starved breast cancer cells prostate cancer cells in vitro, enhances radiation-induced from apoptosis in vitro (51). It is possible that a similar apoptosis, and that a decrease in EGFR phosphorylation mechanism is involved in prostate cancer cell resistance to and signaling may be involved in this effect. As a whole, radiation-induced apoptosis and that blocking NTR1 abro- the data suggest the existence of a cross-talk between EGFR gates the effects of neurotensin. Additional studies are un- and NTR1 and, possibly, a method for prostate cancer to derway to delineate the molecular mechanism(s) responsible modulate responses to radiotherapy. Most importantly, the in for the induction of apoptosis by treatment with SR48692 vivo administration of SR48692 prior to radiotherapy resulted combined with radiation. in a significantly improved tumor response compared with the Previous reports have shown that SR48692 inhibits DNA individual agents in human prostate cancer orthotopic xeno- synthesis and proliferation in serum-starved (quiescent) and grafts. The results presented here propose that targeting NTR1 neurotensin-stimulated PC-3 cells (15, 22). We have confirmed receptor with a selective antagonist, such as SR48692, repre- it here and showed that the drug has a minimal effect on sents a promising new therapeutic strategy for radiosensitiza- cancer cells growing in complete medium with 10% FBS tion of prostate tumors. (Fig. 2). Only when combined with ionizing radiation, SR48692 activity became apparent and biologically significant. Disclosure of Potential Conflicts of Interest These results could also explain the failure of SR48692 in a single-agent clinical trial in patients with lung cancer. No potential conflicts of interest were disclosed. Although SR48692 has been shown to inhibit proliferation in vitro and tumor growth in vivo of small-cell lung cancer (30), Acknowledgments it did not show significant activity in the clinical trial The bioluminescence imaging was carried out using the resources provided (NCT0029095). Our observation could also provide a biological by the UVa Molecular Imaging Core Laboratory. basis for an additional mode of cancer-selective radiosensiti- zation in clinical practice. A systemic treatment with nontoxic Grant Support doses of SR48692, together with spatially targeted delivery of radiotherapy to the particular tumor site, could result in This work was supported, in part, by the Department of Defense PCRP grant W81XWH-08-1-0114 (for G.P. Amorino and J. Dziegielewski) and NASA grant minimal side effects and sparing of the surrounding normal NNX10AC13G (for J. Dziegielewski). The initial stage of the project was funded tissue. This, combined with the specificity afforded by the by UVA Cancer Center pilot project grant P30CA45579 (for G.P. Amorino). The IVIS Spectrum scanner was purchased with funds from the NIH-NCRR, differences in NTR1 expression between normal and cancer 1S10RR025694-01 (principal investigator: S.S. Berr). cells, would significantly improve the therapeutic index of The costs of publication of this article were defrayed in part by the payment prostate tumor radiotherapy. of page charges. This article must therefore be hereby marked advertisement in Recently, several groups have reported on the involvement accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of NTR1 receptor expression and neurotensin stimulation in Received May 12, 2011; revised August 8, 2011; accepted August 19, the proliferation of various cancer cell lines and aggres- 2011; published OnlineFirst September 8, 2011.

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