The Selective Estrogen Receptor Modulator Trioxifene (LY133314) Inhibits Metastasis and Extends Survival in the PAIII Rat Prostatic Carcinoma Model

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[CANCER RESEARCH 63, 6056–6062, September 15, 2003] The Selective Estrogen Receptor Modulator Trioxifene (LY133314) Inhibits Metastasis and Extends Survival in the PAIII Rat Prostatic Carcinoma Model

Blake Lee Neubauer,1 Ann M. McNulty, Marcio Chedid, Keyue Chen, Robin L. Goode, Mac A. Johnson, C. David Jones, Venkatesh Krishnan, Rebecca Lynch, Harold E. Osborne, and Jeremy R. Graff Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285

ABSTRACT that produce androgen ablation but do not induce hypercoagulation or increase the incidence of cerebrovascular accidents. Trioxifene (LY133314) is a selective estrogen receptor modulator Two scientific developments have raised the possibility of revisit- (SERM) with competitive binding activity against estradiol for estrogen ing the use of ER mechanistic-based therapies for treating PCa. First, receptor ␣ (ER␣) and antagonistic activity against ER␣-mediated gene SERMs possess agonist and antagonist activities that are dependent on expression. The PAIII rat prostatic adenocarcinoma (PCa) is an androgen receptor-negative, ER␣- and ER␤-positive, spontaneously metastatic ro- the specific tissue type and interactions of a specific agent with ER dent tumor cell line. After s.c. implantation of 106 PAIII cells in the tail, subtypes (7). Within this pharmacological class, individual SERMs s.c. administration of trioxifene (2.0, 4.0, 20.0, or 40.0 mg/kg-day) for 30 have differing antitumor activities in treating female malignancies. days produced significant (P < 0.05) inhibition of PAIII metastasis from The first-generation SERM, tamoxifen (Fig. 1b) has been widely used the primary tumor in the tail to the gluteal and iliac lymph nodes to treat and prevent breast cancer and osteoporosis, but clinical expe- (maximum nodal weight decreases, 86% and 88% from control values, rience in treating male disorders is limited. Second, a novel ER gene respectively). PAIII metastasis to the lungs was significantly inhibited by product, ER␤ (also known as ER␤1), was cloned from a rat prostatic trioxifene treatment. Numbers of pulmonary foci in PAIII-bearing rats cDNA library and is expressed in murine and human prostate (8–10). were significantly (P < 0.05) reduced by trioxifene administration in a ER␤ has been proposed to act as a ligand-activated tumor suppressor dose-related manner (maximal reduction, 98% from control values). Con- (11). The more recently characterized ER has been designated ER␤, tinual administration of the compound significantly (P < 0.05) extended whereas the classical ER characterized in female accessory sex organs survival of PAIII-bearing rats. Trioxifene inhibited the proliferation of is designated ER␣. PAIII cells at micromolar levels in vitro but did not slow growth of the primary tumor growth in the tail. Trioxifene administration also pro- Trioxifene (LY133314; [3,4-dihydro-2-(4-methoxyphenyl)-1-naphtha- duced regression of male accessory sex organs. In PAIII-tumor-bearing lenyl][4-]2-(L-pyrrolidinyl)ethoxyphenylmethanone, methansulfonic acid animals, trioxifene administration produced a maximal regression of 76% salt; Fig. 1c) is a SERM with activity in rat uterine bioassays (12). In for ventral prostate and 64% for seminal vesicle (P < 0.05 for both). these estrogen-supplemented female rats, the antagonist activity of triox- SERMs may be preferable to estrogens given their efficacy in experimen- ifene was comparable to that of tamoxifen. The inherent estrogenicity of tal PCa models and relative lack of side effects observed in clinical trials. trioxifene in immature female rats was approximately one-third less than Our data support the contention that trioxifene represents a SERM with that of tamoxifen (12). Trioxifene has demonstrated activity in dimethyl- potential antimetastatic efficacy for the treatment of androgen-indepen- benzanthracene-induced mammary carcinoma in rats (13) and human dent, metastatic PCa. breast cancer patients (14). Our laboratory first demonstrated that SERMs such as LY117018 (Fig. 1d; Ref. 15) and raloxifene (Fig. 1e) produce INTRODUCTION significant antiprostatic (16) and antitumor responses in male LW rats that had received s.c. injections of PAIII cells in the tail (17). The Estrogen administration has long been recognized as an effective antitumor activity of SERMs in rat models have recently been extended hormonal ablative therapy to treat disseminated PCa2 (1). However, these to cells and tumors of murine (18) and human origin (19). Given the antitumor effects may result from a centrally mediated decrease in tes- ability to surgically reduce primary tumor bulk and the lack of curative ticular androgens. Clinical responses to estrogens such as diethylstilbes- chemotherapy, antimetastatic agents may be useful to treat disseminated, trol (Fig. 1a) in human prostatic malignancies may not be entirely androgen-independent human PCa after hormonal ablation (20). The attributable to decreased circulating androgens. Doses of synthetic estro- PAIII adenocarcinoma in LW rats is a spontaneously metastatic tumor gens that are efficacious in treating human PCa do not consistently reduce that is useful to evaluate agents to treat disseminated PCa. When PAIII circulating testosterone to castrate levels (2–4). Indeed, estrogens may cells are injected s.c. into the tails of male LW rats, a reproducible, exert direct cytoreductive effects through ER (5) in PCa cells that con- time-dependent, sequential spread of the tumor through the gluteal and tribute to the observed clinical antitumor activity (6). Although estrogens iliac lymph nodes to the lungs is observed (21). The morphology of the such as diethylstilbestrol are effective in treating progressive, metastatic PAIII tumor resembles anaplastic lesions in humans, supporting its utility PCa, this utility is complicated by the risk of cardiovascular side effects. in evaluating cytotoxic and antimetastatic agents in advanced disease Consequently, the use of estrogens in treating advanced PCa has been (22–24). This report characterizes the expression of ER␣ and ER␤ superseded by luteinizing hormone-releasing hormone agonist analogues protein in PAIII rat PCa cells as well as the ER␣/ER␤ selectivity of binding and agonist activity of trioxifene in vitro. Our data show that Received 11/6/02; revised 6/16/03; accepted 7/21/03. trioxifene administration dramatically suppresses PAIII metastasis to the The costs of publication of this article were defrayed in part by the payment of page lymph nodes and lungs, extends survival, produces involution of male charges. This article must therefore be hereby marked advertisement in accordance with accessory sex organs, and provides additional rationale for the use of 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Cancer Research, Lilly SERMs to treat human PCa. Research Laboratories Drop Code 0546, Lilly Corporate Center, Indianapolis, IN 46285. Phone: (317) 276-5675; Fax: (317) 277-3652; E-mail: [email protected]. MATERIALS AND METHODS 2 The abbreviations used are: PCa, prostatic cancer; ER, estrogen receptor; SERM, ␤ selective estrogen receptor modulator; LW, Lobund Wistar; E2,17-estradiol; ERE, ␣ ␤ estrogen response element; AR, androgen receptor; PI, propidium iodide; rh, recombinant ER and ER Binding Assay. The ER competition binding assays with human. purified ER␣ and ER␤ followed modification of previously published proce- 6056

national, Inc., Temecula, CA), ER␤ (1:200 dilution; Upstate Biotechnology, Inc., Lake Placid, NY), AR (1:500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz Biotechnology, CA), and ␤-actin (1:2000; Sigma). Anti-mouse IgG, anti-rabbit IgG (Santa Cruz Biotechnology), and anti-sheep IgG (Upstate Biotechnology) secondary antibodies were used at a 1:2000 dilution. All Western blots were detected by chemiluminescence (Pierce, Rockford, IL) captured with the Lumi-imager and quantitated using the Lumi-Analyst software (Roche Molecular Biochemicals, Indianapolis, IN). PI Cell Proliferation Assay. PI (Sigma) for the proliferation assays was Ϫ stored at a stock concentration of 1.0 mg/ml of H2O( 4°C). Five hundred PAIII cells per well (passage 113–115) in 100 ␮l of growth medium (DMEM with 10% fetal bovine serum) were plated in each well of a 96-well plate. An additional 100 ␮l of growth medium containing either trioxifene or LY117018 was added to each well at 24 h after plating to give a final incubation volume of 200 ␮l. Fresh medium containing drug or DMSO control was added every 2–3 days for a total of 7 days. Replacement of the growth medium with 200 ␮l of PBS and 10-min centrifugation of the plates (2000 rpm) completed the preparation. A working solution of PI was prepared at a final concentration of ␮ ␮ 50 g/ml in distilled H2O. Twenty-five l of working solution was added to each well. Cells were lysed by placing the plates in a Ϫ80°C freezer overnight and thawing to room temperature in a 37°C incubator. Foil-covered 96-well plates were placed on a rotator for 30 min and analyzed using the Victor2 Fig. 1. Chemical structures of diethylstilbestrol (a), tamoxifen citrate (b), trioxifene fluorescent plate reader (Wallac). The plates were read from the bottom with (LY133314; c), LY117018 (d), raloxifene (LY156758; e), and toremifene (f). excitation at 500 nm and emission at 615 nm for 1 s. Raw data were obtained as light units and reported as percentage of control (JMP; SAS Institute, dures developed for cell and tissue lysates (25). The assays were run in buffer Cary, NC). containing 50 mM HEPES (pH 7.5), 1.5 mM EDTA, 150 mM NaCl, 10% PAIII Cell Culture and LW Rats. Dr. Morris Pollard (University of Notre ␮ 3 glycerol, 1 mg/ml ovalbumin, 5 mM DTT, 0.025 Ci/well of [ H]-E2 (118 Dame, South Bend, IN) supplied a stock culture of PAIII rat PCa cells at Ci/mmol, 1 mCi/ml; NEN Life Sciences, Boston, MA), and 10 ng/well of passage 107. This original stock culture was expanded through two passages human recombinant ER␣ or ER␤ protein (PanVera, Madison, WI). Trioxifene and stored in liquid nitrogen with 10% DMSO as a cryoprotectant. The cells and LY117018 (Fig. 1) were synthesized at the Lilly Research Laboratories were kept in liquid nitrogen as 1.0-ml aliquots at a minimum concentration of (Indianapolis, IN) and added at 10 concentrations ranging from 0.3 to 10,000 1.0 ϫ 106 cells/ml. PAIII cells were grown in antibiotic-free MEM with ␮ nM. Nonspecific binding was determined in the presence of 1.0 M E2 (Sigma, Earle’s salts (Life Technologies, Grand Island, NY) supplemented with 10% St. Louis, MO). The binding reaction (140 ␮l) was incubated4hatroom FCS (Hyclone Laboratories, Logan, UT). The cells were grown to confluency temperature and terminated with 70 ␮l of cold dextran-coated charcoal buffer. and harvested with use of 0.06 units/cm2 trypsin (2ϫ recrystallized grade; Dextran-coated charcoal buffer was prepared by adding 0.75g of charcoal Worthington Biochemical Corp., Freehold, NJ). (Sigma) and 0.25g of dextran (Amersham Pharmacia Biotech, Piscataway, NJ) The breeding stock of LW rats was a gift from Dr. Morris Pollard. LW rats per 50 ml of assay buffer. The incubation plates were mixed 8 min on an orbital were maintained as a closed colony at Harlan Industries (Cumberland, IN). shaker at 4°C and then centrifuged at 3000 rpm for 10 min at 4°C. A 120-␮l Male rats weighing 110–125 g were used. Two or three rats were housed in aliquot of the mixture was transferred to a 96-well, white, flat-bottomed plate screen-bottomed cages in a light-controlled environment (lights on: 6:00 a.m., (Costar, Acton, MA), and 175 ␮l of Wallac Optiphase Hisafe-3 (Boston, MA) lights off: 8:00 p.m.). Water and powdered Rodent Laboratory Chow 5001 scintillation fluid was added to each well. The plates were sealed and shaken (Ralston-Purina, St. Louis, MO) were supplied ad libitum. These investigations vigorously on an orbital shaker. Radioactivity was counted in a Microbeta were conducted under practices outlined for the care and use of laboratory

counter (Wallac, Turku, Finland) after an incubation of 2.5 h. The IC50, animals set forth by the NIH and the American Association for Laboratory ␮ 3 inhibition at 10 M, and Kd values for [ H]-E2 were determined by saturation Animal Care. ␣ ␤ binding to ER and ER . The IC50 values for compounds were converted to On day 0, animals were randomized into five or six groups (10 rats/group). Ki values by use of the Cheng-Prusoff equation and the Kd values determined All except one of these groups received injections of PAIII cells. Injections of by saturation binding assay (A-base software; IDBusiness Solution, Ltd., tumor cells and surgical procedures were done under light Metofane (Pittman- Guildford, United Kingdom). Moore, Washington Crossing, NJ) anesthesia. For each injection, 106 PAIII PAIII ER␣-ERE and ER␤-ERE Cotransfection Assay. PAIII cells were cells in a volume of 50 ␮l were injected s.c. into the dorsal surface halfway cultured as described above, plated in 6-well plates (25,000 cells/well) and between the base and tip of the tail with a 25-gauge needle. Control animals transfected using 6 ␮l/well Fugene-6 (Roche, Indianapolis, IN). Human ER␣ received saline injections. Rats not receiving injections of PAIII cells were or ER␤ cDNA was added to the cells with the human estrogen response designated the non-PAIII-bearing (NO PAIII) control group. These studies element ERE-luc (11) at concentrations of 0.5 ␮g/well each. Luciferase activ- were conducted for 28–29 days. In the dose–response studies, PAIII-bearing ity from PAIII cells exposed to trioxifene (0–10 ␮M in 0.1% DMSO) during experimental groups were administered trioxifene (2.0, 4.0, 20.0, and 40.0 the final 24 h of the 72-h transfection was normalized to renilla activity mg/kg) as 0.2-ml single daily s.c. injections. Daily injections were given in the expressed by cotransfected pRLSV40 plasmid. Chemiluminescent activity was morning (8:00 to 9:00 a.m.). Control groups received equivolumetric vehicle measured according to the manufacturer’s protocol (Dual-Luciferase Reporter injections. 1000 Assay System kit; Promega, Madison, WI) and expressed as relative light Trioxifene administered to PAIII-bearing rats was dissolved in ethanol and units compared with that of vehicle (0.1% DMSO) control. diluted to a final ethanol:peanut oil ratio of 1:10. The compound was stored in Western Blot Assays. Protein extracts were taken from PAIII cells and lightproof containers at Ϫ20°C before formulation and in lightproof bottles at analyzed by previously described Western blotting techniques (26). Protein room temperature during compound administration. Trioxifene for injection lysates from PAIII, LNCaP, and LNCaP-derived LNAI T1.16 cells (27) were was formulated for 2-week treatment intervals on a body-weight basis. Initial previously prepared with use of radioimmune precipitation cell lysis buffer dose formulations were calculated based on previously observed 2-week body (New England Biolabs, Cambridge, MA). For Western blots, 30–40 ␮gof weight gains in untreated rats. After 2 weeks of trioxifene treatment, doses in protein extract/lane was electrophoresed, transferred to polyvinylidene mem- each group were adjusted relative to changes in group body weight mean branes (Hybond-P; Amersham Pharmacia Biotech) using the XCell II Mini- values. Cell apparatus (Novex, San Diego, CA), and immunoblotted. The antibodies One-half of the rats in each treatment group were sacrificed on day 28. The used in these studies were as follows: ER␣ (1:250 dilution; Chemicon Inter- remaining animals were sacrificed the following day. Rats were sacrificed by 6057

differences among the group variances for the tail, gluteal and iliac lymph nodal, and pulmonary focal variables; therefore, Dunnett’s test was performed on the rank-transformed data, providing a nonparametric analysis (27).

RESULTS Trioxifene Binds to rhER␣ and rhER␤ Protein and Inhibits ER␣-mediated Luciferase Activity. The competitive activity of tri- 3 ␣ ␤ oxifene for [ H]-E2 binding to rhER and rhER binding are depicted in panels a and b of Fig. 2, respectively. Trioxifene competed with 3 ␣ [ H]-E2 binding to rhER with an IC50 of 203.49 nM and a Ki of 20.84 3 nM. The compound was less potent at displacing [ H]-E2 binding to ␤ rhER , with an IC50 of 1506.04 nM and a Ki of 144.85 nM. An ER␣- and ER␤-ERE-driven luciferase reporter assay was used

to assess the estrogenic activity of trioxifene in PAIII cells. E2 administration induced ER␣- and ER␤-ERE-mediated luciferase transcription in cultured PAIII cells at concentrations Ͼ10 nM and Ͼ100 nM, respectively. Trioxifene was a weak antagonist for ER␣-mediated luciferase transcription in PAIII cells, inhibiting reporter activity at concentrations Ͼ100 nM (Fig. 3). Trioxifene had no activity against ER␤-ERE-mediated luciferase transcription in PAIII cells at any of the concentrations studied. PAIII Cells Express ER␣ and ER␤ but Not AR. Cultured PAIII and LNCaP-derived LNAI T1.16 PCa cells expressed ER␣ (Fig. 4). PAIII cells expressed lower levels of ER␣ than their human PCa cell line comparators. ER␤ expression was detected in PAIII cells, albeit at lower levels than in the parental LNCaP cells. AR protein immunoreactivity was observed in LNCaP but not detected in PAIII rat PCa cells.

3 ␣ Trioxifene Inhibits in Vitro Proliferation of PAIII Cells. PAIII Fig. 2. Trioxifene (LY133314) competes with [ H]-E2 for binding to rhER and ␤ 3 ␣ rhER . In these experiments, trioxifene displaced [ H]-E2 binding for rhER (a) with an cells grown in culture were exposed to trioxifene at concentrations Ϫ10 Ϫ5 IC50 value of 203.49 nM and a Ki of 20.84 nM. Trioxifene was a less potent competitor for ranging from 10 to 3.0 ϫ 10 ␮M in a cell proliferation assay. 3 ␤ [ H]-E2 binding to rhER (b), with an IC50 of 1506.04 nM and a Ki of 144.85 nM. Values are expressed as percentage of DMSO vehicle and are representative of duplicate assays. Under these conditions, trioxifene inhibited PAIII cell proliferation ϭ ␮ For experimental details, see “Materials and Methods” section. (IC50 4.6 M). The positive reference SERM, LY117018 (11), was less active than trioxifene for inhibiting PAIII cellular proliferation ϭ ␮ (IC50 14.7 M; Fig. 5). In vivo administration of trioxifene failed to

CO2 asphyxiation, body weights was recorded, and tails were amputated 1 inch produce cytoreductive effects against primary PAIII tumor growth in from the base and weighed. The gluteal and iliac lymph nodes were also the tail (Table 1). excised and weighed. The lungs were removed and inflated through the trachea with ϳ5.0 ml of Bouin’s fixative. The lungs were placed in Bouin’s fixative for 24 h and stored in 70% ethanol. Pleural lesions on the lung surface were counted under a dissecting microscope by methods published previously (21). Effects of Trioxifene on Survival. The effects of trioxifene on PAIII- bearing LW rat survival were subsequently evaluated in studies using a 40.0 mg/kg-day dose of the compound. Rats were divided into six experimental groups. Five groups received injections of PAIII cells as described above, and one experimental group received injections of 50 ␮l of isotonic saline in the tail. Three of the PAIII-bearing experimental groups were administered trioxifene (40.0 mg/kg-day) as described above. After 28 days, the PAIII- and non-PAIII-bearing control groups and one of the three trioxifene-treated groups were sacrificed as described above. At this time, compound administration was discontinued in one group of trioxifene-treated PAIII-bearing rats. These animals were administered vehicle until death. The remaining trioxifene-treated group continued to receive the compound (40.0 mg/kg-day) until death. The rats were checked each morning, and moribund animals were sacrificed if their condition indicated that they would not live through the day. These animals were necropsied, and tissues were processed as described above. Statistical Analysis. Numerical data from the cell proliferation studies were expressed as mean percentage of control Ϯ SE from triplicate observa- Fig. 3. Trioxifene antagonizes ER␣- but not ER␤-mediated luciferase reporter activity tions during three separate experiments. Differences in the mean values among in PAIII rat prostatic adenocarcinoma cells. Human ER␣ or ER␤ cDNA was cotransfected different treatment groups were compared using Dunnett’s test (JMP; SAS with the human estrogen response element ERE-luc and pRLSV40 plasmid into PAIII Institute). Differences with P values Ͻ0.05 were considered statistically sig- cells. Firefly luciferase chemiluminescence was assayed in PAIII cells exposed to triox- ␮ nificant. The data from the in vivo experiments were analyzed using Dunnett’s ifene (0–10 M) during the final 24 h of the 72-h transfection. The firefly luciferase signal was normalized to renilla activity expressed in PAIII cells. Values are expressed as multiple comparison procedure. Dunnett’s test assumes equal group variances. percentage of DMSO-vehicle control-treated PAIII cells and are representative of tripli- Analyses with Bartlett’s test determined that there were significant (P Ͻ 0.001) cate assays. Bars, SE. For experimental details, see “Materials and Methods” section. 6058

bearing rats produced significant (P Ͻ 0.05) dose-related decreases in final-to-initial body weight ratios (Table 1). Maximum inhibition of body weight gain was 31% from nontreated controls at a daily trioxifene dose of 40.0 mg/kg-day.

DISCUSSION

The results of the present study demonstrate that the ER␣-selective Fig. 4. Western blot analyses of ER␣ and -␤ gene products and AR in 20 ␮l of PAIII SERM trioxifene inhibits the in vitro cellular proliferation and in vivo cell lysates containing 30–40 ␮g of protein extract/lane. The blots were reprobed with metastasis of the androgen-independent PAIII metastatic PCa cell line anti-␤-actin to control for loading and transfer. ER␣ and ER␤ were detectable at relatively in rats. PAIII cells express measurable levels of both the ␣ and ␤ low levels, but AR expression was not detected in PAIII cells. For experimental details, see “Materials and Methods” section. isoforms of ER and are AR negative. Unlike most human prostatic malignancies, PAIII and other rat metastatic PCa cells are AR negative (28, 29). Despite this phenotypic difference, the PAIII model has Antimetastatic Effects in PAIII PCa Tumor-Bearing LW Rats. utility for evaluating the potential role of ER␣ and ER␤ in mediating The PAIII prostatic adenocarcinoma metastasizes in a reproducible, tumor progression and metastasis. Our results with trioxifene confirm time-dependent manner through lymphatic channels from the tail to previously published findings of antimetastatic activity with the gluteal and iliac lymph nodes and subsequently to the lungs (21). LY117018 (15) and raloxifene (17) in this rat PCa preclinical model. Histological evaluation of tissues from untreated PAIII-bearing LW In dimethylbenzanthracene-treated female rats, trioxifene adminis- rats revealed a time-dependent expansion of neoplastic cellular foci in tered in the dose range used in our PAIII studies was less effective the subcapsular sinuses of the lymph nodes draining the tail (21). than tamoxifen in preventing the development of mammary tumors After 28 days, the lymph nodes of untreated rats contained large (13). The antimetastatic activity of tamoxifen in the PAIII model is masses of carcinoma cells. Although reduced in size, histological inferior to trioxifene.3 In contrast, the highest trioxifene dose tested in evaluation revealed that the lymph nodes trioxifene-treated PAIII- the PAIII model (40.0 mg/kg-day, or ϳ240.0 mg/m2) was comparable bearing LW rats were composed entirely of tumor cells and indistin- to an active dose of the drug (200.0 mg/m2) evaluated in human breast guishable from vehicle-treated control tissues.3 Trioxifene-treated rats cancer patients (30). Our data add to a growing body of evidence that exhibited significant (P Ͻ 0.05) regression of gluteal and iliac lymph suggests potential therapeutic utility of SERMs such as trioxifene in node weights relative to PAIII-bearing controls (Fig. 6). Maximum PCa patients with androgen-independent metastatic disease. A recent inhibition of PAIII lymphatic metastasis to the gluteal and iliac lymph publication demonstrated the AR-independent activation of caspase- nodes was 86 and 88%, respectively. In addition to suppressing lymph 9-related apoptotic activity of a related SERM, raloxifene, in LNCaP nodal metastases, trioxifene inhibited metastasis to the lungs. Lung cells. These data have provided part of the preclinical rationale for colony numbers were significantly reduced (P Ͻ 0.05) by trioxifene ongoing human clinical trials with raloxifene in PCa patients (19). administration. Maximum inhibition of pulmonary metastasis was Trioxifene is an active competitor for human ER␣, binding with 98% with a daily trioxifene dose of 40.0 mg/kg-day (Fig. 7). approximately 20-fold selectivity over the ER␤ isoform. The com- Trioxifene Extends Survival in PAIII-Bearing LW Rats. In the pound is antagonistic for ER␣-mediated luciferase activity in PAIII survival study, administration of trioxifene (40.0 mg/kg-day) for 28 cells at concentrations exceeding 100 nM. Trioxifene did not markedly days produced significant (P Ͻ 0.05) antimetastatic activity as evi- alter ER␤-mediated luciferase activity at any concentration tested in denced by reductions in gluteal (Ϫ94%) and iliac (Ϫ91%) lymph these same cells. Given these in vitro responses, trioxifene can be node weights (Fig. 8) and pulmonary foci counts (Ϫ87%; Fig. 9). considered a SERM with selective ER␣-antagonistic properties in a Trioxifene failed to reduce the primary tumor weights in the tail (data malignant prostatic epithelial cellular environment. PAIII rat prostatic not shown). PAIII-bearing rats administered trioxifene (40.0 mg/kg-day) for 28 days followed by vehicle treatment until death (mean survival, 50.11 Ϯ 1.32 days) lived significantly (P Ͻ 0.05) longer than vehicle- treated control animals (mean survival, 41.78 Ϯ 0.88 days; Fig. 10). Administration of trioxifene to PAIII-bearing rats from the day after PAIII cell injection until death significantly (P Ͻ 0.05) extended survival (mean survival, 64.44 Ϯ 2.99 days) beyond the vehicle control and trioxifene (28 days) plus vehicle treatment groups. In Vivo Accessory Sex Organ Regression and Testicular and Body Weight Effects. Trioxifene administration to PAIII-bearing rats for 28 days produced significant (P Ͻ 0.05) dose-related regression of normalized (for body weight changes) ventral prostatic weights with maximum inhibition of 76% (Table 1). Trioxifene also produced decreases in normalized seminal vesicular weights with maximum inhibition of 64% at a daily dose of 40.0 mg/kg-day. Trioxifene administration to PAIII-bearing rats produced significant (P Ͻ 0.05) increases in normalized (per 100 g of body weight) testicular weights (Table 1) over PAIII control levels. In these ani- Fig. 5. Trioxifene inhibits the in vitro proliferation of PAIII cells. PAIII cells were plated at a density of 500 cells/microplate well, and trioxifene or LY117018 was added mals, the absolute testicular wet weights were not significantly after 24 h of incubation. Fresh medium containing compound or DMSO was added every changed from control values. Trioxifene administration to PAIII- 2–3 days for a total of 7 days. PAIII proliferation was determined by PI staining of cellular DNA. Values represent the percentage of DMSO-treated control PAIII cell cultures, and data are representative of multiple experiments. Bars, SE. For experimental details, see 3 B. L. Neubauer, unpublished observations. “Materials and Methods” section. 6059

Table 1 Trioxifene effects on body weight gain, ventral prostatic and seminal vesicle involution, testicular weights, and primary tumor growth in the tails of male LW rats inoculated with PAIII cellsa Final/Initial body Tail weight Testicular weight Ventral prostatic weight Seminal vesicle weight Treatmentb weight ratio (g/100 g body wt) (g/100 g body wt) (mg/100 g body wt) (mg/100 g body wt) PAIII ϩ vehicle (8) 1.888 Ϯ 0.064 2.222 Ϯ 0.057 43.69 Ϯ 3.25 (1015.89 Ϯ 24.77)c 82.2 Ϯ 3.1 (185.0 Ϯ 4.7)c 77.4 Ϯ 3.0 (174.78 Ϯ 9.2)c No PAIII ϩ vehicle (9) 1.790 Ϯ 0.051 1.823 Ϯ 0.024d 46.73 Ϯ 1.32 (1004.0 Ϯ 56.97)c 90.0 Ϯ 4.1 (195.9 Ϯ 9.2)c 81.8 Ϯ 1.8 (178.22 Ϯ 5.7)c PAIII ϩ trioxifene (mg/kg-day) 2.0 (8) 1.459 Ϯ 0.046e 2.389 Ϯ 0.075 56.61 Ϯ 0.58e (964.12 Ϯ 17.61)c 60.9 Ϯ 2.6e (99.8 Ϯ 5.8)c,e 45.5 Ϯ 2.7e (75.4 Ϯ 6.0)c,e 4.0 (7) 1.371 Ϯ 0.037e 2.426 Ϯ 0.071 54.01 Ϯ 1.24e (934.00 Ϯ 58.28)c 51.9 Ϯ 6.8e (71.9 Ϯ 8.9)c,e 39.4 Ϯ 5.5e (55.2 Ϯ 7.4)c,e 20.0 (9) 1.357 Ϯ 0.039e 2.152 Ϯ 0.054 57.97 Ϯ 2.80e (888.00 Ϯ 21.25)c 44.3 Ϯ 5.1e (85.1 Ϯ 11.0)c,e 34.0 Ϯ 4.3e (64.4 Ϯ 8.4)c,e 40.0 (10) 1.296 Ϯ 0.021e 2.199 Ϯ 0.035 56.01 Ϯ 5.32e (873.25 Ϯ 90.44)c 29.9 Ϯ 2.4e (46.0 Ϯ 4.4)c,e 18.7 Ϯ 3.0e (28.9 Ϯ 4.8)c,e a For experimental details, see “Materials and Methods” section. b Number of observations in parentheses. c Mean Ϯ SE of the absolute organ wet weight (mg). d Significantly different from PAIII ϩ vehicle-treated controls (P Ͻ 0.05) by Dunnett’s test on ranked data. e Significantly different from PAIII ϩ vehicle-treated controls (P Ͻ 0.05) by Dunnett’s test.

LY117018 and raloxifene in PAIII tumors, may be mediated by the ER␣ antagonistic properties of these SERMs. In a limited series of human androgen-independent and metastatic PCa specimens, Bonkhoff et al. (34) demonstrated an association between increased levels of endogenous ER␣ and expression of the androgen-independent metastatic PCa phenotype. The antiproliferative effects seen with trioxifene in cell culture conditions were not extended in vivo to a reduction of primary PAIII tumor in the tail. The observed antimetastatic effects of trioxifene may result from the action of ER␣ as an antagonist to processes involved with tumor metastasis (34) rather than as an inhibitor of growth of the metastatic lesions. Although these studies lacked sufficient numbers of PAIII-bearing rats to demonstrate a statistical response based on Kaplan–Meier analysis,3 trioxifene-induced decreases in metastatic tumor burden were sufficient to significantly extend the life span of PAIII-bearing rats. Survival is the key preclinical end point for the assessment of a potential antimetastatic agent for human trials (35). These data support the contention that trioxifene treatment reduces lymphatic and Fig. 6. Inhibitory effects of trioxifene on the gluteal and iliac lymph nodal metastasis pulmonary tumor burden and extends life span, producing significant of the PAIII prostatic adenocarcinoma in male LW rats. LW rats received s.c. injections of 106 PAIII cells in the tail, and trioxifene (0–40.0 mg/kg-day) was administered by the statistical and meaningful clinical responses in tumor-bearing animals. s.c. route for 28 days beginning the day after tumor cell inoculation. Gluteal and iliac Despite the variability in untreated control absolute PAIII tumor significantly different from burdens, trioxifene produced consistent antimetastatic responses in ,ء .lymph nodes were dissected at necropsy and weighed PAIIIϩ vehicle (P Ͻ 0.05) by Dunnett’s test on ranked data. Numbers of observations are listed in parentheses. Bars, SE. For details, see “Materials and Methods” section.

adenocarcinoma cells express both ER␣ and ER␤ but are AR negative, as evidenced by the Western blot immunoreactivity described here and the lack of in vivo responsiveness to castration of the tumor-bearing host (15). Our data from PAIII cell lysates correlate with the findings of Lau et al. (31), who demonstrated variable expression of ER␤ and ER␣ in the AR-negative human PCa cell lines DU-145 and PC-3. We have demonstrated an approximate 50-fold difference in trioxifene concentrations needed to compete for ER␣ binding and ER␣- mediated luciferase gene activation as well as the antiproliferative effects of the compound in PAIII cell cultures. This differential potency between the biochemical and PAIII cellular responses may reflect the cellular trafficking of trioxifene or the potential for this agent and other SERMs to impinge on off-target-mediated cellular pathways at concentrations in excess of levels relevant to their binding to the two ER gene products. ER-independent high-affinity binding sites for SERM binding have been demonstrated in a variety of models (32, 33); however, the role of these binding proteins in mediating estrogenic and SERM cellular responses is poorly under- Fig. 7. Inhibitory effects of trioxifene on metastasis of the PAIII prostatic adenocarcinoma to the lungs of male LW rats. LW rats received s.c. injections of 106 PAIII cells stood. in the tail, and trioxifene (0–40.0 mg/kg-day) was administered by the s.c. route for 28 Trioxifene treatment significantly inhibited PAIII metastasis days beginning the day after tumor cell inoculation. Tumor foci were counted on the significantly different from ,ء .pleural surface of Bouin’s-fixed lungs removed at necropsy through lymphatic channels and reduced the spread of the tumor to the PAIIIϩ vehicle (P Ͻ 0.05) by Dunnett’s test on ranked data. Numbers of observations are lungs. One mechanism for the antimetastatic action of trioxifene, like listed in parentheses. Bars, SE. For details, see “Materials and Methods.” 6060

patient responses to the early-generation SERMs, toremifene and tamoxifen, when they were used to treat advanced androgen-independent PCa (36–44). In these trials, the early-generation SERMs were administered to patients whose disease may have progressed to the point where the tumors were refractory to therapy. In our studies, trioxifene was administered to PAIII-bearing LW rats beginning the day after tumor cell inoculation. The antimetastatic activity of other SERMs in the PAIII model is diminished when therapy is delayed.3 Early intervention with toremifene has been shown to prevent carci- nogenesis in TRAMP mice (18) and is presently being evaluated as a chemopreventive agent in presurgical patients with the diagnosis of high-grade prostatic intraepithelial neoplasia, a presumed PCa precur- sor lesion. Unlike toremifene, which increased rodent ventral prostatic weights, trioxifene produced regression of the ventral prostate and seminal vesicles consistent with its SERM activity in the male accessory sex organs (16). The degree of trioxifene-induced accessory sex organ regression was similar to the effects of comparable tamoxifen doses in rats.3 Trioxifene-induced reductions in rodent accessory sex Fig. 8. Inhibitory effects of trioxifene (40.0 mg/kg-day; 28-day interim analysis of organ weights raise the possibility that this agent might be useful in survival study) on the gluteal and iliac lymph nodal metastasis of the PAIII prostatic treating androgen-sensitive benign and malignant neoplasms in addi- adenocarcinoma in male LW rats. LW rats received s.c. injections of 106 PAIII cells in the tail, and trioxifene (40.0 mg/kg-day) was administered by the s.c. route for 28 days tion to androgen-independent metastatic PCa. Apparent increases in beginning the day after tumor cell inoculation. Gluteal and iliac lymph nodes were normalized testicular weights in trioxifene-treated rats were largely significantly different from PAIIIϩ vehicle ,ء .dissected at necropsy and weighed (P Ͻ 0.05) by Dunnett’s test on ranked data. Numbers of observations are listed in attributable to slight, nonsignificant organ weight decreases in the parentheses. Bars, SE. For details, see “Materials and Methods.” presence of diminished body weight gains. Dose-related reductions in final-to-initial body weight ratios observed in these pubescent rats treated with trioxifene, although being statistically significant and substantial (Ϫ31%), were not the result of systemic compound tox- icity. Estrogen agonists and SERMs produce significant, albeit non- toxic body weight reductions in rodents through their hypothalamic- mediated inhibition of feeding behavior (45, 46). Although trioxifene appeared to exert its antitumor activity in the PAIII model through ER␣, modulation of the related gene product, ER␤, may provide an additional approach to the treatment of PCa and other malignancies. Using human PCa xenograft models, Corey et al. (47) have proposed that the antitumor activity of estrogenic agents in PCa xenograft models is mediated exclusively through the ER␤ isoform. These issues will be resolved as ER␣- and ER␤-selective modulators become available for study in PCa cell and tumor models. The data presented here support the contention that trioxifene and related compounds could be useful in the treatment of androgen- independent metastatic PCa. Trioxifene was well tolerated and showed activity in human breast cancer studies (14). To the extent that

Fig. 9. Inhibitory effects of trioxifene (40.0 mg/kg-day; 28-day interim analysis of survival study) on metastasis of the PAIII prostatic adenocarcinoma to the lungs of male LW rats. LW rats received injections of 106 PAIII cells s.c. in the tail, and trioxifene (40.0 mg/kg-day) or vehicle was administered by the s.c. route for 28 days beginning the day after tumor cell inoculation. Tumor foci were counted on the pleural surface of Bouin’s- significantly different from PAIIIϩ vehicle ,ء .fixed lungs removed at necropsy (P Ͻ 0.05) by Dunnett’s test on ranked data. Numbers of observations are listed in parentheses. Bars, SE. For details, see “Materials and Methods.”

multiple in vivo studies. It should be noted that trioxifene-treated PAIII-bearing rats were not free of metastatic disease. Total lymphatic and pulmonary tumor burden was markedly reduced but not elimi- nated by 28 days of trioxifene treatment, and residual lesions persisted that were apparently resistant to the antimetastatic effects of the compound. On the basis of these studies and the findings of others, a Fig. 10. Extended survival of PAIII-bearing male LW rats administered trioxifene potential therapeutic strategy for PCa may involve the adjunctive use (40.0 mg/kg-day for 28 days or continuously until death). LW rats received s.c. injections of 106 PAIII cells in the tail, and trioxifene (40.0 mg/kg-day) or vehicle was administered of trioxifene or other SERMs in combination with hormonal ablation by the s.c. route beginning the day after tumor cell inoculation. Animals were treated with significantly different from PAIIIϩ ,ء .or cytoreductive therapy (19). trioxifene for 28 days or continuously until death ␣ vehicle (P Ͻ 0.05); #, significantly different from PAIIIϩ trioxifene (28 days; P Ͻ 0.05) Given the ER -selective pharmacological activity of trioxifene, the by Dunnett’s test. Number of observations are listed in parentheses. Bars, SE. For details, caveat imparted by the evidence is that there were no significant see “Materials and Methods.” 6061

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2003 American Association for Cancer Research. The Selective Estrogen Receptor Modulator Trioxifene (LY133314) Inhibits Metastasis and Extends Survival in the PAIII Rat Prostatic Carcinoma Model

Blake Lee Neubauer, Ann M. McNulty, Marcio Chedid, et al.

Cancer Res 2003;63:6056-6062.

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