On Human Endometrial Cancer Growth in Athymic Mice1

Vol. 7, 4149–4155, December 2001 Clinical Cancer Research 4149 Effects of the New Selective Estrogen Receptor Modulator LY353381.HCl (Arzoxifene) on Human Endometrial Cancer Growth in Athymic Mice1 Rita C. Dardes,2 David Bentrem, viously) EnCa101 endometrial tumors were treated with Ruth M. O’Regan, Jennifer MacGregor Schafer, Tam or Arzox, we observed a stimulatory effect of both compounds in these models. and V. Craig Jordan3 Conclusions: The results indicate that Arzox may be a Robert H. Lurie Comprehensive Cancer Center [R. C. D., J. M. S., good first-line agent, but it may be ineffective at controlling V. C. J.], Division of Hematology Oncology [R. M. O. ], and Department of Surgery [D. B.], Northwestern University Medical the growth of endometrial cancer after exposure to Tam. School, Chicago, Illinois 60611 Our data suggest that Arzox stimulates endometrial tumor growth to at least the same extent as Tam, thereby suggest- ing a limited role as a second-line agent for the patient on ABSTRACT Tam who develops occult endometrial cancer. Purpose: Arzoxifene (Arzox) is a novel benzothiophene analogue with selective estrogen receptor modulator activity similar to raloxifene. Arzox is being developed as a treat- INTRODUCTION ment for breast cancer and has a predominantly antiestro- Endometrial carcinoma is the most common malignancy of genic effect on the rodent uterus. Our objectives were to the female genital tract, and it is estimated to account for verify whether the novel selective estrogen receptor modu- approximately 36,100 new cases and more than 6,500 deaths in lator, Arzox, can be a good first-line agent and also be 2000 in the United States (1). Several risk factors for the effective at controlling the growth of endometrial cancer development of endometrial carcinoma are related to an ex- after exposure to tamoxifen (Tam). tended exposure to unopposed estrogen action, such as in nul- Experimental Design: We compared the effects of Tam liparity, late menopause, obesity, diabetes mellitus, estrogen and Arzox on the growth of estrogen responsive ECC-1 replacement therapy, and Tam4 treatment. Tam has a low inci- endometrial cancer cells in vitro, and we determined their dence of serious side effects, however, it has a partial estrogen antitumor effects on ECC-1 and EnCa101 endometrial car- agonist effect in the human uterus (2). The National Surgical cinoma growth in athymic mice. Adjuvant Breast and Bowel Project-P1 study showed that Tam Results: We observed that estrogen receptor protein does not increase the risk of endometrial cancer in premeno- expression is down-regulated by Arzox to the same extent as pausal women, but it does increase the risk by 3- to 4-fold in raloxifene, whereas 4-hydroxytamoxifen, the active metab- postmenopausal women (3). It is important to stress, however, olite of Tam, does not affect estrogen receptor protein levels. that the stage and grade of endometrial cancers observed in Tam and Arzox inhibit the growth of Tam-naı¨ve ECC-1 postmenopausal women were the same as in the general popu- tumors in athymic mice. However when Tam-stimulated or lation (4). estrogen-stimulated (which had been treated with Tam pre- The mechanism of uterine carcinogenesis caused by Tam is unknown. Two possible mechanisms have been proposed: (a) conversion of the drug to electrophilic metabolites that damage cellular DNA; and (b) an estrogen-agonist action on the uterus, promoting endogenous lesions. In vitro studies with human Received 4/25/01; revised 8/7/01; accepted 9/19/01. tissues or cells do not show significant DNA adduct formation The costs of publication of this article were defrayed in part by the by Tam in endometrium. However, in vivo studies have dem- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to onstrated contradictory results with DNA adduct formation in indicate this fact. human endometrium (5). It seems most likely that, in women 1 These studies were supported by NIH Specialized Programs of Re- treated with Tam, the increased incidence of endometrial cancer search Excellence in Breast Cancer 1P50 CA89018-01 (to V. C. J.), is related to its estrogenic effects promoting uterine cell prolif- Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior Schol- arship-Research Program from the Ministry of Education-Brasilia, DF, eration and stimulating the growth of preexisting endometrial Brazil (to R. C. D.), a grant from Eli Lilly, Indianapolis, Indiana (to cancers, because 5 years is not enough time to complete the V. C. J.), the generous support of the Lynn Sage Breast Cancer Foun- carcinogenic process (6). In addition, women with both previous dation of Northwestern Memorial Hospital, and the Avon Products estrogen replacement therapy exposure and higher body mass Foundation. 2 Present address: Department of Gynecology, Federal University of Saõ Paulo, 740 Botucatu CEP: 04023-900, Saõ Paulo-Brazil. 3 To whom requests for reprints should be addressed, at Northwestern University Medical School, Robert H. Lurie Comprehensive Cancer Center, 303 East Chicago Avenue, 8258 Olson Pavilion, Chicago, IL 4 The abbreviations used are: Tam, tamoxifen; ER, estrogen receptor; 60611. Phone: (312) 908-5148; Fax: (312) 908-1372; E-mail: vcjordan@ Arzox, arzoxifene; Ral, raloxifene; SERM, selective estrogen receptor ␤ nwu.edu. modulator; E2,17 -estradiol; 4-OHT, 4-hydroxytamoxifen. Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2001 American Association for Cancer Research. 4150 Arzoxifene and Human Endometrial Carcinoma human endometrial carcinoma. The association between occult endometrial cancer and breast cancer is well known (18–20). If this paradigm is correct, then it is essential to evaluate the potential endometrial safety of a new SERM in the laboratory. We have used several different laboratory models of endometrial cancer to represent the application of Arzox treatment in the clinic. (a) endometrial cancer (ECC-1) never exposed to Tam; (b) an endometrial cancer initially exposed to chronic Tam treatment but passaged in E2-treated athymic mice for 3 years (EnCa101-E2); and (c) EnCa101-Tam endometrial carcinoma, which is stimulated by Tam. These models represent: (a) patients never treated with Tam; (b) patients previously treated with Tam and now requiring a second-line endocrine therapy years after adjuvant Tam was stopped; and (c) patients treated with a second-line agent. Using our unique models of human endometrial carcinoma, we determine whether Arzox would be a viable agent for first- or second-line breast cancer in patients who develop occult endometrial cancer. Fig. 1 Structures of SERMs referred to in the text. MATERIALS AND METHODS The ECC-1 human endometrial cancer cell line was de- rived from a primary well-differentiated, ER-positive endometrial adenocarcinoma (21). ECC-1 cells were maintained in index have the highest risk of Tam-associated endometrial can- flasks with phenol red containing DMEM supplemented with cer (6). 10% fetal bovine serum and 1% L-glutamine, 1% nonessential Tam (Fig. 1) is the endocrine adjuvant treatment of choice amino acids, 100 units/ml penicillin, and 100 mg/ml streptomy- for all ER-positive breast cancers (7). Not surprisingly, long- cin. Before experiments, cells were maintained in phenol red- term adjuvant Tam treatment does not control the recurrence of free DMEM with stripped fetal serum with the addition of 1% all ER-positive breast cancers, and drug resistance can develop L-glutamine, 1% nonessential amino acids, 100 unit/ml penicil- (8). In fact, some of these tumors seem to be stimulated by Tam lin, and 100 mg/ml streptomycin for at least 4 days. Cells were (9, 10). Several molecular and cellular mechanisms have been grown at 37°C in a humidified 5% CO2 atmosphere. proposed as contributors to the acquisition of this resistant Growth Assay. The ECC-1 cells were harvested after phenotype (11, 12), including the selection of ER mutants, trypsinization and seeded into each well of 24-well plates at a alterations in the intracellular pharmacology and metabolism, concentration of 1.5 ϫ 104 cells (in 1 ml of estrogen-free ligand-independent ER-mediated transcription, and perturbation media). The growth assay was performed as described previ- of the interaction of ER with corepressors of transcription. ously (22), with doses of compounds ranging from 10Ϫ10 to Ϫ6 However, most of these Tam-stimulated tumors retain expres- 10 M. After 5 days of treatment, media was aspirated, 1 ml of sion of the ER (13) and respond to a second-line endocrine 0.1ϫ solution of hypotonic calcium, magnesium-free Hanks’ therapy (14). In addition to this problem, concerns about the balanced saline solution was added to each well, and cells were uterine safety of Tam (3, 4) have naturally provoked a search for chilled at 4°C for 10 min. The cells were sonicated, and 100 ␮l new first- or second-line agents that control the growth of both of the sonicated cell solution was transferred to an assay tube. A breast and endometrial carcinomas. standard DNA curve was completed using a stock concentration Arzox is a new benzothiophene analogue that is structur- of DNA (20 ␮g of calf thymus DNA/ml in hypotonic calcium, ally related to Ral (Fig. 1). Like Ral, Arzox is classified as a magnesium-free Hank’s balanced saline solution with 0.2 mg/ml SERM, based on its differential estrogenic/antiestrogenic effects BSA). The cell solution was incubated for at least1hin1.5ml in vivo on estrogen-target tissues (15). Arzox was designed of 1:200 solution of Hoechst dye (Polysciences, Inc.) and PES to improve the bioavailability of Ral and provide sustained [2 M NaCl, 50 mM Na2HPO4,and1mM EDTA (pH 7.4)] per antiestrogenic blockade in the treatment of breast cancer.

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