Lack of Efficacy of the Statins Atorvastatin and Lovastatin in Rodent Mammary Carcinogenesis

Cancer Prevention Research

Ronald A. Lubet,1 Daniel Boring,1 Vernon E. Steele,1 J. Michael Ruppert,2 M. Margaret Juliana2 and Clinton J. Grubbs2

Abstract The statins are highly effective in lowering cholesterol by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase. Recently, there has been conflicting epidemiologic data indicating that statins decrease the incidence of certain types of cancer, including breast cancer. Ator- vastatin and lovastatin, statins with different lipophicilities, were administered in diet either as single agents or in combination with suboptimal doses of tamoxifen or the retinoid X receptor agonist bexarotene were evaluated for prevention of estrogen receptor–positive mammary cancers induced in the rat with methylnitrosourea. Atorvastatin (125 or 500 mg/ kg diet) alone did not significantly alter cancer incidence or multiplicity. Suboptimal doses of tamoxifen (0.4 mg/kg diet) or bexarotene (80 mg/kg diet) reduced cancer multiplicity from 3.8 (control) to 2.9 and 0.9, respectively. Combining atorvastatin (500 mg/kg diet) with either of these effective agents minimally altered their efficacy. Although this dose of atorvastatin did not decrease serum triglyceride levels in control rats, it significantly decreased triglyceride levels that had been increased in bexarotene-treated rats. Experiments done with a second statin, lovastatin (100 and 400 mg/kg diet), yielded similar results: (a) limited activity when administered alone, (b) no obvioussynergywith bexarotene, and (c ) an ability to decrease bexarotene-induced increases in serum triglycerides. Thus, the statins had minimal activity in thismodel of mammary cancer in which approximately half of the cancersare mutated in the Ha Ras oncogene. Similarly, atorvastatin failed to alter the development of estrogen receptor–negative mammary carcinomas in a new animal model using bitransgenic mice (MMTV-Neu+/−/p53KO+/−), whereasbexarotene (250 mg/kg diet) waseffective.

The statins reduce serum cholesterol by inhibiting the Ki Ras, and proteins such as RHO A,B,C and CEBPE/F. Many upstream enzyme 3-hydroxy-3-methylglutaryl CoA (HMG- of these are either known to be or are hypothesized to contrib- CoA) reductase. Recently, there have been epidemiologic ute to the oncogenic process (8). In addition, the statins have reports that this class of agents may reduce the incidence of been shown, at least in vitro, to increase cellular levels of the cancer (1–3). Although perhaps the most convincing epidemi- two cyclin-dependent kinase inhibitors p21 and p27, which di- ologic data have been associated with colon cancer (2) and minish cell proliferation (7). prostate cancer (3), there are data implying that statins may The methylnitrosourea (MNU)–treated Sprague-Dawley also alter the incidence of breast cancer (4–6). One early hy- rats develop multiple estrogen receptor–positive (ER+) can- pothesis was that by inhibiting cholesterol synthesis, the sta- cers that have a histopathology (9) and gene expression profile + tins would decrease the production of farnesyl PPi and (10) similar to highly differentiated ER breast cancer in geranyl PPi (7). These metabolites are used in the prenylation women. Tumors in this model have proven to be highly sus- and activation of a wide variety of proteins. Included among ceptible to a wide variety of antihormonal agents (e.g., tamox- the prenylated proteins are the known oncogenes Ha Ras and ifen, aromatase inhibitors) that are effective against human ER+ breast cancer. In addition, the cancers are susceptible to other agents such as retinoid X receptor (RXR) agonists (11–13) and epidermal growth factor receptor inhibitors (14) Authors' Affiliations: 1Chemopreventive Agent Development Research Group, that may not work directly by inhibiting the hormonal axis. Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland 2 Unlike human breast cancer where Ras mutations are rarely and Departments of Surgery, Medicine, and Genetics, University of Alabama at observed, approximately half of the MNU-xinduced cancers Birmingham, Birmingham, Alabama Received 07/07/2008; revised 09/23/2008; accepted 11/20/2008. have Ha Ras mutations (15). Tumors with Ha Ras mutations Grant support: National Cancer Institute contract HHSN261200433001C are particularly sensitive to the preventive and therapeutic ef- and grant R01 CA127405. fects of the farnesyl transferase inhibitor tipifarnib (16). Be- Requests for reprints: Ronald A. Lubet, National Cancer Institute, Executive cause statins should decrease prenylation, the presence of Plaza North, Suite 2110, 6130 Executive Boulevard, Bethesda, MD 20852. Phone: 301-594-0457; Fax: 301-402-0553; E-mail: [email protected]. Ha Ras in 50% of MNU-induced mammary tumors (15) ©2009 American Association for Cancer Research. should have made this model particularly sensitive to preven- doi:10.1158/1940-6207.CAPR-08-0134 tion by statins.

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As indicated earlier, chemically induced cancers in rats are time period) at 63, 94, and 126 d after the administration of the car- primarily ER+ (17), whereas many of the mammary cancer cinogen to determine serum triglyceride levels. For the study that − models in transgenic mice yield ER tumors. The mouse mam- evaluated lovastatin, blood was collected only at termination of the mary tumor virus (MMTV)-Neu transgenic model uses over- study. In both studies, blood was collected from rats not receiving the carcinogen. After centrifugation of the blood, the serum was fro- expression of NEU under the control of a MMTV promoter to − zen at −85°C until it was analyzed for triglycerides (22). The Infinity induce the development of multiple ER mammary carcino- triglyceride assay kit was purchased from Thermo DMA. mas. However, the majority of the resulting tumors have a +/− +/− mutation in the transmembrane domain in Neu unlike most MMTV-Neu /p53KO mammary cancer model − − human Neu-expressing tumors (18). By making a bitransgenic Female MMTV-Neu+/ /p53KO+/ mice were placed on a diet sup- animal that expresses the MMTV-Neu transgene and that also plemented with atorvastatin (200 mg/kg diet) or bexarotene (250 mg/ has an alteration in the p53 tumor suppressor gene, the result- kg diet) beginning at 60 d of age. The numbers of mice of group were ing tumors overexpress Neu and have an altered p53 (19). as follows: controls, 31; atorvastatin, 27; and bexarotene, 28. The ani- However, the resulting tumors do not have a mutation in mals remained on the diet until the termination of the study (at 310 d the transmembrane domain. This lack of mutation in Neu of age). The mice were weighed weekly, palpated for mammary tu- − mors twice per week, and checked daily for signs of toxicity. At nec- and an alteration in p53 are characteristic of human ER tu- ropsy, all mammary lesions were removed and histologically mors that overexpress Neu. evaluated (20). In the present experiments, the ability of two structurally distinct statins (lovastatin and atorvastatin) to prevent the de- Statistical analysis + velopment of ER cancers in rats was evaluated. The statins Chemopreventive effects of the statins on cancer incidence and la- were tested either alone or in combination with suboptimal tency were determined using the log-rank analysis, and differences in doses of tamoxifen or the RXR agonist bexarotene. Both of cancer multiplicity were determined using the Armitage test (23, 24). these agents have proven to be highly effective in inhibiting The Student's t test was used to compare differences in animal body tumor development (multiplicity and incidence) in this model weights and serum triglyceride levels. when used at higher doses. In addition, we examined the ability of the statins to modulate serum triglyceride levels in nor- Results mal rats as well as in rats treated with bexarotene. Finally, the In an initial study (data not shown), the efficacy of lovastat- − efficacies of atorvastatin and bexarotene to prevent ER mam- in and atorvastatin against MNU-induced mammary cancers − − mary cancers in heterozygous MMTV-Neu+/ /p53KO+/ bi- were evaluated at relatively low doses: 100 and 125 mg/kg transgenic mice were also determined. diet, respectively. Because these dose levels did not alter tumor multiplicity or incidence, additional studies using higher Materials and Methods doses of the agents were done. Statin, tamoxifen, or the combination of these agents did not alter the body weight gain of Experimental animals and chemicals the animals in any of the studies >7% from the respective con- Female Sprague-Dawley rats were obtained from Harlan Sprague- − trols. Atorvastatin was given at 500 mg/kg diet beginning Dawley, Inc., at 4 wk of age. The bitransgenic (MMTV-Neu+/ /p53 − 5 days after MNU and continuing for the duration of the studies KO+/ ) female mice were generated in the Chemoprevention Center (Table 1; Fig. 1). The number of mammary cancers at the end at the University of Alabama at Birmingham. The p53-deficient line p53N5-T was purchased in a C57BL background (Taconic) and back- of the study in the MNU-treated-only group was 3.8 per rat. crossed at least five times onto a FBV/N background (The Jackson Atorvastatin caused a 30% increase in tumor number, whereas Laboratory). MMTV-Neu transgenic mice [strain FVB/N-Tg bexarotene (80 mg/kg diet) and tamoxifen (0.4 mg/kg diet) (MMTVneu) 202Mul/J] were purchased from The Jackson Laboratory. caused 76% and 24% decreases, respectively. The combina- − − − − For generation of MMTV-Neu+/ /p53KO+/ females, p53 / males tions of atorvastatin with bexarotene or tamoxifen did not were crossed to MMTV-Neu+/+ females (20, 21). All animals were alter the preventive efficacy from that observed when bexaro- housed in groups of five per cage in a room maintained at 22 ± 2°C tene or tamoxifen were given alone. The effects of the agents and artificially lighted 12 h/d. Atorvastatin, lovastatin, and bexaro- on serum triglyceride levels were determined at three time in- tene were obtained from the National Cancer Institute Prevention Re- tervals during the study (at 63, 94, and 126 days after initiating pository. Tamoxifen was purchased from Sigma Chemical Company, treatment in rats not receiving the carcinogen). As indicated in and MNU was from the National Cancer Institute Chemical Carcino- gen Repository. All agents evaluated for prevention activity were in- Table 2, bexarotene greatly increased triglycerides when given corporated into the diet by mixing with mash feed using a liquid-solid alone. Atorvastatin treatment did not reduce triglyceride le- blender with intensifier bar (Patterson-Kelly). vels in rats when given alone, but diminished the increase in triglycerides by bexarotene when the two agents were given in MNU mammary cancer model combination. Tamoxifen, as expected, did not modify trigly- When rats were 50 d of age, they were injected i.v. (via the jugular cerides either when given alone or in combination with ator- vein) with MNU (75 mg/kg body weight) as previously described vastatin. (13). Rats (15 per group) were given the statins, bexarotene, and/or In the second experiment, lovastatin (400 mg/kg diet) and tamoxifen (either as a single agent or in combination) beginning 5 d bexarotene (80 mg/kg diet) were given alone or in combina- after MNU. Rats were weighed weekly, palpated for mammary tu- tion to determine their effects on mammary cancer prevention. mors twice per week, and checked daily for signs of toxicity. The studies were terminated 126 d after MNU. Mammary tumors were In this study, the control group developed 3.6 cancers per rat removedatnecropsyoftheratsandexaminedhistopathologically with a mammary cancer incidence of 93% (Table 3; Fig. 2). as previously described (9). Lovastatin treatment caused a slight increase (19%) in cancer During the study that evaluated atorvastatin, blood (0.5 mL) was multiplicity. As seen with atorvastatin, the combination of lov- collected from the jugular vein of anesthetized rats (5 per group per astatin with bexarotene was no more or less effective than the

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Table 1. Effectsof atorvastatin, bexarotene, and tamoxifen (alone and in combination) in the prevention of MNU-induced mammary cancers

Group Carcinogen* Treatment† Mammary adenocarcinomas‡ Incidence (%) Multiplicity

1 MNU None 93 3.8 ± 0.7 2 MNU Atorvastatin, 500 mg/kg diet 93 4.9 ± 0.7 (29%↑)§,∥ 3 MNU Bexarotene, 80 mg/kg diet 60 0.9 ± 0.2 (76%↓)¶,** 4 MNU Bexarotene, 80 mg/kg diet plusatorvastatin,500 mg/kg diet 47 ¶ 0.9 ± 0.3 (76%↓)¶ 5 MNU Tamoxifen, 0.4 mg/kg diet 73 2.9 ± 0.8 (24%↓)∥,†† 6 MNU Tamoxifen, 0.4 mg/kg diet plusatorvastatin,500 mg/kg diet 73 2.2 ± 0.6 (42%↓)∥

*Female Sprague-Dawley ratsreceived MNU at 50 d of age. †Chemopreventive agentsadministeredwhen the ratswere 55 d of age. ‡Study terminated 126 d after MNU. §Valuesare mean ± SE ( n = 15 ratsper group). The number in parenthesisisthe percent difference from control group. ∥No statistical difference from group 1; P > 0.05. ¶Statistically different from group 1; P < 0.05. **No statistical difference from group 4; P > 0.05. ††No statistical difference from group 6; P > 0.05.

administration of the RXR agonist alone. Lovastatin also pre- the statins is the relatively recent and somewhat conflicting vented the large increase in serum triglycerides caused by bex- epidemiology reports regarding the efficacy of this class of arotene but did not alter these levels from controls when given agents in preventing breast cancer (4–7). In the present ex- alone (Table 3). periments, the statins were first examined as potential pre- Finally, atorvastatin (and a positive control bexarotene), ventive agents in a chemically induced model of mammary − − when given as single agents to MMTV-Neu+/ /p53KO+/ bi- carcinogenesis in rats. Each of the statins was initially tested transgenic mice, was evaluated for its preventive efficacy at lower doses (atorvastatin, 125 mg/kg diet, and lovastatin, − against ER mammary cancers. Other lesions (e.g., skin cancers, lymphomas) were less than three per group and were not related to treatment. As seen in Fig. 3, atorvastatin had minimal effects on the development of mammary cancers when compared with control bitransgenic mice, whereas bexarotene was effective in preventing the appearance of tumors (P < 0.05).

Discussion A carcinogen-induced model of mammary cancer in rats was developed five decades ago by Huggins and coworkers (17). The resulting mammary cancers appear histologically similar to invasive ductal adenocarcinoma in women. Recent gene array analyses of these tumors have shown them to be similar to highly differentiated human ER+ tumors (10). As expected, these tumors are responsive to a wide variety of agents that are effective in prevention and/or therapy of human cancers, including selective ERmodulators (SERM),aromatase inhibitors, and pregnancy (25, 26). Because of its relative ease of performance, this model has been used to screen for potential chemopreventive efficacy using the widest range of agents. Various nonhormonal agents (e.g., RXR agonists and, more recently, farnesyltransferase inhibitors and epidermal growth Fig. 1. Effectsof atorvastatin,bexarotene, and tamoxifen (alone and in factor receptor inhibitors) have all been shown to be highly combination) on the time of appearance of mammary cancersinduced with – active (11 14). MNU. The groups were as follows: atorvastatin (500 mg/kg diet), • ; bexarotene Lovastatin and atorvastatin are two small-molecule inhibi- (80 mg/kg diet), ▵; tamoxifen (0.4 mg/kg diet), □; atorvastatin (500 mg/kg diet) tors of HMG-CoA reductase that alter cholesterol metabo- plusbexarotene (80 mg/kg diet); ▴; atorvastatin (500 mg/kg diet) plus tamoxifen (0.4 mg/kg diet),▪; none,○. The latency of the cancersdeveloping lism (1). Both have KIs in the low nanomolar range when in the bexarotene and the atorvastatin plus bexarotene groups was used against purified HMG-CoA. The impetus for testing statistically different from the controls (P < 0.05).

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Table 2. Effects of atorvastatin, bexarotene, and tamoxifen on serum triglyceride levels in female Sprague- Dawley rats

Group Treatment Serum triglycerides (mg/dL)* 63 d 94 d 126 d

1 None 40 ± 5† 27 ± 3 65 ± 6 2 Atorvastatin, 500 mg/kg diet, 41 ± 9 25 ± 6 53 ± 12 3 Bexarotene, 80 mg/kg diet 211 ± 36‡ 150±24‡ 264 ± 34‡ 4 Bexarotene, 80 mg/kg diet plusatorvastatin,500 mg/kg diet 89 ± 17 § 69 ± 11§ 141 ± 23§ 5 Tamoxifen, 0.4 mg/kg diet 39 ± 4 47 ± 5 80 ± 9 6 Tamoxifen, 0.4 mg/kg diet plusatorvastatin500 mg/kg 24 ± 4 20 ± 3 49 ± 3

*Serum triglycerideswere determined at the indicated time periodsafter initiating treatment with the chemopreventive agents. †n = 5. Valuesare mean ± SE. ‡Statistically different from group 1; P < 0.05. §Statistically different from group 3; P < 0.05.

100 mg/kg diet) and found to have minimal effects on tu- However, the evaluation of how lipophilicity might influ- mor incidence and multiplicity. They were subsequently ence statin chemoprevention is complex. Lipophilicity rank- tested at higher, albeit nontoxic, doses (atorvastatin, 500 ing of compounds is routinely expressed by the use of mg/kg diet, and lovastatin, 400 mg/kg diet), which similar- either experimentally determined or computed log P values ly failed to significantly decrease cancer multiplicity (Tables (log of the n-octanol/water partition coefficient). Although 1 and 3). In fact, increases in tumor multiplicity of 20% to an approximate log P value is readily generated, extrapola- 30% were observed; however, these increases were not sta- tion of the measured log P in a highly complex organism is tistically significant. The results were somewhat unexpected fraught with questions. The log P value is premised on the because of one unusual characteristic of the model: ∼50% of basis of a neutral molecule partitioning between two sepa- the MNU-induced cancers have mutations in the Ha Ras rate but chemically homogenous static solvent systems. oncogene, specifically at codon 12 (15). We have previously When ionizable groups are present in a molecule, the pro- shown that the Ha Ras–mutated tumors are highly sensitive tonation state will change with pH. Therefore, the log P val- to the preventive and therapeutic activity of the farnesyl- ue must be adjusted to reflect the pH of the medium and transferase inhibitor tipifarnib (R115777; ref. 16). This is pre- sumably due to blocking of farnesylation of Ha Ras, which is necessary for activation of Ras proteins. Because statins would be expected to block the prenylation process upstream of an FTI inhibitor, one might expect these cancers to be sensitive to prevention by statins. Finally, atorvastatin − and bexarotene were tested as single agents in an ER model of mammary cancers in bitransgenic mice. The MMTV/ Neu model was developed almost 15 years ago by Mueller and colleagues (21). The specific bitransgenic mice used in this study were heterozygous both for the MMTV-Neu transgene and KO of p53. The resulting mice developed mammary carcinomas that overexpress Neu and have an al- − teration in p53 (19), which is similar to human ER cancers expressing Neu. As observed in the rat, no effect of atorvastatin on cancer formation was found but bexarotene (at the dose used) reduced tumor multiplicity by ∼60%. The efficacy of bexarotene is even more striking given that only 23% of the bexarotene-treated bitransgenic mice had developed a tumor at 310 days, whereas 50% of control mice had developed tumors at 274 days of age. We chose the two specific statins (lovastatin and atorvastatin) because they both have been commonly used in humans and because atorvastatin is considered relatively lipophilic Fig. 2. Effectsof lovastatinand bexarotene (alone and in combination) on the and lovastatin less lipophilic. Some investigators had pro- time of appearance of mammary cancersinduced with MNU. The groupswere posed that relatively lipophilic statins (e.g., atorvastatin) as follows: lovastatin (400 mg/kg diet),○ ; bexarotene (80 mg/kg diet),▵ ; lovastatin (400 mg/kg diet) plus bexarotene (80 mg/kg diet), □; none,• . The might exhibit significantly greater chemopreventive activity latency of the cancersdeveloping in the bexarotene and the lovastatinplus compared with less lipophilic statins (7). bexarotene groups was statistically different from the controls (P < 0.05).

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Table 3. Efficacy of lovastatin and bexarotene (alone or in combination) in the prevention of MNU-induced mammary cancers

Group Carcinogen* Treatment† Mammary Serum triglyceride levels (mg/dL)§ adenocarcinomas‡ Incidence (%) Multiplicity

1 MNU None 93 3.6 ± 0.5 23 ± 3 2 MNU Lovastatin, 400 mg/kg diet 93 4.3 ± 0.9 (19%↑)∥ 24 ± 4 3 MNU Bexarotene, 80 mg/kg diet 67 1.3 ± 0.3 (64%↓)¶ 225±18¶ 4 MNU Bexarotene, 80 mg/kg diet ± 67 1.2 ± 0.3 (67%↓)¶,** 97 ± 9 ¶,†† Lovastatin 400 mg/kg diet

*Female Sprague-Dawley ratsreceived MNU at 50 d of age. n = 15 ratsper group. †Chemopreventive agentsadministeredwhen the ratswere 55 d of age. ‡Study terminated 126 d after MNU. §Serum triglyceridesdetermined at the end of study( n = 5). Valuesare mean ± SE. ∥The number in parenthesis is the percent difference from control group. ¶Statistically different from group 1; P < 0.05. **No statistical difference from group 3; P > 0.05. ††Statistically different from group 3; P < 0.05.

the ionization constant of the acid. Thus, a standard log P tone and open acid can be interconverted spontaneously both value does not capture true lipophilicity of ionizable com- through pH-dependent chemical hydrolysis and enzymatic pounds such as the statins. The log D value (log D =log actions. Each individual form undergoes separate binding P− [1 + 10 (pH-pKa)] for an acid) is a more accurate measure and partitioning with specific lipophilicity influences operat- of true lipophilicity. However, even with the addition of pH, ing on each form. These many issues are illustrated in the factors such as multiple phases, temperature fluxes, specific Scheme and highlight the complexity involved in attempting drug receptors, transport mechanisms, multiple ionized spe- to correlate lipophilicity with tissue concentrations and relative cies, and drug metabolism are not modeled by the log P or efficacy. log D equation. Although atorvastatin and lovastatin given alone were in- Another factor that complicates interpreting the true influ- effective, we evaluated the combination of statins with other ence of lipophilicity on the activity of the statins in a biological agents with known preventive efficacy. This was to test for matrix is the ability of statins to equilibrate between a cyclic possible enhanced or decreased efficacy of the combinations lactone (non-ionized) and free acid (ionized) structure. The lac- of agents, as well as to determine whether the expected

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1 and 3, no increase in efficacy was observed when combining a suboptimal dose of bexarotene with either of the statins. Bexarotene and various RXR agonists strongly increase triglyceride levels (29). We, therefore, determined whether the combination of bexarotene and either lovastatin or atorvastatin would diminish the increase in triglyceride levels induced by bexarotene. As shown in Tables 2 and 3, both lovastatin and atorvastatin significantly decreased the bexarotene-induced increase in serum triglycerides. These studies also showed that the statins do not interfere with the efficacy of this RXR agonist. This is important because the present clinical use of bexarotene in cutaneous T-cell lymphoma has used this agent in combination with a statin. In summary, the statins were not effective by themselves in this model of breast cancer despite the fact that the doses were relatively high. In contrast, these doses were effective in de- creasing azoxymethane-induced colon cancer in rats (30). Our data demonstrating lack of efficacy of the statins in two mammary models is in contrast to a prior study examining statins in transgenic mice (31), which showed significant, albe- Fig. 3. Effectsof atorvastatinand bexarotene on the time of appearance of − − it limited, activity of lovastatin. The dose used was lower than mammary cancers occurring spontaneously in MMTV-Neu+/ /p53KO+/ . The groups were as follows: atorvastatin (200 mg/kg diet), □; bexarotene (80 mg/kg our dose on a mg/kg body weight basis. However, the meth- diet),○ ; none,•. Statistical analysis of tumor latency indicated no difference ods of administration (diet in the present study versus i.p.) between atorvastatin and controls but a statistical difference between make it difficult to directly compare because i.p. administra- bexarotene and controls( P < 0.05). The mean timesof tumor appearance for each of the groups were as follows: controls, 217 d; atorvastatin, 232 d; and tion circumvents typical concerns of absorption and hepatic bexarotene, 250 d. metabolism. Similarly, two articles examining the effects of statins on growth of grafted tumor cells (32, 33) also used i. p. administration of the statins and showed limited efficacy. This may contribute to the different results obtained in the physiologic effect of statins (decreased serum triglycerides) various studies. The present experiments, however, clearly show the lack of efficacy of these statins following dietary dos- was achieved. In these studies, the statins were combined in vivo with suboptimal doses of two agents, the SERM tamoxifen ing in two models of mammary cancer used routinely and the RXR agonist bexarotone. Tamoxifen is a SERM that in screening for preventive agents. functions as an ERα antagonist in the breast (27). Tamoxifen is highly effective in prevention of ER+ breast cancer, both Disclosure of Potential Conflicts of Interest in this animal model and in human cancer. As seen (Table No potential conflicts of interest were disclosed. 1), there was no improvement in efficacy by combining atorvastatin with a low suboptimal dose of tamoxifen. The second agent used was the RXR agonist bexarotene. Bexar- Acknowledgments otene and related RXR agonists have proven to be highly We thank Jeanne Hale and Mary Jo Cagle for editorial services; Tom + − effective in preventing ER tumors as well as ER mamma- Morgan, Bonnie Mould, and Caroline Kirkner for performing all aspects of the ry tumors in rodent models (11–13, 28). As shown in Tables animal studies; and Julie Gray for analytic determinations on diets and serum.

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