Combination Chemoprevention of HER2/neu-Induced Breast Cancer Using a Cyclooxygenase-2 Inhibitor and a Retinoid X Receptor-Selective Retinoid

Combination chemoprevention of HER2/neu-induced breastcancer using a COX-2 inhibitor and an RXR-selective retinoid

Powel H. Brown1, Kotha Subbaramaiah2, Amoi P. Salmon3,5, Rebecca Baker3,5, Robert A.Newman6, Peiying Yang6, Xi Kathy Zhou4, Reid P. Bissonnette7, Andrew J. Dannenberg2,and Louise R. Howe3,51 Breast Center, Departments of Medicine and Molecular and Cellular Biology, Baylor College ofMedicine, Houston, TX 770302 Department of Medicine, Weill Medical College of Cornell University, New York, NY 100653 Departments of Cell and Developmental Biology, Weill Medical College of Cornell University, NewYork, NY 100654 Public Health, Weill Medical College of Cornell University, New York, NY 100655 Strang Cancer Research Laboratory, Rockefeller University, New York, NY 100656 Pharmaceutical Development Center, MD Anderson Cancer Center, Houston, TX 770547 Department of Molecular Oncology, Ligand Pharmaceuticals Inc., San Diego, CA 92121

AbstractThe inducible prostaglandin synthase isoform cyclooxygenase-2 (COX-2) is overexpressed in ~40%of human breast carcinomas, and in precancerous breast lesions, particularly in association withoverexpression of human epidermal growth factor receptor 2 (HER2/neu). Experimental breastcancer can be suppressed by pharmacological inhibition or genetic ablation of Cox-2, suggestingpotential clinical utility of COX-2 inhibitors with respect to breast cancer. Importantly, severalclinical trials have found reduced colorectal adenoma formation in individuals administered selectiveCOX-2 inhibitors. However, such trials also identified increased cardiovascular risk associated withCOX-2 inhibitor use. The goal of this research was to test whether improved chemopreventiveefficacy could be achieved by combining submaximal doses of a selective COX-2 inhibitor and aretinoid X receptor-selective retinoid (rexinoid). The rate of HER2/neu-induced mammary tumorformation was substantially delayed by coadministration of the COX-2 inhibitor celecoxib (500 ppmin diet) and the rexinoid LGD1069 (10 mg/kg body weight; oral gavage) to MMTV/neu mice. Mediantime to tumor formation was increased from 304 days to >600 days (P<0.0001). The combinationwas substantially more effective than either drug individually. Similarly, potent suppression ofaromatase activity was observed in mammary tissues from the combination cohort (44% of control;P<0.001). Regulation of aromatase expression and activity by COX-derived prostaglandins is wellestablished. Interestingly however, single agent LGD1069 significantly reduced mammaryaromatase activity (71% of control; P<0.001) without modulating eicosanoid levels. Our datademonstrate that simultaneous blockade of COX/prostaglandin signaling and retinoid X receptor-dependent transcription confers potent anticancer efficacy, suggesting a novel avenue for clinicalevaluation.

Requests for reprints: Louise R. Howe, Ph.D., Department of Cell & Developmental Biology, Weill Cornell Medical College, Box 60,1300 York Avenue, New York, NY 10065, E-mail: [email protected].

NIH Public AccessAuthor ManuscriptCancer Prev Res (Phila). Author manuscript; available in PMC 2009 August 1.

Published in final edited form as:Cancer Prev Res (Phila). 2008 August ; 1(3): 208–214. doi:10.1158/1940-6207.CAPR-08-0021.

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KeywordsHER2/neu; Cox-2; breast cancer; celecoxib; RXR-selective retinoid

IntroductionThe inducible prostaglandin (PG) synthase cyclooxygenase-2 (COX-2) is strongly implicatedin breast neoplasia (1,2). COX-2 is overexpressed in approximately 40% of human breastcarcinomas, and in 60–80% of preinvasive ductal carcinoma in situ lesions (1,2). COX-2overexpression in breast carcinomas correlates with poor prognosis, and with severalassociated clinical variables, including overexpression of HER2 (human epidermal growthfactor receptor 2; also called neu and c-ERBB2) (3–8). Epidemiologic data are broadlyconsistent with a protumorigenic role for COX enzymes. Several studies have identifiedreduced breast cancer incidence associated with the use of COX-inhibiting drugs, includingaspirin, non-steroidal antiinflammatory drugs (NSAIDs), and selective COX-2 inhibitors (9–17).

Consistent with the human expression data, Cox-2 is also upregulated in rodent mammarytumors (18–22), and thus rodent breast cancer models have proven useful for analyzing thecontribution of Cox-2 to mammary neoplasia. Numerous studies have shown that experimentalbreast cancer can be suppressed by inhibiting Cox activity using either conventional NSAIDsor selective Cox-2 inhibitors (COXibs) (1,2). Given the observed correlations betweenCOX-2 and HER2/neu overexpression in human breast tumors (3–6), HER2/neu-driven modelsare of particular interest. Both we and others have shown that HER2/neu-induced tumorformation is significantly delayed by administration of the selective COX-2 inhibitor celecoxib(20,23). Furthermore, knocking out Cox-2 significantly decreases HER2/neu-inducedmammary tumor formation in mice (24). Conversely, transgenic overexpression of COX-2 issufficient to induce mammary neoplasia in multiparous mice, providing direct evidence of thein vivo oncogenicity of COX-2 (25). Mechanistically, the antitumor action of Cox-2 inhibitionhas been ascribed to induction of apoptosis, suppression of proliferation, and modulation oftumor cell invasiveness and immune surveillance (2). Additionally, genetic manipulations ofCox-2 gene dosage suggest an important role for Cox-2 in regulating both angiogenesis andthe activity of the estrogen synthase aromatase in breast tissues (24,26–28).

Together, the above datasets identify COX-2 as a potential target for inhibiting breastcarcinogenesis, stimulating the development of several clinical trials designed to evaluateCOX-2 inhibitors in breast cancer patients (http://www.cancer.gov/clinicaltrials). In supportof this approach, randomized clinical trials have shown that COX-2 inhibitors can reduce theincidence of colorectal adenomas (29,30). Less propitiously, some studies reported anincreased risk of cardiovascular events associated with COXib use, reducing enthusiasm forCOX-2 inhibitors as cancer chemopreventive agents (29,30). Nevertheless, the demonstratedrole of COX/PG signaling in neoplasia identifies this pathway as an important target. Thus, itbehooves us to identify anticancer strategies capable of suppressing this signaling axis withminimal associated toxicity. We hypothesized that enhanced anti-breast cancer activity anddiminished side effects could be achieved by using a COXib in combination with a drug froma distinct class. Such combination approaches offer the potential to achieve substantial cancersuppression while minimizing collateral toxicity by combining submaximal doses of two ormore agents (31–33). To test this concept, here we have assessed the chemopreventive efficacywith respect to HER2/neu-induced breast cancer of celecoxib in combination with the retinoidLGD1069.

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http://www.cancer.gov/clinicaltrials

Retinoids have been extensively evaluated as cancer chemopreventive agents (34,35). Earlyclinical trials established the utility of retinoids for preventing cancer, using Vitamin Aderivatives such as 13-cis-retinoid acid, all-trans retinoic acid and 9-cis-retinoic acid.However, these trials also identified unacceptable toxicity associated with high dose regimensof these naturally-occurring retinoids. Limiting toxicities include teratogenicity,hepatotoxicity, severe headaches, and mucocutaneous toxicity. More recently, compoundswith greater selectivity, and hence decreased toxicity, have been developed through increasedunderstanding of retinoid receptor biology (31,36). Two distinct classes of retinoid receptorhave been identified, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Whileboth RARs and RXRs are ligand-regulated transcription factors of the steroid/thyroid hormonereceptor superfamily, the two classes are distinguished by their differential ligand affinities,and by the spectrum of proteins with which they interact. Importantly, RXR-selective retinoids,or rexinoids, have markedly diminished toxicities relative to panretinoids and RAR ligands(37), and thus offer considerable promise as anticancer agents. Of particular interest, bothLGD1069 (bexarotene, Targretin) and LG100268 have robust chemopreventive efficacy withrespect to experimental breast cancer, including HER2/neu-induced tumorigenesis (31,36,38–40).

Here we have evaluated the combination of the selective COX-2 inhibitor celecoxib and theRXR-selective retinoid LGD1069 for preventing HER2/neu-induced mammary tumorigenesis.We show that celecoxib and LGD1069 in combination mediate greater-than-additivesuppression of mammary tumor formation in HER2/neu transgenic mice. Furthermore, paralleldecreases in mammary aromatase activity were observed, suggesting that aromatasemodulation may contribute to tumor suppression mediated by the celecoxib/LGD1069combination.

Materials and MethodsReagents and Chemicals

Celecoxib (Celebrex; 4-[5-(4-methylphenyl)-3-trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide) was purchased from LKT Laboratories, Inc. (St. Paul, MN). LGD1069(Targretin; bexarotene) was provided by Ligand Pharmaceuticals (San Diego, CA).

Mouse Experimental ProceduresFVB/N-Tg(MMTVneu)202Mul/J mice (MMTV/neu) were obtained from The JacksonLaboratory and bred to produce multiple litters. Virgin female offspring were used in threeseparate studies.

Study I—Study I has been previously reported (20), and tumor latency data from that studyare provided in Table 4 for comparative purposes only. To briefly recapitulate the study design,females were randomly assigned to one of two groups at three weeks of age (Vehicle orCelecoxib), both of which were fed Laboratory Autoclavable Rodent Diet 5010 ad libitum.The diet of mice in the Celecoxib group was supplemented with 500 ppm celecoxib.

Study II—In Study II, females were randomly assigned to one of four groups at eight weeksof age as shown in Table 1 (Group 1, Vehicle; Group 2, Celecoxib; Group 3, LGD1069; Group4, Celecoxib and LGD1069). All mice were fed AIN-76A diet (Research Diets Inc., NewBrunswick, NJ) ad libitum. The diet of groups 2 and 4 was supplemented with 500 ppmcelecoxib. Animals in groups 1 and 2 were gavaged five days per week with 100μl sesame oil(Croda, Inc., Edison, NJ). Animals in groups 3 and 4 were gavaged five days per week withLGD1069 (10 mg/kg body weight) in 100μl sesame oil. Mild dermatitis was observed in asubset of animals in each group, predominantly manifesting as auricular irritation and alopecia

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on dorsal surfaces in the neck and scapular region. Onset occurred between 7 and 15 monthsof age (Group 1, n=1; Group 2, n=2; Group 3, n=5; Group 4, n=6). Mouse weights weremeasured weekly. No significant difference in animal weight was observed in any treatmentgroup relative to the control group, indicative of the absence of drug-induced cachexia. Thus,all drug regimens were well-tolerated with minimal side-effects. Expression of the neutransgene was unaltered by any of the treatment regimens (data not shown).

Study III—In Study III, females were randomly assigned to one of two groups at three weeksof age (Vehicle or Celecoxib) and fed control AIN-76A diet or AIN-76A diet supplementedwith 500 ppm celecoxib, respectively.

In all three studies, mice were palpated twice weekly for mammary gland tumors, and the ageat appearance of the first tumor was recorded (tumor latency). In Studies II and III, animalswere sacrificed two months after initial tumor detection, and the number of tumors of at least0.5 cm diameter was scored (tumor multiplicity). Non-tumor-bearing animals were maintainedfor their natural lifespan, or up to 98 weeks, whichever was shorter. All animals wereeuthanized by CO2 asphyxiation, in accordance with the recommendations of the Panel onEuthanasia of the American Veterinary Medical Association. Mammary tissues were harvestedfrom sacrificed animals, snap-frozen in liquid nitrogen and stored at −80oC. Additionally, aportion of mammary tissues and tumors were formalin-fixed and embedded in paraffin. Tumorswere confirmed by histological evaluation. Non-tumorous mammary glands were used forsubsequent analyses.

Determination of Eicosanoid LevelsResected mammary glands were homogenized, and eicosanoids were extracted and assayedusing LC-MS-MS as previously described (41).

Assay of Celecoxib LevelsBlood was collected from 5.5 month old animals by retroorbital bleeding, and plasma levelsof celecoxib were assayed by LC-MS as previously described (20).

Aromatase Activity AssaysResected mammary glands were homogenized, and mammary aromatase activity was assayedby measuring the release of 3H2O from [1-3H]androstenedione as described previously (28).

Statistical AnalysisKaplan-Meier curves of tumor-free survival in control and drug-treated cohorts were comparedusing a log-rank test (two-tailed) (Prism 4.0 for Macintosh, GraphPad Software Inc., San Diego,CA). Poisson regression analysis was used to compare tumor multiplicity in each group, basedon the assumption that the number of tumors per animal would follow a Poisson distribution.Mean tumor multiplicity was compared in a pairwise manner, and P-values were corrected formultiple comparisons using Tukey’s method. Repeated measure ANOVA was used to comparethe mean mouse weights in each experimental group over time. Mammary eicosanoid data,shown as mean±SD, were compared by one-way ANOVA, and further analyzed by Dunnett’stest to effect comparisons between each treatment group and the control group adjusting formultiple comparisons. Aromatase activity data, shown as mean+SD, were evaluated forstatistical significance using one-way ANOVA, followed by pairwise testing using Tukey’smethod to adjust for multiple comparisons.

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Results and DiscussionCelecoxib and LGD1069 mediate greater-than-additive suppression of mammarytumorigenesis

The primary goal of this research was to compare the chemopreventive efficacy of the COX-2inhibitor celecoxib and the RXR-selective retinoid LGD1069 alone and in combination, in abreast cancer model. Our ultimate aim was to determine whether greater protection could beachieved by combining drugs from two distinct classes than could be achieved using eitherdrug individually. This combination chemoprevention approach offers the possibility ofincreased preventive efficacy with minimal associated toxicity through utilization ofsubmaximal doses of individual agents. Thus, drug doses for our study were selected based onthe following information. Celecoxib delays tumor formation in MMTV/neu mice whenadministered in food at either 500 or 900 ppm, while 1500 ppm celecoxib induces cachexiaand weight loss in this mouse strain (20,23). LGD1069 has dose-dependent activity in theMMTV/neu model over the range 10–100mg/kg body weight (40). Therefore, we selected 500ppm celecoxib and 10 mg/kg LGD1069 as submaximal doses for use in our study.

Virgin female MMTV/neu mice were randomly assigned to one of four experimental groupsat 8 weeks of age, as shown in Table 1. Tumor incidence was measured as a function of timein all four groups. The rate of formation of mammary tumors was significantly delayed in theLGD1069 cohort relative to the Vehicle group (Fig. 1, Table 2), consistent with the previouslyreported chemopreventive action of LGD1069 with respect to HER2/neu-induced breast cancer(40). Mammary tumors were detected in 50% of control animals by 304 days of age vs. 420days of age in LGD1069-treated mice (T50 = 304 days vs. T50 = 420 days; P=0.0085).Unexpectedly, celecoxib alone failed to delay tumor onset in this study (Fig. 1,Table 2), incontrast to previous reports (20,23). Failure to achieve comparable circulating celecoxib levelsin this study relative to our earlier study would provide a trivial explanation for the lack ofeffect of celecoxib in the present study. However, serum celecoxib levels in this experimentwere 2.8±0.7μM (mean±SD; range 2.2–3.6μM), and thus extremely similar to those achievedin our previous study (mean, 2.4μM) (20). We hypothesize that the variable protective efficacyof celecoxib between studies may be attributable to the use of different diets in each experiment(discussed below).

The combination of celecoxib and LGD1069 was markedly more effective at suppressingtumor formation than either drug alone (Fig. 1, Table 2). Tumor latency was more than doubledin the Celecoxib/LGD1069 cohort relative to the untreated group (Group 1, T50 = 304 days vs.Group 4, T50 > 600 days; P=0.0001). Tumor multiplicity was also significantly reduced in thecombination group relative to the control cohort (Table 2, P=0.041). Celecoxib, althoughineffective as a single agent in this study, substantially enhanced the delay in tumor onsetelicited by LGD1069 (LGD1069 alone, T50 = 420 days vs. LGD1069/Celecoxib, T50 > 600days; P=0.0125). Importantly, serum celecoxib levels were not elevated in the combinationgroup relative to the Celecoxib cohort, excluding the possibility that increased efficacyreflected elevated circulating celecoxib levels (Group 2, 2.8±0.7μM; Group 4, 1.6±0.8μM;P=0.12). A similar greater-than-additive antitumor effect of celecoxib and LGD1069 was alsoobserved in the C3(1)-SV40 T antigen breast cancer model (data not shown), in which we havepreviously demonstrated single agent activity of LGD1069 (42). Thus, enhanced suppressionof mammary tumorigenesis can be achieved by combining the selective COX-2 inhibitorcelecoxib and the RXR-selective retinoid LGD1069.

Drug-mediated peturbations of mammary eicosanoid levelsWe previously reported a reduction of approximately 50% in mammary PGE2 levels incelecoxib-treated mice (20). In the present study, celecoxib administration resulted in a modest

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34% decrease in mean mammary PGE2 levels and a 49% decrease in mean mammary PGD2levels (Table 3), although neither of these changes achieved statistical significance.Interestingly, substantially increased levels of leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12-HETE) were observed in mammary tissues from celecoxib-treated animals (Table 3). Mean levels of mammary LTB4 and 12-HETE were increased to337% and 183%, respectively, of those in mammary tissues from vehicle-treated mice(P<0.001 and P=0.11, respectively). These data suggest that a metabolic shunt is occurring inmammary tissues from celecoxib-treated animals, with arachidonic acid being metabolized by5-lipoxygenase to LTA4 and subsequent byproducts, and by 12-lipoxygenase to 12-HETE,rather than by Cox-2.

Although LGD1069 has been shown to downregulate Cox-2 expression both in vitro in humanbreast cancer cell lines and in vivo in mammary glands from MMTV/neu mice (43,44),LGD1069 alone was ineffective at modulating mammary PG levels in vivo (Table 3).Unexpectedly, LGD1069 coadministration appeared to suppress the celecoxib-mediatedarachidonic acid shunt to 5-lipoxygenase and 12-lipoxygenase. These data demonstrate thatLGD1069 has complex effects on eicosanoid metabolism, elucidation of which will requirefurther studies.

Mouse diet modulates susceptibility to celecoxib-mediated tumor suppressionThe failure of celecoxib to delay tumor formation in MMTV/neu mice in the present studycontrasts with previous data from our group and from other investigators (20,23). Threealterations in experimental design relative to our earlier study were implemented in the presentstudy, any of which could provide a basis for the discrepant observations. Firstly, the age atinitiation of drug administration was changed from three weeks in our first study (Study I) toeight weeks in the present study (Study II). Secondly, the diet was switched from 5010 mousechow to AIN-76A, a purified diet. Thirdly, all animals received 100μl sesame oil by gavagefive days per week. To evaluate the influence of timing of drug initiation on celecoxib efficacy,a third study was performed (Study III) in which animals were fed AIN-76A, as in Study II,and celecoxib administration was initiated at three weeks, as in Study I. Tumor latency in theVehicle cohort was similar to that observed in Study II, and the rate of tumor formation wasnot delayed by celecoxib administration (Table 4). Comparison of tumor latencies in controlcohorts in all three experiments suggests that the alteration in diet could account for the lackof concordance between these studies. Specifically, tumors formed substantially more rapidlyin the Vehicle cohort in Study I than in Study II or III (for example, T50 = 32 weeks in StudyI vs. T50 = 43 weeks in Study II; Table 4), suggesting that tumor formation is accelerated inMMTV/neu mice fed 5010 mouse chow compared with those fed AIN-76A diet, presumablydue to differences in nutritional constituents. Since tumor latency was similar in the Vehiclecohort in Studies II and III, but only animals in Study II received sesame oil, we infer thatsesame oil administration in Study II is unlikely to account for the increased tumor latencyobserved in Studies II/III relative to Study I.

Comparisons of tumor latencies in Vehicle-treated animals in the three studies, and of therelative chemopreventive efficacy of celecoxib, thus support the following conclusions. Firstly,the rate of mammary tumor formation is accelerated in MMTV/neu mice fed 5010 mouse chowrelative to those consuming AIN-76A purified diet (Study I vs. Studies II/III). Secondly,consumption of 5010 mouse chow is associated with susceptibility to celecoxib-mediatedchemoprotection against breast cancer (Study I vs. Studies II/III). Thirdly, initiating drugtreatment at three weeks of age in AIN-76A-fed mice is insufficient to confer susceptibility tocelecoxib-mediated protection (Study II vs. III).

Since HER2/neu-induced breast neoplasia is suppressed by either pharmacological inhibitionor genetic ablation of the PG synthase Cox-2 (20,23,24), increasing mammary PG levels would

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be predicted to accelerate HER2/neu-driven mammary tumor formation. We speculate that5010 chow may contain components that increase mammary eicosanoid levels. Consistent withthis notion, dietary administration of celecoxib reduced mammary PGE2 by only 34% inanimals consuming AIN-76A (Table 3), compared with the 47% reduction we previouslyreported for 5010-fed animals (20), suggesting that animals consuming 5010 chow may haveelevated basal mammary eicosanoid levels.

Celecoxib and LGD1069 suppress mammary aromatase activityOur next goal was to investigate potential mechanisms by which celecoxib and LGD1069 mightinduce greater-than-additive suppression of HER2/neu-induced mammary tumor formation.Substantial evidence supports a functional relationship between COX/PG signaling and theestrogen synthase aromatase. Correlations have been identified between expression ofcyclooxygenase enzymes and the aromatase cytochrome P450 in human breast cancers (45,46). These correlations are believed to reflect a causal relationship since PG signaling canstimulate transcription of the CYP19 gene encoding aromatase (28,47–51). Furthermore,aromatase activity is reduced in mammary tissues from Cox-2 knockout mice and, conversely,is increased by transgenic COX-2 overexpression (27,28). Therefore, we focused on aromatasemodulation as a potential effector of celecoxib/LGD1069-mediated chemoprotection.

Aromatase activity was compared in mammary tissues from all four experimental groups ofthe celecoxib/LGD1069 combination chemoprevention experiment (Study II). Celecoxib alonecaused a 17% decrease in mammary aromatase activity relative to that in the Vehicle cohort(P<0.001; Fig. 2). The magnitude of the celecoxib effect was substantially less than theapproximately 60% reduction in mammary aromatase activity we had previously observed inCox-2 knockout mice (28), consistent with the comparatively modest celecoxib-mediatedreductions in mammary PGE2 levels in the current experiment (Table 3).

Mammary aromatase activity in LGD1069-treated animals was reduced by 29% (P<0.001;Fig. 2). The rexinoid LG101305 has previously been shown to reduce transcription from theCYP19 promoter in cultured human breast adipose cells (52). Our data provide the first invivo correlate for these tissue culture data. The mechanism by which rexinoids suppressaromatase transcription has not yet been elucidated, but may be independent of prostanoidmetabolism since LGD1069 alone did not significantly affect in vivo mammary eicosanoidlevels (Table 3).

Strikingly, the combination of celecoxib and LGD1069 potently suppressed mammaryaromatase activity, causing a greater-than-additive reduction (56%, P<0.001 compared withVehicle, Fig. 2). The relative potency of the three drug regimens for depressing mammaryaromatase activity was: celecoxib/LGD1069 > LGD1069 > celecoxib. Since the drug regimensranked in the same order of efficacy with respect to tumor suppression, we speculate thatinhibition of aromatase expression may contribute to the observed chemopreventive activity.While frank tumors in MMTV/neu mice lack estrogen receptor (ER) expression, ER isexpressed in normal-appearing and hyperplastic MMTV/neu mammary tissues (40,53).Importantly, tumor formation in the MMTV/neu strain is antagonized by the selective ERmodulators (SERMs) arzoxifene and acolbifene, indicating that estrogen signaling contributesto tumorigenesis in this model (39). Thus, we conclude that drug-mediated alterations inmammary aromatase activity may contribute to the observed chemopreventive activity of thecelecoxib/LGD1069 combination.

ConclusionsThe major goal of this study was to compare the chemopreventive efficacy of celecoxib andLGD1069 in combination with that of each of the two drugs individually, in a breast cancer

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model. The MMTV/neu strain was used for this experiment, since we had previouslydemonstrated chemopreventive efficacy of both celecoxib and LGD1069 as single agents inthis model (20,40). We observed a potent, greater-than-additive action of celecoxib andLGD1069 in suppressing mammary tumor formation (Fig. 1). Interestingly, we detectedcorresponding decreases in mammary aromatase activity (Fig. 2), leading us to conclude thataromatase suppression may be a component of the anticancer action of the celecoxib/LGD1069combination.

Our data have potential implications for the clinical management of breast cancer. Combinationapproaches are thought likely to afford greater benefit than can be achieved using single agents,with the additional benefit of minimizing collateral toxicity (31–33). For example,simultaneous targeting of COX-2 and HER-2/neu, using celecoxib and anti-HER-2 antibodies,has been shown to inhibit experimental colorectal carcinoma growth more effectively thaneither agent alone (54). This combination approach has been extended to experimental breastcancer. Rexinoid/SERM combinations, for example, have striking synergistic efficacy inrodent breast cancer models (31,39). Furthermore, clinical trials are ongoing to evaluatecelecoxib in combination with aromatase inhibitors as neoadjuvant therapy. LGD1069 is FDA-approved for treating refractory advanced stage cutaneous T-cell lymphoma, and is currentlybeing evaluated in numerous clinical studies, including breast cancer-related trials.Additionally, several rexinoids with even greater RXR selectivity and hence diminished sideeffects have been developed, including LG100268 and UAB30 (31,36,55). These agents havedemonstrated cancer preventive activity and may be useful candidates for future clinicaldevelopment. The results of our present study suggest that drug combinations targeting COX/PG signaling and RXR-dependent transcription may have clinical utility for breast cancerprevention.

AcknowledgmentsPreliminary statistical support for this study was provided by Paul Christos.

Grant Support: This work was supported by NIH Grants R03 CA105458 (L.R.H.), R03 CA119273 (L.R.H.), R01CA078480 (P.H.B.), a Breast Cancer Alliance Young Investigator Grant (L.R.H.), the Breast Cancer ResearchFoundation (P.H.B., A.J.D.), and the Irving Weinstein Foundation, Inc. (L.R.H.).

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Fig. 1. Suppression of mammary tumorigenesis by Celecoxib and LGD1069Female MMTV/neu transgenic mice were subjected to the following drug regimens, startingat 8 weeks of age. Group 1 were fed control diet and gavaged 5 days per week with 100μlsesame oil (Vehicle; black squares). Group 2 were fed diet containing 500 ppm celecoxib andand gavaged 5 days per week with 100μl sesame oil (Celecoxib; yellow diamonds). Group 3received control diet and were gavaged 5 days per week with 10 mg/kg body weight LGD1069in 100μl sesame oil (LGD1069; red triangles). Group 4 were fed celecoxib-containing diet andgavaged with LGD1069 (Both; blue circles). Statistical comparisons of tumor latency wereeffected using the log-rank test, and data are shown in Table 2. The median time to tumorformation was significantly delayed by LGD1069 administration. Celecoxib alone wasineffective, but significantly increased the delay in tumor formation mediated by LGD1069.



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Fig. 2. Celecoxib and LGD1069 suppress mammary aromatase activityAromatase activity was measured in mammary glands harvested from mice from each of thefour experimental groups in the experiment shown in Fig. 1 (Study II). Aromatase activity inall three drug-treated groups was significantly lower than that in the control group (*; P<0.001).Celecoxib caused a modest reduction in mammary aromatase activity (83% of control).Aromatase activity was decreased to 71% in tissues from mice treated with LGD1069 alone,and to 44% in tissues from mice treated with both celecoxib and LGD1069. Aromatase activityin tissues from the celecoxib/LGD1069 combination group was significantly less than that insamples from animals treated with LGD1069 alone (‡, P<0.001). Enzyme activity is expressedas fmol/μg protein/h. Data shown are mean + SD, n=6 per group.



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Table 1

Experimental Groups

Group Diet, ad libitum Gavage, 5 days per week

1. Vehicle AIN-76A Sesame oil

2. Celecoxib AIN-76A containing Celecoxib (500 ppm) Sesame oil

3. LGD1069 AIN-76A LGD1069 in Sesame oil (10 mg/kg body weight)

4. Both (Celecoxib and LGD1069) AIN-76A containing Celecoxib (500 ppm) LGD1069 in Sesame oil (10 mg/kg body weight)


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Table 2

Effect of celecoxib and LGD1069 on tumor latency and multiplicity in MMTV/neu mice

Drug Regimen Median time to tumor formation, days P Tumor Multiplicity (mean ± SD) P

Vehicle 304 days (n=32) - 1.31 ± 1.00 (n=32) -

Celecoxib (500 PPM) 285 days (n=29) 0.26 * 2.03 ± 1.38 (n=29) 0.13 ‡

LGD1069 (10 mg/kg) 420 days (n=26) 0.0085 * 1.27 ± 0.87 (n=26) 1.0 ‡

Celecoxib and LGD1069 > 600 days (n=26) <0.0001 * 0.58 ± 0.78 (n=24) 0.041 ‡

0.0125 $ 0.068 #

*P value obtained by comparison with vehicle-treated cohort (log-rank test)

$P value obtained by comparison with LGD1069-treated cohort (log-rank test)

‡P value obtained by comparison with vehicle-treated cohort (Poisson regression, corrected for multiple comparisons using Tukey’s method)

#P value obtained by comparison with LGD1069-treated cohort (Poisson regression, corrected for multiple comparisons using Tukey’s method)


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Table 3

Effect of celecoxib and LGD1069 on mammary eicosanoid levels in MMTV/neu mice

Drug Regimen PGE2, ng/mg protein(P=0.09) *

PGD2, ng/mg protein(P=0.15) *

LTB4, ng/mg protein(P<0.001) *

12-HETE, ng/mg protein(P=0.19) *

Vehicle 1.29±0.50 2.86±1.55 0.070±0.029 7.85±2.99

Celecoxib 0.85±0.49 (P=0.46) ‡ 1.45±0.65 (P=0.17) ‡ 0.236±0.082 (P<0.001) ‡ 14.36±7.20 (P=0.11) ‡

LGD1069 1.50±0.93 (P=0.91) ‡ 2.63±1.82 (P=0.98) ‡ 0.097±0.049 (P=0.78) ‡ 9.04±5.55 (P=0.96) ‡

Celecoxib and LGD1069 0.62±0.37 (P=0.18) ‡ 1.53±0.57 (P=0.20) ‡ 0.102±0.064 (P=0.69) ‡ 9.79±4.31 (P=0.86) ‡

*P value obtained by one-way ANOVA analysis across the different treatment groups

‡P value obtained by comparison with vehicle-treated cohort (using Dunnett’s test and correcting for multiple comparisons)


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Table 4

Diet affects tumor latency and the protective efficacy of celecoxib in MMTV/neu mice

Experiment Mouse Diet (gavage) Age at drug initiation Tumor Latency

Vehicle Celecoxib

Study I $ 5010 (no gavage) 3 weeks 32 weeks 40 weeks *

Study II AIN-76A (sesame oil) 8 weeks 43 weeks 41 weeks‡

Study III AIN-76A (no gavage) 3 weeks 41 weeks 39 weeks ¶

$Data for Study I were previously reported in Howe et al. (2002) Cancer Research, 62: 405–7 (20)

*P=0.003, compared to vehicle-treated cohort

‡P=0.26, compared to vehicle-treated cohort

¶P=0.17, compared to vehicle-treated cohort


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Combination Chemoprevention of HER2/neu-Induced Breast Cancer Using a Cyclooxygenase-2 Inhibitor and a Retinoid X Receptor-Selective Retinoid