Endometrial Profile of Tamoxifen and Low-Dose Estradiol ... · protect the endometrium from adverse...

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Published OnlineFirst January 26, 2010; DOI: 10.1158/1078-0432.CCR-09-1541

Cancer Therapy: Preclinical Clinical
Cancer
Research
Endometrial Profile of Tamoxifen and Low-Dose EstradiolCombination Therapy
Charles E. Wood1, Jay R. Kaplan1, M. Babette Fontenot2, J. Koudy Williams1, and J. Mark Cline1

Abstract

Authors' AMedicine, WNorth CarResearchLouisiana

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doi: 10.115

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Purpose: Combination estrogen + progestin therapy has been associated with increased breast cancerrisk in postmenopausal women. Selective estrogen receptor modulators (SERM) are potential alternativesto progestins, although the endometrial safety of estrogen + SERM co-therapies is not known. The goal ofthis study was to evaluate the endometrial profile of low-dose estradiol and the SERM tamoxifen aloneand in combination.Experimental Design: Twenty-four postmenopausal female cynomolgus macaques were randomized

by social group to receive placebo, low-dose micronized estradiol (E2; 0.25 mg/1,800 kcal), the SERMtamoxifen (Tam; 20 mg/1,800 kcal), or E2 + Tam for 4 months in a parallel-arm design.Results: Tamoxifen alone resulted in overlapping but distinct effects compared with E2. Both E2 and

Tam increased uterine weight and endometrial thickness, whereas only E2 increased endometrial prolif-eration. Morphologic effects were similar for Tam and E2 + Tam, which both induced stromal fibrosis andcystic change. Tamoxifen inhibited E2-induced proliferation and expression of genes related to cell cycleprogression while exhibiting mixed agonist and antagonist effects on gene markers of estrogen receptoractivity. The gene expression profile for E2 + Tam was distinct from either E2 or Tam alone but dominatedby the Tam effect for estrogen-regulated genes. Tam also attenuated E2 effects on both vaginal maturationand cervical epithelial height.Conclusions: These findings characterize a novel phenotype resulting from estrogen + SERM co-

therapy. The predominance of Tam effects on endometrial proliferation, morphology, and transcrip-tional profiles suggests that endometrial risks for E2 + Tam may be similar to Tam alone. Clin Cancer

Res; 16(3); 946–56. ©2010 AACR.

Estrogen exposure is an important risk factor for endo-metrial cancer (1, 2). Many key risk factors for endometrialcancer risk relate to lifetime exposure to endogenous estro-gens (2), and long-term unopposed estrogen therapy (ET)results inmarkedly increased risk of endometrial hyperplasiaand cancer in postmenopausal women (1). Progestogensprotect the endometrium from adverse estrogen-inducedeffects (1) and, for this reason, are given with estrogen incombined postmenopausal hormone therapy regimens.Progestogens lack similar protective effects against breastcancer, however. Results from the Women's Health Initia-tive randomized clinical trials (3, 4) and other observa-

ffiliations: 1Department of Pathology/Section on Comparativeake Forest University School of Medicine, Winston-Salem,

olina and 2Division of Behavioral Sciences, New IberiaCenter, University of Louisiana at Lafayette, Lafayette,

lementary data for this article are available at Clinical Cancernline (http://clincancerres.aacrjournals.org/).

ding Author: Charles E. Wood, Department of Pathology/Comparative Medicine, Wake Forest University School ofedical Center Boulevard, Winston-Salem, NC 27157-1040.

-716-1636; Fax: 336-716-1515; E-mail: [email protected].

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tional studies (5) indicate that long-term use of estrogen +progestin therapy (EPT) results in a modest but significantincrease in invasive breast cancer incidence among post-menopausal women, above that seenwith ET. This evidencehas generated interest in progestin alternatives that selec-tively block estrogen actions in both the endometriumand breast but not in other tissues such as bone, urogenitaltract, and brain.The most promising candidates for this application are

selective estrogen receptor modulators (SERM), which ex-hibit tissue-specific estrogen agonist and antagonist effects(6). Tamoxifen (Tam) is a first-generation SERMwidely usedin the treatment and prevention of estrogen-responsivebreast cancer. Tam metabolites competitively bind estrogenreceptors (ER) and inhibit the growth-promoting activity ofendogenous estrogens in the breast (6). In clinical trials, Tamtreatment decreases the incidence of ER-positive breast can-cer by 30% to 60% over 5+ years in women at high risk forthe disease (7, 8). However, Tam is also associated with ad-verse side effects related to estrogen deficiency, most notablymenopausal symptoms andurogenital atrophy (9–11),whichnegatively affect quality of life in breast cancer survivors andmany other postmenopausal women. These observationshave contributed to the idea that the combination of low-dose estrogen and Tam may provide a unique safety and

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Translational Relevance

The addition of a progestin to estrogen therapy hasbeen associated with increased breast cancer risk inpostmenopausal women. Recently, selective estrogenreceptor modulators (SERM) have been proposed asprogestin alternatives. Endometrial safety is a majorconcern, however, for both SERM and estrogen thera-pies. In this preclinical study, we investigate for the firsttime the endometrial profile of an estrogen + SERM co-therapy. Our findings show a dominant effect of theSERM tamoxifen (Tam) over oral estradiol (E2) onmea-sures of endometrial morphology, proliferation, andtranscriptional profiles, suggesting that long-term risksassociated with E2 + Tam may be similar to those seenwith Tam alone rather than E2 alone. This informationshould be useful for postmenopausal women who aretaking or considering estrogen + progestin therapy formenopausal symptoms and for future trials of estrogen +SERM co-therapies.

Endometrial Profile of Tamoxifen + Estradiol


therapeutic profile, particularly for women at high risk ofbreast cancer (12–15). Data from observational studies ofTam-treated women provide support for this idea, suggest-ing that Tammay reduce breast cancer risk even when givenalongside hormone therapy (16, 17).Tamoxifen and certain other SERMs elicit diverse and

poorly understood effects in the human endometrium.Tam-associated changes include increased endometrialthickening, stromal fibrosis, cystic change, and polyp for-mation (18, 19). In addition, Tam increases the incidence ofendometrial carcinoma from∼1 to 2 cases per 1,000womenper year (7, 20, 21) and of uterine sarcoma from 0.04 to0.17 cases per 1,000womenper year (22). Tam also inducescertain markers of ER activity in the endometrium (23, 24),suggesting that estrogen agonist activity may contribute toTam-associated cancer risk (25). Endometrial profiles ofTam and other SERMs given with ET are not well known,despite recent interest in these co-therapies (12–15, 26–31).Thepurposeof this studywas to evaluate the endometrial phe-notype of low-dose oral estradiol and Tam in the postmeno-pausal endometrium.

Materials and Methods

Study design and treatments. This study followedaparallel-arm design inwhich 24 ovariectomized female cynomolgusmacaques (Macaca fascicularis) with amean age of 14.7 ± 0.7 ywere randomized to receive one of the following four treat-ments for 4mo: (a) placebo (control; n = 6); (b)micronized17β-estradiol (E2; Estrace, Mylan Pharmaceuticals) at adose of 16.7 μg/kg body weight (0.25 mg/1,800 kcal; n =6); (c) the SERM tamoxifen (Tam; Nolvadex, AstraZenecaPharmaceuticals LP) at a dose of 1.3 mg/kg body weight(20 mg/1,800 kcal; n = 6); or (d) E2 + Tam (n = 6). Dose

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equivalents approximated a low ET dose of oral E2 in post-menopausal women (ref. 32; standard dose is 1.0 mg/d)and a standard maintenance dose of Tam following breastcancer diagnosis (7). In a previous study in this model, se-rum concentrations of 4-hydroxytamoxifen (one of theprimary active metabolites of Tam) for the 20 mg/1,800kcal dose were 5 ± 1 ng/mL, similar to those reported inwomen (33).Hormone treatments were given in standard control

diets with casein + lactalbumin as the protein source andmacronutrient composition based on a typical NorthAmerican humandiet. Other than E2 and/or Tam treatments,groupdietswere the same inmacronutrients, cholesterol, cal-cium, and phosphorus. Animals were fed 60 kcal/kg bodyweight (+10% extra to account for waste) twice daily. DailyE2 and Tam doses were scaled to 1,800 kcal of diet (the esti-mated daily intake for a U.S. woman) to account for differ-ences in metabolic rates between monkeys and humansubjects. All animals were originally imported from theInstitut Pertanian Bogor in Bogor, Indonesia, and housedin stable social groups of three to four animals each. Allanimals were considered multiparous based on historicaldata from the original breeding colony, in which >90% ofthe adult females have had 2+ live births, and on myome-trial evidence of prior pregnancy (expansion of venousadventitia).Macaques are anthropoid primates with a high overall

genetic coding sequence identity to humans, including im-portant genes related to cancer susceptibility (34). Priorwork from our lab and others has shown similarities be-tweenmacaque and human endometrial biology, includingresponses to exogenous estrogen and SERMs, sex steroid re-ceptor expression, and the presence of hyperplastic lesions(35, 36).All procedures involving these animals were conducted

in compliance with State and Federal laws, standards ofthe U.S. Department of Health and Human Services, andguidelines established by the Wake Forest University Ani-mal Care and Use Committee. The facilities and laboratoryanimal program of Wake Forest University are fully accre-dited by the Association for the Assessment and Accredita-tion of Laboratory Animal Care.Serum estradiol concentrations. To confirm dietary intake

of treatments, serum E2 concentrations were measured inblood samples collected by femoral venipuncture. Estradiolconcentrations were measured during each month of treat-ment by RIA using a commercially available kit and proto-col from Diagnostic Systems Laboratories (E2, DSL-4800ultra-sensitive). Assays were done at the Yerkes NationalPrimate Research Center Endocrinology Laboratory. Cali-bration standards ranged from 5 to 750 pg/mL.Endometrial tissue collection and processing. At the end of

the treatment period, animals were sedated with ketamineand euthanized using sodium pentobarbital (100 mg/kg,i.v.), as recommended by the Panel on Euthanasia of theAmerican Veterinary Medical Association. Euthanasia wasdone for data collection related to cardiovascular andbrain end points, to be described elsewhere. Uteri were

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collected and weighed. Samples of endometrium were di-vided into two portions; the first part was snap-frozen inliquid nitrogen and stored at −70°C for gene expressionanalyses, whereas the remaining part was fixed for histol-ogy and immunohistochemistry. Fixed tissues were placedin fresh 4% paraformaldehyde solution at 4°C, transferredto 70% ethanol 24 h later, and then sectioned transverselyimmediately proximal to the uterotubal junction. All fixedsamples were paraffin embedded, sectioned at 5 μm, andstained with H&E using standard histologic procedures.Histomorphometry and histology. Endometrial thickness,

stromal collagen, glandular area, cervical epithelial cellheight, vaginal epithelial thickness, and vaginal keratinthickness were quantified by histomorphometric methodssimilar to those described previously (37). Briefly, H&Eslides were digitized using a Labophot 3 light microscope(Nikon Instruments) and Infinity 3 digital camera (Lume-nera), and measurements were taken with Image Pro-Plussoftware (Media Cybernetics). For each measure, six mi-croscopic fields were randomly selected and examined at200× magnification. Endometrial collagen content was de-termined from slides stained with Masson's trichrome(containing Weigert's iron hematoxylin, Crocein ScarletMOO, 5% aqueous phosphomolybdic acid, and anilineblue; Fisher Scientific and Sigma); pale blue–stained areas(representing collagen and/or ground substance) weredigitally measured using selective color-based analysis onImage Pro-Plus and values were averaged for each animal.Endometrial edema was quantified in a similar manner bydigitally selecting and measuring clear or white areas with-in endometrial stroma. Cystic space was measured bymanually tracing luminal area of endometrial glands. Ep-ithelial area was determined by digital quantification ofred positive staining for the glandular epithelial marker cy-tokeratin 18 (CK18; see below). Endometrial sectionsstained with H&E were evaluated for evidence of complexhyperplasia, neoplasia, and other histologic lesions by twoboard-certified veterinary pathologists (C.E.W. and J.M.C.).All histomorphometric measures were made blinded totreatment group.Immunohistochemistry. Fixed endometrial sections were

immunostained using commercially available primarymonoclonal antibodies for the proliferation markerKi67 (Ki67/MIB1, Dako), the apoptosis marker cleavedcaspase-3 (Cell Signaling Technologies), the glandularepithelial marker CK18 (clone DC10, Lab Vision), andestrogen receptor-α (ESR1; NCL-ER-6F11, Novocastra).Antibodies were diluted 1:50 for Ki67 and cleaved cas-pase-3 and 1:100 for CK18 and ESR1 in 1× AutomationBuffer (Biomeda) containing 0.5% casein (Sigma). Im-munostaining procedures included antigen retrieval withcitrate buffer (pH 6.0), biotinylated rabbit anti-mouseFc antibody as a linking reagent, alkaline phosphatase–conjugated streptavidin as the label, and Vector Red asthe chromogen (Vector Laboratories). Negative controlslides were run for each immunostain using the same pro-tocol as for study slides except with nonimmune serum(from the same species as primary antibody) in place of

Clin Cancer Res; 16(3) February 1, 2010


the primary antibody. Nuclear cell labeling for Ki67 andESR1 was quantified by a computer-assisted counting tech-nique using a grid filter to select cells for counting and ourmodified procedure of cell selection, described previously(38). For endometrial glands and stroma, 200 cells werecounted in both superficial and basal compartments. Im-munolabeling counts were conducted blinded to experi-mental treatment and analyzed as a percentage of thetotal number of cells examined. Cytoplasmic CK18 label-ing was quantified digitally as percent positive area acrosssix microscopic fields per slide and values were averagedfor each individual.Gene microarray analyses. Endometrial total RNA was

extracted from frozen samples using Tri Reagent (MolecularResearch Center), purified using RNeasyMini kit (QIAGEN),and quantitated using a NanoDrop ND-1000 UV-Vis spec-trophotometer (NanoDrop). Nucleic acid intactness andquality were confirmed using an Agilent 2100 Bioanalyzer(Agilent Technologies). Biotinylated cRNA sampleswere pre-pared according to the standard Enzo Bioarray protocol(Enzo Life Sciences) and hybridized using the standard Affy-metrix protocol for eukaryotic samples. Biotinylated cRNAfrom each sample was hybridized to Affymetrix GeneChipRhesus Macaque Genome Arrays, washed, and stained inan Affymetrix GeneChip Fluidics Station, and then scannedwith an Affymetrix GeneChip Scanner 3000. Intensity datawere extracted from scanned images and checked for qualityusing Affymetrix GeneChip Operating Software and Expres-sion Console (MAS5 algorithm). Microarray assays weredone at Cogenics, a Division of Clinical Data (Morrisville,NC). Microarray data are publicly available on the NationalCenter for Biotechnology InformationGene ExpressionOm-nibus database (accession no. GSE14518).Microarray data analyses were done using the GeneSifter

software program (Geospiza). Intensity data were RMAnormalized, converted to a log 2 scale, screened for hetero-geneity among samples and groups, and evaluated usingsupervised ANOVA and pairwise comparisons betweentreatments. Principal components analysis, pattern naviga-tion, cluster analysis, heatmapping, and KEGG pathwayanalyses were done on filtered data subsets, as describedin Results. Differences in gene numbers altered by eachtreatment were compared using either Fisher's exact testor χ2 test. Euclidean distances (representing the numericaldifference between treatment vectors) were calculated aspart of hierarchical clustering dendrograms using averagelinkage. Pathways related to cell proliferation were eval-uated using z-scores generated in KEGG analyses; a z-score>2.0 was considered significant overrepresentation of genesin a particular pathway. All P values were corrected whenpossible for multiple comparisons using the Benjaminiand Hochberg method (Padj; ref. 39), which derives a falsediscovery rate estimate from the raw P values (40). Repre-sentation of differentially expressed genes within specificfunctional categories was evaluated using Ingenuity Path-way Analysis software v6 (Ingenuity Systems). Significanceof gene numbers represented within a given category wasdetermined in Ingenuity Pathway Analysis using Fisher's

Clinical Cancer Research





exact test with Benjamini and Hochberg correction and ex-pressed as −log10 (P value) for gene numbers within eachtreatment group.Quantitative gene expression. Expressions of genes asso-

ciated with proliferation (MKI67, Ki67 antigen), matrix re-modeling (OVOS2, ovostatin 2), and ER activity [ESR1,estrogen receptor-α; TFF1, trefoil factor 1, also known aspS2; STC2, stanniocalcin 2; IGFBP2, insulin-like bindingprotein 2; PGR, progesterone receptor; and CXCL12, che-mokine (C-X-C motif) ligand 12, also known as SDF1]were measured in endometrial samples using quantitativereal-time reverse transcriptase PCR (qRT-PCR). Macaque-specific qRT-PCR primer-probe sets for internal controlgenes [glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and β-actin (ACTB)] were generated throughthe Applied Biosystems (ABI) TaqMan Assay-by-Designservice. Sources for target primer-probe sets are given inSupplementary Table S1. All probes spanned an exon-exonjunction to eliminate genomic DNA contamination. qRT-PCR reactions (20 μL volume) were done on an ABI Prism7000 Sequence Detection System using standard TaqManreagents and thermocycling protocol (41). Relative expres-sion was determined using the ΔΔCt method described in



ABI User Bulletin #2 (available online). The Ct values forthe control genes GAPDH and ACTB were averaged for usein internal calibration, whereas reference premenopausalbreast tissue RNA was run in parallel for plate-to-plate cal-ibration. Calculations were done using ABI Relative Quan-tification SDS Software v1.1.Statistical analysis. Data were analyzed using the SAS

statistical package (version 8, SAS Institute). All data wereevaluated for normal distribution and homogeneity of var-iances among groups. A general linear model was used todetermine mean values and calculate group differences forbody weight, age, serum E2, uterine weight, endometrialmorphometric measures, Ki67 immunolabeling, and qRT-PCR expression data. Immunolabeling data for ESR1 andcleaved caspase-3 were evaluated using a nonparametricKruskal-Wallis test followed by two-sided Wilcoxon ranksum pairwise analysis. Gene expression data were log trans-formed to improve distribution, and data were then retrans-formed to original scale and reported as fold-change ofcontrol with 90% confidence interval. All other data arereported as mean ± SE. One animal randomized to theTamgroupwas excluded fromall analyses based on repeatedbaseline serum E2 values >30 pg/mL, indicating ectopic

Fig. 1. Tamoxifen increases uterine weight and endometrial thickness while antagonizing E2-induced proliferation. A, uterine weight and endometrialthickness were higher in all treatment groups. B and C, endometrial proliferation, determined by immunolabeling and relative gene expression for the Ki67marker, was greater only in the E2 group. The addition of Tam antagonized E2-induced proliferation (B). Images of Ki67 labeling (C) were taken at 200×magnification. Gene expression values were measured by qRT-PCR, corrected for internal control gene expression, and expressed relative to control groupvalues. Vertical bars indicate 90% confidence intervals (gene expression) or SEs (other measures). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with therespective control group values; # P < 0.05, compared with the E2 group.




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and/or remnant ovarian tissue. Final group sizes were thusn = 6 for Con, E2, and E2 + Tam and n = 5 for Tam for all endpoints. All pairwise P values were adjusted for the numberof pairwise tests using a Bonferroni correction. A two-tailedsignificance level of 0.05 was chosen for all comparisons.

Results

Treatment group characteristics. No treatment groupdifferences were noted in age or body weight at baseline(P>0.1 for both; Supplementary Table S2).During4monthsof treatment, no significant differences in body weight orbody weight changes among groups were noted. Serum E2was higher in the E2 and E2 + Tam groups compared withcontrol in eachmonthof treatment (P<0.01 for all),whereasno significant differences were observed between the E2 andE2 + Tam groups.Tamoxifen and estradiol effects on endometrial thickness

and proliferation. Uterine weight and endometrial thick-ness were at least 2-fold higher in the E2, Tam, and E2 +Tamgroupscomparedwithplacebo(P<0.01forall;Fig. 1A). Endometrial thickness was marginally higherin the E2 + Tam group compared with the E2 alone group(P = 0.06). In contrast, endometrial proliferation, indicatedbyMKI67 gene expression and Ki67 immunolabeling withinglandular and stromal compartments, was greater only in the



E2 alone group (P < 0.05 for all; Fig. 1B and C). The additionof Tam significantly abolished E2-induced stromal and epi-thelial Ki67 expression (P < 0.05 for E2 + Tam comparedwithE2; Fig. 1B). No significant treatment effects were seen on en-dometrial apoptosis, measured by the expression of themarker cleaved caspase-3 (data not shown). No neoplasticor complex/atypical hyperplastic lesions were noted onhistology.Tamoxifen and estradiol effects on endometrial gene ex-

pression profiles. Gene microarrays were used to further in-vestigate treatment effects on endometrial proliferation.Global expression profiles showed greater numbers ofgenes altered by E2 compared with Tam but greater percentoverlap between Tam and E2 + Tam. For example, amongsignificantly altered (named) genes with fold-change >2(ANOVA Padj < 0.05), E2 (n = 634) had 38% overlap withTam (n = 364) and 45% overlap with E2 + Tam (n = 498),whereas Tam had 67% overlap with E2 and 84% overlapwith E2 + Tam (Fig. 2A). Supervised hierarchical clusteringalso indicated that Tam and E2 + Tamwere the most closelyassociated groups with a Euclidean distance of ∼15 forgenes significantly altered at fold-change >2 (Fig. 2A). Thenumber of genes significantly altered only in the E2 groupwas greater than that for the Tam and E2 + Tam groups at allfold-change values <5 (P < 0.001 byχ2 test). This divergenceof E2 from Tam and E2 + Tamwas evident qualitatively from

Fig. 2. Tamoxifen and E2 exhibit divergent effects on gene expression profiles in the endometrium related to cell proliferation and cell cycle. A, Venn diagram(top) and hierarchical clustering dendrogram (bottom) show greater percent overlap and tighter clustering between the Tam and E2 + Tam groups for alldifferentially expressed genes at fold-change (FC) >2. Dendrogram axis values represent Euclidean distances between groups. B and C, principalcomponent analyses (B) and heatmaps (C) of differentially regulated genes related to cell proliferation (n = 461) and cell cycle (n = 563) show a distinctpattern for E2 with close overlap of vectors for the Tam and E2 + Tam groups. D, functional categories with significant overrepresentation of genesincluded cancer, cell growth and proliferation, and cell cycle. Treatment with E2 resulted in the largest number of differentially altered genes among thesecategories, whereas Tam and E2 + Tam resulted in lower number of genes represented. Diagrams in B and C correspond to significantly altered genes(ANOVA P < 0.05) within cell proliferation and cell cycle ontology categories. Diagrams in A and D correspond to the following gene filter: FC >2 in at least onegroup versus control, adjusted ANOVA P < 0.05, and quality >2.






principal components analysis vectors and heatmaps forboth overall altered genes and altered genes specifically relat-ed to cell proliferation and cell cycle (based onontology clas-sification; Fig. 2B and C). A similar pattern was seen whenaltered genes were sorted by functional category, whichshowed significant overrepresentation of genes (−log P >1.2) related to cancer, cell cycle, and cell proliferation func-tions in all groups, with the greatest representation in the E2group (Fig. 2D).Complementary pathway analyses evaluated the represen-

tation of altered genes at fold-change >2 in nine preselectedKEGG pathways related to cell proliferation. Cell cycle wasthe only one of these pathways to have a significant z-score(Table 1). Fourteen of the 18 cell cycle genes identified wereupregulated in the E2 group; in 13 of these 14 genes (the ex-ception being cyclin D1), E2 had a greater fold-change effectthan Tam and E2 + Tam (Table 2). Three of the four down-regulated genes were cyclin-dependent kinase inhibitors in-volved in negative regulation of cell cycle progression.Expanding the filter to genes significantly altered at fold-change >1.2 provided cell cycle z-scores of 5.27 (53 genes re-presented out of 89 on array) on KEGG analysis and 7.75on ontology analysis (308 genes represented out of 686 onarray). Of the KEGG genes, 37 of 53 were upregulated inthe E2 group, and 34 of these 37 genes had the greatestmagnitude of change in the E2 group (P < 0.0001 comparedwith Tam and E2 + Tam by Fisher's exact test), indicatingpartial or full antagonism of Tam on E2 effects related to cellcycle gene expression. A complete list of all significantlyaltered genes related to cell cycle (KEGG and ontology)and proliferation (ontology) is provided in SupplementaryTable S3A to C.



To examine genes altered specifically in the E2 + Tamgroup, differentially expressed transcripts (ANOVA Padj <0.05) were screened for a pattern of fold-change >2.0 inthe E2 + Tam group but not in the E2 or Tam group. A totalof 169 transcripts (with GenBank accession numbers)were identified (Supplementary Table S3D). Notable genesin this list included androgen receptor (AR; ↓ 2.0×), retinoicacid receptor–related orphan receptor B (RORB; ↓ 2.2×),V-erb-a erythroblastic leukemia viral oncogene homolog4 (ERBB4; ↑ 2.6×), trefoil factor 3 (TFF3; ↑ 2.3×), tumor pro-tein D52 (TPD52; ↑ 2.3×), myosin heavy chains 1 (MYH1;↑ 3.0×) and 2 (MYH2; ↑ 7.1×), and breast carcinoma ampli-fied sequence 1 (BCAS1; ↑ 2.2×).Divergent effects of estradiol and tamoxifen on endometrial

morphology. Endometrial morphometric measures wereused to evaluate changes contributing to the increased uter-ine weight and endometrial thickness. In the Tam and E2 +Tam groups, these effects were due in part to greater endo-metrial fibrosis (P < 0.001 for both groups compared withcontrol), whichwas evident on histology and confirmed us-ing morphometry on sections stained with Masson's tri-chrome for collagen (Fig. 3A and B). This change hasbeen noted previously in the human endometrium in re-sponse to Tam and other SERMs andmay contribute to for-mation of polyps (18, 19).To explore gene expression changes related to the Tam

effect on endometrial collagen, we evaluated pathways

Table 1. Representation of significantly alteredgenes in preselected pathways related tocell proliferation

KEGG pathway
List Array z-score
Cell cycle
18 89 3.90 mTOR signaling pathway 5 35 1.17 TGF-β signaling pathway 5 46 0.52 Jak-STAT signaling pathway 10 103 0.36 ErbB signaling pathway 6 68 0.03 Insulin signaling pathway 9 105 −0.06 Wnt signaling pathway 9 106 −0.09 VEGF signaling pathway 4 49 −0.14 Notch signaling pathway 2 34 −0.59 MAPK signaling pathway 9 156 −1.35
NOTE: Microarray gene expression data were screened us-ing a threshold fold-change >2.0 (in at least one group),Benjamini and Hochberg-adjusted ANOVA P value < 0.05,and quality setting >2. The resulting gene set (n = 2,065)was then subjected to KEGG pathway analysis in theGenesifter software program.

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Table 2. Treatment effects on relative expres-sion (fold-change versus control) of individualgenes involved in cell cycle regulation onmicroarray analysis

Gene ID

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GenBank ID

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E2

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socia

Tam

(3) Febr

tion for

E2 + Tam

BUB1 (+/−)
AF043294 4.3 1.4 1.5 BUB1B (+/−) NM_001211 5.3 1.4 1.6 CCNB1 (+) BE407516 3.9 1.4 1.5 CCNB2 (+) NM_004701 5.1 1.5 1.8 CCND1 (+) BC000076 1.8 2.2 2.7 CDC14B (+/−) AK024886 0.5 0.6 0.5 CDC2 (+) NM_001786 3.9 1.1 1.1 CDC20 (+) NM_001255 2.0 1.0 1.2 CDC6 (+) NM_001254 3.8 1.2 1.3 CDK6 (+) AW274756 2.6 2.1 2.4 CDKN1B (−) BC001971 0.4 0.5 0.5 CDKN1C (−) R78668 0.4 0.5 0.5 CDKN2B (−) AW444761 0.3 0.4 0.5 CHEK1 (−) NM_001274 3.4 1.8 2.1 MAD2L1 (+/−) AF394735 2.2 1.4 1.2 MCM4 (+) AI936566 2.3 1.2 1.2 PTTG1 (+) NM_004219 3.5 1.1 1.2 SKP2 (+) BC001441 2.2 1.6 1.4
NOTE: (+) and (−) indicate the respective effects on cellcycle progression.

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involved inmatrix remodeling. Among significantly alteredgenes with fold-change >2 in the Tam and E2 + Tam groups(but not in E2 group) on microarray analysis, no significantontology or KEGG pathways directly related to extracellularmatrix or collagen remodelingwere identified. Similarly, noeffects specific to the Tam and E2 + Tam groups were ob-served for geneswithin related classes such as collagens,ma-trix metalloproteinases, fibrogenic cytokines, and tissueinhibitors of matrix metalloproteinases. However, marked-ly increased gene expression of the protease inhibitor ovos-tatin 2 (OVOS2) was noted in the Tam (24×) and E2 + Tam(12×) groups (P < 0.01 for both compared with controlgroup; Fig. 3A). Whereas the exact role of ovostatin 2 is un-determined, the highly similar ovostatin 1 protein is a po-tent inhibitor of matrix metalloproteinases, includingcollagenase (42). An incidental but potentially relatedchange noted on histology was distinctive thickening ofglandular basement membranes in the Tam and E2 + Tamgroups.A second contributing factor to increased endometrial

thickness in the Tam and E2 + Tam groups was cystic dila-tion of glands, indicated by greater glandular luminal area



(P < 0.01 for both compared with control group; Fig. 3C).The addition of E2 to Tam had modest, if any, abrogatingeffects on fibrosis or cystic changes. In contrast to Tam, E2effects on endometrial thickness were due largely to super-ficial stromal edema, which was marginally higher in theE2 but not in the Tam or E2 + Tam group (ANOVA P =0.06; Fig. 3C). Glandular epithelium measured by CK18expression was higher in the E2, Tam, and E2 + Tam groupsfor deep, but not for superficial, endometrial glands butdid not contribute substantially to overall endometrialthickness, occupying <3% of the sectional area (data notshown).Estrogen agonist and antagonist effects of tamoxifen on

endometrium. Nuclear expression of ESR1 protein was de-tected within endometrial glands and stroma. No signifi-cant treatment effects were seen for ESR1 immunolabeling(Supplementary Fig. 1A) or gene expression (data notshown). Two major patterns of expression were seen forgene markers of ER activity. In the first pattern, E2-inducedgenes such as TFF1, STC2, and IGFBP2 were higher in alltreatment groups (P < 0.01 for all compared with control;Supplementary Fig. 1B), consistent with an ER agonist

Fig. 3. Effects of Tam + E2 on endometrial morphology are dominated by Tam. A, increased endometrial fibrosis in Tam-treated groups corresponded withincreased gene expression of the proteinase inhibitor ovostatin 2 (OVOS2). B, representative images of superficial endometrium stained with Masson'strichrome show increased stromal collagen (pale blue) in the Tam and E2 + Tam groups; images were taken at 100× magnification. C, the Tam and E2 + Tamgroups showed greater luminal area within endometrial glands, indicative of cystic change noted on histology, whereas stromal edema was marginallyhigher only in the E2 group. Morphometric measures are expressed as percent total endometrial area measured. Vertical lines indicate 90% confidenceintervals (gene expression) or SEs (other measures). **, P < 0.01; ***, P < 0.001, compared with the respective control group values. ##, P < 0.01; ###,P < 0.001, compared with the E2 group.






effect of Tam. Other ER-driven genes such as PGR andCXCL12 showed a similar pattern, although thesechanges were not significant on qRT-PCR (ANOVA P >0.05; data not shown). In the second pattern, E2-inducedgenes were higher only in the E2 group, with full or partialantagonism of E2 by Tam (Table 3). As with MKI67 andmany of the cell cycle targets (Table 2), E2-induced gene ex-pression for E2 + Tam seemed to be dominated by Tam rath-er than by E2.Antagonism of estradiol effects by tamoxifen in the genital

tract. Treatment with E2 resulted in ∼3-fold greater vaginalepithelial thickness (Fig. 4A and B), vaginal keratin thick-ness (Fig. 4C), and cervical gland height (Fig. 4D; P < 0.01for all compared with control). Tam had no effects on anyof these measures when given alone, completely antago-nized E2 effects on vaginal maturation, and partially antag-onized E2 effects on cervical epithelial height (Fig. 4A-D).

Discussion

Estrogen + SERM co-therapy is an emerging alternativeto traditional EPT, especially for postmenopausal womenconcerned about the promotional effects of progestins onbreast cancer. The endometrial safety of estrogen + SERMcombinations is not known, however. The primary goal ofthis study was to evaluate the endometrial profile of low-dose E2 and Tam alone and in combination. Our findingsreveal an endometrial phenotype for E2 + Tam character-ized by increased endometrial thickness, stromal fibrosis,and cystic change with reduced epithelial and stromal



proliferation compared with E2 alone. A divergent effectof Tam was observed for specific ER activity markers andendometrial proliferation, suggesting that Tam effects onendometrial cancer risk may not relate exclusively topartial estrogen agonist activity. Despite clear effects oflow-dose E2 alone, the profile for E2 + Tam based on endo-metrial morphology, proliferation, and transcriptionalprofile was dominated by Tam, suggesting that long-termrisks associated with this combination may be similar tothose seen with Tam alone.Previous studies evaluating ET or EPT alongside Tam are

limited. In a recent small clinical trial, Tam at a low doseof 5 mg/d increased endometrial thickness, but not Ki67expression, when given with EPT; no significant increasesin vasomotor symptoms were noted compared with EPTalone (14). In a second larger trial, Tam at 20 mg/d tendedto attenuate ET/EPT effects on vasomotor symptoms inwomen at increased risk of breast cancer (15). A more re-cent study also found no benefit for vasomotor symptomsfrom adding ET or EPT to Tam at 20 mg/d (43). Findingsfrom the current study indicate that Tam may also inhibitbeneficial estrogen effects on urogenital atrophy. Collec-tively, these data indicate that Tam may override two ofthe primary indications for ET and EPT and is thus not asuitable SERM co-therapy for estrogen.Other SERMs investigated as estrogen co-therapies include

raloxifene and bazedoxifene. A small clinical trial reportedthat raloxifene at 60 mg/d given with oral E2 at 1 mg/d im-proved menopausal symptoms but increased endometrialthickness comparedwith baseline and treatmentwith ralox-ifene alone (28). Similar findings were noted in an earlierpilot study using transdermal estradiol (27). One additionalstudy examining vaginal atrophy found no adverse attenuat-ing effects of raloxifene on the efficacy of an estradiol-releasingvaginal ring in postmenopausal women (26). Preclinicaldata indicate that the third-generation SERM bazedoxifenemay antagonize estrogen effects on both mammary glandand uterine measures while maintaining estrogen effectson vaginal maturation (29–31). These latter effects includedose-dependent attenuation of estrogen-induced prolifera-tion of ER-positive MCF-7 breast cancer cells in culture (29)and uterine weight gain in mice (30, 31).Endometrial safety is an important concern in the devel-

opment and long-term use of SERMs. Preclinical and clin-ical evaluation of endometrial SERM effects is often basedon markers of estrogen agonist activity. Previous studieshave shown that Tam induces estrogen-responsive markersin the endometrium (23, 24) and increases endometrialthickness and cancer risk in postmenopausal women (7,20, 21), supporting the idea that carcinogenic effects ofTam are due to ER agonist signaling. In this study, Tam in-duced a subset of ER-driven genes in the endometriumwhileantagonizing others, indicating that Tam is not simply actingas a weak ER agonist relative to E2 but instead exhibitingmore complex mixed patterns of ER transactivation. Giventhe heterogeneity of tissue samples used for gene expressionanalyses, it is possible that Tam estrogen agonist/antagonisteffects may even differ within specific compartments (e.g.,

Table 3. Treatment effects on relative expres-sion (fold-change versus control) of selectedestrogen-induced genes on microarrayanalysis

Gene ID
GenBank ID E2 Tam E2 + Tam
Tamoxifen agonist pattern
STC2 BC000658 2.9 2.1 2.4 IGFBP2 NM_000597 2.9 2.8 3.4 CDH1 L08599 2.2 2.5 2.0 EGFR S75916 3.0 2.3 2.4 MUC1 AI610869 2.0 2.8 3.6
Tamoxifen antagonist pattern
MKI67 AU132185 2.6 1.0 1.1 PCNA NM_002592 1.4 1.0 1.0 TOP2A AL561834 5.3 1.5 1.8 PTEN U96180 1.6 1.3 1.1 PTTG1 NM_004219 3.5 1.1 1.2
NOTE: Microarray gene expression data were screenedusing a threshold fold-change >2.0 (in at least one group),Benjamini and Hochberg–adjusted ANOVA P value < 0.05,and quality setting >2. Known estrogen-induced genesshown were selected from the resulting gene set.




Wood et al.

954


stroma and epithelium) of the same tissue. Of note, Tam hasalso been shown to induce certain estrogen-responsive mar-kers in normal mammary gland (44) and breast cancer cells(45) despite having well-documented ER antagonisteffects. This information suggests that individual estrogen re-sponse markers are not necessarily the best predictors ofSERM effects or risk profile.Increased proliferation is associated with the develop-

ment of many cancers and provides a useful biomarkerof potential cancer-promoting effects. In the uterus, epi-thelial cell proliferation serves as an important prognosticmarker in human endometrial cancers (46) and may helppredict risk associated with different hormone therapies(47). In this study, Tam decreased E2-induced Ki67 label-ing and inhibited or partially inhibited the expression ofnumerous E2-induced genes related to proliferation. Manyof the proliferation-related genes with this pattern directlyinvolve cell cycle progression. These findings, in combina-tion with phenotypic features dominated by Tam, suggest



that E2 + Tam may be associated with cancer risk estimatesmore similar to that of Tam than of E2 alone.Numerous studies have shown that standard doses of

unopposed ET (for oral E2, 1 or 2 mg/d) increase endome-trial hyperplasia and cancer risk in postmenopausal women(1, 2). The endometrial effects of newer low-dose ETs areless clear, however. A small clinical trial previously reportedincreased endometrial thickness following oral E2 doses of0.5 and 1.0 mg/d but not 0.25 mg/d (32), whereas a sepa-rate trial using oral conjugated equine estrogens noted adose-related increase in endometrial hyperplasia incidencefrom 3% for 0.3 mg/d to 27% for the standard 0.625 mg/ddose after 2 years (48). Although no neoplastic or complexhyperplastic lesions were observed in the current study, ourresults suggest that lower doses of oral E2 (≤0.5 mg/d) maystill exert a clear stimulatory effect, increasing endometrialweight, thickness, glandular area, and proliferation. It isalso worth noting that the peak serum E2 concentrationsin the current study (40-80 pg/mL) were comparable to

Fig. 4. Tamoxifen antagonizes E2 effects on lower reproductive tract measures. A, representative images of vaginal epithelium show increased overallepithelial maturation and keratinization (superficial laminar zone) following E2 and antagonism of this effect by Tam. Images were taken at 100×magnification; H&E stain. B to D, Tam fully antagonized E2 effects on overall epithelial thickness (B) and keratin thickness (C) and partially antagonizedstimulatory E2 effects on cervical glandular height (D). Vertical lines indicate SEs. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the respective controlgroup values. ##, P < 0.01, compared with the E2 group.






the steady-state concentrations reported in postmenopausalwomen receiving E2 via vaginal ring at 150 μg/d (49) or trans-dermal patch at 50 μg/d (50). Serum E2 concentrations (andpharmacodynamics) vary widely for different E2 formulationsand routes of administration, however, and it is unclearwhether Tam or other SERMs may exert similar effects tothose seen in the present study when given alongside lowerdoses of parenteral E2. Dose-dependent endometrial effectsmay also be seen with Tam (19), and additional studies areneeded to determine whether lower Tam doses than thoseused here (<20 mg/d human equivalent) would similarlyinfluence E2 effects.The ideal postmenopausal hormone therapy would pro-

vide estrogen agonist effects in tissues such as bone and uro-genital tract while minimizing the risk of breast andendometrial cancer. Estrogen + SERM combinations havebeenproposed recently as a potential way to achieve this pro-file. Data from this study provide an initial step in profilingthe uterine effects of these therapies. Our results show that aSERM given at a standard dose may dominate the estrogenphenotype in the endometrium, at least for lower doses oforal E2. In the case of Tam, this profile may still be associatedwith adverse long-term effects, including cancer risk. Never-theless, the dominance of the SERM signature suggests thatother SERMs with more favorable profiles in the endo-metriumand elsewheremaybeused as progestin alternatives



in future postmenopausal therapies. Further studies directlycomparing different SERM-estrogen combinations areneeded to identify the safest SERM for this purpose.

Disclosure of Potential Conflicts of Interest

J.M. Cline, J.R. Kaplan, and C.E. Wood are unpaid co-investigators on aninvestigator-initiated preclinical study funded by Wyeth Pharmaceuticals(now part of Pfizer) on the effects of the SERM bazedoxifene.

Acknowledgments

We thank Lisa O'Donnell, Joseph Finley, Hermina Borgerink,Jean Gardin, Diana Swaim, Dewayne Cairnes, Jamie Fox, and BrianMcCollough for their technical contributions.

Grant Support

NIH NationalCenter for ResearchResources (NCRR) grant K01RR021322-04 and National Heart, Lung, and Blood Institute (NHLBI) grant R01 HL49085. The contents are solely the responsibility of the authors and do notnecessarily represent the view of the NCRR, NHLBI, or NIH.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Received 6/16/09; revised 11/11/09; accepted 11/22/09; publishedOnlineFirst 1/26/10.

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2010;16:946-956. Published OnlineFirst January 26, 2010.Clin Cancer Res Charles E. Wood, Jay R. Kaplan, M. Babette Fontenot, et al. Combination TherapyEndometrial Profile of Tamoxifen and Low-Dose Estradiol

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