Estrogen Metabolites Are Not Associated with Colorectal Cancer Risk in Postmenopausal Women

Published OnlineFirst June 23, 2015; DOI: 10.1158/1055-9965.EPI-15-0541

Null Results in Brief Cancer Epidemiology, Biomarkers Estrogen Metabolites Are Not Associated with & Prevention Colorectal Cancer Risk in Postmenopausal Women Roni T. Falk1, Cher M. Dallal2, James V. Lacey Jr3, Douglas C. Bauer4, Diana S.M. Buist5, Jane A. Cauley6, Trisha F. Hue7, Andrea Z. LaCroix8, Jeffrey A. Tice4, Ruth M. Pfeiffer9, Xia Xu10, Timothy D. Veenstra11, and Louise A. Brinton1, for the BFIT Research Group

Abstract

Background: A potential protective role for estrogen in colon at the C-2, C-4, or C-16 position) and by ratios of the groupings carcinogenesis has been suggested based on exogenous hor- using Cox proportional hazards regression models. mone use, but it is unclear from previous studies whether Results: No signiﬁcant associations were seen for estrone endogenous estrogens are related to colorectal cancer risk. (HRQ4 vs. Q1 ¼ 1.15; 95% CI, 0.69–1.93; Ptrend ¼ 0.54), estradiol These few prior studies focused on parent estrogens; none (HRQ4 vs. Q1 ¼ 0.98; 95% CI, 0.58–1.64; Ptrend > 0.99), or total EM evaluated effects of estrogen metabolism in postmenopausal (the sum of all EM; HRQ4 vs. Q1 ¼ 1.35; 95% CI, 0.81–2.24; Ptrend ¼ women. 0.33). Most metabolites in the 2-, 4-, or 16-pathway were unre- Methods: We followed 15,595 women (ages 55–80 years) lated to risk, although a borderline trend in risk was associated enrolled in the Breast and Bone Follow-up to the Fracture Inter- with high levels of 17-epiestriol. vention Trial (BFIT) who donated blood between 1992 and Conclusion: Circulating estrogens and their metabolites were 1993 for cancer through December 2004. A panel of 15 estrogen generally unrelated to colorectal cancer risk in postmenopausal metabolites (EM), including estradiol and estrone, were measured women. in serum from 187 colorectal cancer cases and a subcohort of 501 Impact: Additional studies are needed to understand how women not using exogenous hormones at blood draw. We exogenous estrogen may prevent colorectal cancer. Cancer Epidemiol examined EM individually, grouped by pathway (hydroxylation Biomarkers Prev; 24(9); 1–4. 2015 AACR.

Introduction affinity, but at least one estrogen metabolite (EM), 17-epiestriol, shows a binding preference for ERb (7). We speculated that Exogenous hormones are linked to colorectal cancer risk reduc- selective binding of EM to ERa or ERb may influence colon tion (1, 2), yet risks associated with circulating estrogens are carcinogenesis. Thus, we investigated the association between a elevated (3, 4), or null (5). In normal colonic tissue, ERa enhances panel of EM and colorectal cancer in the Breast and Bone Follow- cell growth and is expressed at low levels, while the antiprolifera- up to the Fracture Intervention Trial (BFIT). tive ERb is abundant (6). In neoplastic colonic tissue, this is reversed. Estradiol binds to both receptors with comparably high Materials and Methods FIT, a randomized trial designed to test alendronate, screened 22,695 postmenopausal women ages 55 to 80 from 1Hormonal and Reproductive Epidemiology Branch, Division of Can- cer Epidemiology and Genetics, National Cancer Institute, Bethesda, 1992 to 1993, who provided a bone mineral density (BMD) Maryland. 2Department of Epidemiology and Biostatistics, University scan, blood and information on demographic, lifestyle, and of Maryland School of Public Health, College Park, Maryland. 3Division reproductive factors through a self-administered question- of Cancer Etiology, Department of Population Sciences, Beckman naire. BFIT comprises 15,595 of the FIT screenees followed Research Institute of the City of Hope, Duarte, California. 4Department of Medicine, University of California, San Francisco, California. 5Group for incident cancer through December 2004. An additional Health Research Institute, Seattle, Washington. 6Department of Epi- questionnaire covering subsequent cancer diagnoses was sent demiology,Graduate School of Public Health, University of Pittsburgh, 7 to these participants (8). Pittsburgh, Pennsylvania. Department of Epidemiology and Biosta- fi tistics, University of California San Francisco, San Francisco,California. Self-reported cancers were con rmed by medical records and/ 8Division of Epidemiology, University of California San Diego, La Jolla, or cancer registries. Additional cases were identified from cancer 9 California. Biostatistics Branch, Division of Cancer Epidemiology and registries. Vital status was determined by the National Death Genetics, National Cancer Institute, Bethesda, Maryland. 10Laboratory of Proteomics and Analytical Technologies, Advanced Technology Index. All participants provided written informed consent, and Program, Leidos-Frederick (formerly SAIC-Frederick), Inc., Frederick Institutional Review Board approval was obtained from all clin- 11 National Laboratory for Cancer Research, Frederick, Maryland. C2N ical sites and the National Cancer Institute. Diagnostics, St. Louis, Missouri. This analysis was part of a case–cohort study within BFIT Corresponding Author: Roni T. Falk, National Cancer Institute, 96096 Medical examining EM in relation to colorectal cancer, breast, endome- Center Drive, Room 7-E224, Bethesda, MD 20892. Phone: 240-276-7329; Fax: trial, and ovarian cancer (8). We studied 187 colorectal cancer 240-276-7838; E-mail: [email protected] cases (10 in the subcohort) and 501 subcohort women. Fifteen doi: 10.1158/1055-9965.EPI-15-0541 EM were measured in serum: estrone, estradiol, 2-hydroxyestrone, 2015 American Association for Cancer Research. 2-methoxyestrone, 2-hydroxyestradiol, 2-methoxyestradiol,

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2-hydroxyestrone-3-methyl ether, 4-hydroxyestrone, 4-methox- (n ¼ 491). Covariates examined included body mass index (BMI), yestrone, 4-methoxyestradiol, 16a-hydroxyestrone, estriol, 17- parity, smoking, postmenopausal hormone use, and ages at epiestriol, 16-ketoestradiol, and 16-epiestriol. menarche, birth of the first child, and menopause. The final EM were examined individually, by pathway (C-2, C-4, or C-16 model adjusted for clinic site, trial participation status, age at hydroxylation, with EM in each pathway summed), and by ratios blood collection, and total BMD. No adjustment was made for of pathways. Cox proportional hazards regression models with multiple comparisons. robust variance adjustment were used to estimate hazard ratios (HR) and 95% confidence intervals (CI). For cases not in the subcohort, follow-up began 6 months prior to colorectal cancer Results diagnosis and ended at diagnosis. For the subcohort, follow-up Descriptive characteristics and HR for lifestyle/reproductive risk began at baseline and ended at colorectal cancer diagnosis for factors are presented in Table 1. Compared with the subcohort, cases (n ¼ 10), and death or end of the follow-up for noncases cases were older at study entry, followed for fewer years, and had

Table 1. Select characteristics and HRsa for lifestyle and reproductive/hormonal risk factors; colorectal cancer (CRC) case–cohort study, BFIT Characteristics CRC cases Subcohort P Caucasian, n (%) 182 (97) 475 (95) Years follow-up, mean (SD) 5.4 (3.1) 10.2 (2.2) <0.001 Age at blood draw, mean (SD) 69.8 (5.6) 67.3 (6.2) <0.001 Years postmenopausal at blood draw, mean, (SD) 22.5 (8.2) 20.7 (8.8) 0.015 Bone mineral density, femoral neck g/cm, mean (SD) 0.74 (0.1) 0.77 (0.1) 0.030

CRC cases Subcohort HR 95% CI Ptrend Lifestyle risk factors BMI at blood draw (kg/m2) <25 78 209 1.00 Referent 25–29 72 157 1.18 (0.78–1.77) 30–34 21 83 0.68 (0.39–1.21) 35þ 14 47 0.81 (0.40–1.65) 0.232 Regular exercise program, yes 72 228 0.98 (0.81–1.17) 0.975 Cigarette use Never 111 259 1.00 Referent Former 53 185 1.03 (0.86–1.25) Current 20 53 1.38 (1.06–1.81) 0.849 Recent alcohol consumption None (past 30 days) 95 210 1.00 Referent <3 days/week 50 138 0.97 (0.88–1.22) 3–6 days/week 27 116 1.18 (0.95–1.46) Daily 15 37 1.13 (0.83–1.53) 0.127 Hormonal and reproductive risk factors Age at menarche <11 27 77 1.00 Referent 12, 13 93 280 0.94 (0.55–1.61) 14þ 58 125 1.36 (0.77–2.42) 0.615 Age at ﬁrst live birth Nulliparous 24 47 1.00 Referent <20 23 50 0.87 (0.41–1.85) 20–24 71 216 0.66 (0.36–1.21) 25–29 52 128 0.82 (0.44–1.52) 30þ 18 60 0.50 (0.24–1.07) 0.136 Parity Nulliparous 24 47 1.00 Referent 1 15 60 0.35 (0.16–0.77) 2 49 143 0.67 (0.35–1.26) 3 47 115 0.86 (0.46–1.63) 4þ 52 136 0.75 (0.40–1.39) 0.564 Ever breastfeed, yes 103 274 1.04 (0.73–1.47) 0.984 Age at menopause <40 27 82 1.00 Referent 40–44 27 81 1.23 (0.64–2.35) 45–49 62 176 1.16 (0.65–2.08) 50–54 60 145 1.48 (0.84–2.60) 55þ 11 17 2.51 (1.08–5.85) 0.064 Postmenopausal estrogen use Never 129 334 1.00 Referent <1 year 12 49 0.74 (0.37–1.47) 1–4 years 30 64 1.25 (0.74–2.10) 5–9 years 4 27 0.35 (0.12–1.04) 10þ years 10 23 0.94 (0.41–2.15) 0.608 aHRs are adjusted for clinic, trial participation status, age at blood collection, and total BMD.

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Estrogen Metabolites and Colorectal Cancer Risk

ABHormone Cases HR (95% CI) P trend 2-Pathway Q1 56 1.00 (referent) 0.48 Q2 42 0.68 (0.42–1.11) Hormone Cases Q3 39 0.67 (0.40–1.11) HR (95% CI) P trend Q4 50 0.83 (0.51–1.36) Total EM Q1 43 1.00 (referent) 0.33 2-Hydroxyestrone Q2 48 1.12 (0.68–1.85) Q3 41 1.01 (0.60–1.72) Q1 57 1.00 (referent) 0.27 Q4 55 1.35 (0.81–2.24) Q2 46 0.90 (0.5t6–1.47) Q3 34 0.54 (0.32–0.90) Estrone Q4 50 0.88 (0.54–1.42) Q1 44 1.00 (referent) 0.54 Q2 47 0.96 (0.58–1.58) 2-Hydroxyestradiol Q3 43 1.02 (0.61–1.17) Q4 53 1.15 (0.69–1.93) Q1 52 1.00 (referent) 0.88 Q2 50 1.06 (0.65–1.71) Estradiol Q3 30 0.86 (0.51–1.46) Q1 42 1.00 (referent) 1.00 Q4 55 1.02 (0.62–1.70) Q2 47 1.01 (0.61–1.67) Q3 54 1.08 (0.66–1.76) 2-Methoxyestrone Q4 44 0.98 (0.58–1.64) Q1 44 1.00 (referent) 0.99 Q2 56 1.36 (0.84–2.19) 0.1 1 10 Q3 33 0.80 (0.46–1.38) Q4 54 1.16 (0.71–1.91)

2-Methoxyestradiol Q1 55 1.00 (referent) 0.88 Q2 38 0.67 (0.40–1.10) Q3 39 0.71 (0.42–1.19) Q4 55 1.02 (0.63–1.66)

2-Hydroxyestrone-3-methyl ether Q1 49 1.00 (referent) 0.67 Q2 42 0.84 (0.51–1.38) Q3 48 0.99 (0.61–1.62) Q4 48 1.06 (0.65–1.76)

0.1 1 10

C Hormone Cases HR (95% CI) P trend

16-Pathway Q1 49 • 1.00 (referent) 0.55 Q2 39 0.87 (0.53–1.43) Q3 49 1.12 (0.69–1.84) Q4 50 1.08 (0.67–1.77)

16Alpha-hydroxyestrone Q1 56 1.00 (referent) 0.32 D Q2 39 0.69 (0.43–1.14) Hormone Cases Q3 50 0.86 (0.53–1.40) Q4 42 0.72 (0.44–1.18) HR (95% CI) P trend 4-Pathway Estriol Q1 56 1.00 (referent) 0.43 Q2 45 0.74 (0.45–1.22) Q1 46 1.00 (referent) 0.54 Q3 34 0.53 (0.31–0.90) Q2 41 0.95 (0.57–1.57) Q4 52 0.89 (0.54–1.47) Q3 49 1.21 (0.74–1.97) Q4 51 1.09 (0.67–1.79) 4-Hydroxyestrone Q1 52 1.00 (referent) 0.52 17-Epiestriol Q2 50 1.00 (0.62–1.62) Q1 44 1.00 (referent) 0.06 Q3 30 0.53 (0.31–0.91) Q2 37 0.93 (0.54–1.58) Q4 55 0.99 (0.61–1.62) Q3 56 1.41 (0.84–2.38) 4-Methoxyestrone Q4 50 1.49 (0.88–2.52) Q1 47 1.00 (referent) 0.81 Q2 40 0.81 (0.48–1.37) 16-Ketoestradiol Q3 54 1.18 (0.73–1.90) Q1 51 1.00 (referent) 0.78 Q4 46 0.94 (0.56–1.58) Q2 46 0.91 (0.57–1.48) Q3 38 0.77 (0.47–1.28) 4-Methoxyestradiol Q4 52 1.13 (0.71–1.82) Q1 52 1.00 (referent) 0.78 Q2 38 0.66 (0.40–1.09) Q3 45 0.88 (0.53–1.47) 16-Epiestriol Q4 52 0.89 (0.60–1.60) Q1 43 1.00 (referent) 0.28 Q2 45 1.08 (0.65–1.81) Q3 53 1.42 (0.86–2.34) 0.1 1 10 Q4 46 1.21 (0.73–2.00)

0.1 1 10

Figure 1. A–D, HRs and 95% CIs, estrogen metabolites, and colorectal cancer [parent estrogens (A), 2-pathway (B), 16-pathway (C), and 4-pathway EM (D)]. Time scale for HR estimates is age at entry (baseline) through age at diagnosis or censoring (exit). For cases not in the subcohort, follow-up began 6 months prior to their colorectal cancer diagnosis. HRs are adjusted for clinic, age at blood draw, bone mineral density, and trial participation.

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lower BMD at baseline. Current smokers experienced a 40% studies of endogenous and exogenous estrogens are not consistent increased risk, and women with a late age at menopause had (1, 2). >2-fold excess risk, but HR for other factors were not statistically In summary, although our apparent lack of association significant. between endogenous estrogens is at odds with findings of reduced Women with higher total EM were not at elevated risk com- risk associated with exogenous hormone use, additional well- pared with those in the lowest quartile (Fig. 1A). HR for estrone powered studies are needed to improve our understanding of the and estradiol were not significant, and there was no evidence of exogenous hormone and colorectal cancer association. dose response. HR and trends were not significant for 2- or 4-pathway metabolites, except for reduced risks for moderately Disclosure of Potential Conflicts of Interest fl high 2-hydroxyestrone (HRQ3 vs. Q1 ¼ 0.54; 95% CI, 0.32–0.90; No potential con icts of interest were disclosed. Fig. 1B) and 4-hydroxyestrone (HRQ3 vs. Q1 ¼ 0.53; 95% CI, 0.31– 0.91; Fig. 1D). For the 16-pathway, a trend of increasing risk with Authors' Contributions higher 17-epiestriol was suggested, but results for other metabo- Conception and design: R.T. Falk, C.M. Dallal, J.V. Lacey Jr, T.D. Veenstra, lites were null (Fig. 1C). HR for quartiles of pathway ratios (2- vs. L.A. Brinton Development of methodology: R.T. Falk, J.V. Lacey Jr, T.D. Veenstra, 4-pathway, 2- vs. 16-pathway, and 4- vs. 16-pathway) showed no L.A. Brinton association (results not shown). Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.V. Lacey Jr, D.S.M. Buist, T.F. Hue, J.A. Tice, X. Xu, L.A. Brinton Discussion Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.T. Falk, C.M. Dallal, J.V. Lacey Jr, D.C. Bauer, This first prospective study of EM and colorectal cancer risk J.A. Tice, R.M. Pfeiffer, T.D. Veenstra, L.A. Brinton found no evidence for associations with these hormones. There Writing, review, and/or revision of the manuscript: R.T. Falk, C.M. Dallal, was a suggestion of a slight effect for 17-epiestriol, but this may J.V. Lacey Jr, D.C. Bauer, D.S.M. Buist, J.A. Cauley, A.Z. LaCroix, J.A. Tice, have occurred by chance given the number of comparisons. Our R.M. Pfeiffer, T.D. Veenstra, L.A. Brinton lack of association is consistent with results from one study (5), Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.T. Falk, C.M. Dallal, D.C. Bauer, D.S.M. Buist, but not others (3, 4), showing modest elevated risks for estradiol T.F. Hue, L.A. Brinton or estrone. Reasons for these disparate findings are not clear. Study supervision: J.V. Lacey Jr, D.S.M. Buist, A.Z. LaCroix Women in our study were older (mean age 67 vs. 60–64 in prior studies), but all involved postmenopausal women not using Acknowledgments menopausal hormones at blood draw. The studies were of com- The authors thank Stephanie Litwack-Harrison, MPH, for study manage- parable size; however, unlike others, BMI was not associated with ment; Eric Boyd and Vicky Chia, PhD, for assistance in data management; and colorectal cancer in our study. Additionally, immunoassays for the B FIT participants for their contributions to this study. estrogens in prior studies may not detect the low levels in Grant Support postmenopausal women. The original FIT study was supported by Merck Research Laboratories. BFIT Estrogen is not central to the etiology of colorectal cancer, but b was supported by the National Cancer Institute (contract # N02-CP; D.C. Bauer the change from predominantly ER expression in the healthy and J.A. Tice). colon to ERa in neoplastic tissue suggests that it may afford protection in the healthy colon, but promote tumorigenesis once Received May 20, 2015; accepted June 5, 2015; published OnlineFirst June 23, neoplastic changes occur. However, results from observational 2015.

References 1. Lin KJ, Cheung WY, Lai JY, Giovannucci EL. The effect of estrogen vs. 5. Lin JH, Zhang SM, Rexrode KM, Manson JE, Chan AT, Wu K, et al. Association combined estrogen-progestogen therapy on the risk of colorectal cancer. between sex hormones and colorectal cancer risk in men and women. Clin Int J Cancer 2012;130:419–30. Gastroenterol Hepatol 2013;11:419–24. 2. Tsilidis KK, Allen NE, Key TJ, Bakken K, Lund E, Berrino F, et al. Oral 6. Zhao C, Dahlman-Wright K, Gustafsson JA. Estrogen receptor b: an overview contraceptives, reproductive history and risk of colorectal cancer in the and update. Nucl Recept Signal 2008;6:e003. European prospective investigation into cancer and nutrition. Br J Cancer 7. Zhu BT, Han GZ, Shim JY, Wen Y, Jiang XR. Quantitative structure- 2010;103:1755–9. activity relationship of various endogenous estrogen metabolites for 3. Clendenen TV, Koenig KL, Shore RE, Levitz M, Arslan AA, Zeleniuch- humanestrogenreceptora and b subtypes: Insights into the structural Jacquotte A. Postmenopausal levels of endogenous sex hormones and risk determinants favoring a differential subtype binding. Endocrinology of colorectal cancer. Cancer Epidemiol Biomarkers Prev 2009;18:275–81 2006;147:4132–50. 4. Gunter MJ, Hoover DR, Yu H, Wassertheil-Smoller S, Rohan TE, Manson JE, 8. Dallal CM, Tice JA, Buist DS, Bauer DC, Lacey JV Jr, Cauley JA, et al. Estrogen et al. Insulin, insulin-like growth factor-I, endogenous estradiol, and risk of metabolism and breast cancer risk among postmenopausal women: a case– colorectal cancer in postmenopausal women. Cancer Res 2008;68:329–37. cohort study within BFIT. Carcinogenesis 2014;35:346–55.

OF4 Cancer Epidemiol Biomarkers Prev; 24(9) September 2015 Cancer Epidemiology, Biomarkers & Prevention

Estrogen Metabolites Are Not Associated with Colorectal Cancer Risk in Postmenopausal Women

Roni T. Falk, Cher M. Dallal, James V. Lacey, Jr, et al.

Cancer Epidemiol Biomarkers Prev Published OnlineFirst June 23, 2015.

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