Published OnlineFirst March 9, 2010; DOI: 10.1158/1078-0432.CCR-09-2481 Published Online First on March 9, 2010 as 10.1158/1078-0432.CCR-09-2481 Clinical Human Cancer Biology Cancer Research Intratumoral Estrogen Disposition in Breast Cancer Ben P. Haynes1, Anne Hege Straume3,4, Jürgen Geisler6, Roger A'Hern7, Hildegunn Helle5, Ian E. Smith2, Per E. Lønning3,5, and Mitch Dowsett1 Abstract Purpose: The concentration of estradiol (E2) in breast tumors is significantly higher than that in plas- ma, particularly in postmenopausal women. The contribution of local E2 synthesis versus uptake of E2 from the circulation is controversial. Our aim was to identify possible determinants of intratumoral E2 levels in breast cancer patients. Experimental Design: The expression of genes involved in estrogen synthesis, metabolism, and sig- naling was measured in 34 matched samples of breast tumor and normal breast tissue, and their corre- lation with estrogen concentrations assessed. Results: ESR1 (9.1-fold; P < 0.001) and HSD17B7 (3.5-fold; P < 0.001) were upregulated in ER+ tumors compared with normal tissues, whereas STS (0.34-fold; P < 0.001) and HSD17B5 (0.23-fold; P < 0.001) were downregulated. Intratumoral E2 levels showed a strong positive correlation with ESR1 expression in all patients (Spearman r = 0.55, P < 0.001) and among the subgroups of postmenopausal (r = 0.76, P < 0.001; n = 23) and postmenopausal ER+ patients (r = 0.59, P = 0.013; n = 17). HSD17B7 expression showed a significant positive correlation (r =0.59,P < 0.001) whereas HSD17B2 (r = −0.46, P = 0.0057) and HSD17B12 (r = −0.45, P = 0.0076) showed significant negative correlations with intratumoral E2 in all patients. Intratumoral E2 revealed no correlation to CYP19, STS, and HSD17B1 expression. Multivariate models comprising ESR1 and plasma E2 predicted between 50% and 70% of intratumoral E2 variability. Conclusion: Uptake due to binding to the ER, rather than intratumoral estrogen synthesis by aroma- tase or sulfatase, is the single most important correlate and a probable determinant of intratumoral E2.An increased expression of HSD17B7 may explain the increased ratio of E2 to estrone (E1) in breast tumors compared with normal tissue. Clin Cancer Res; 16(6); 1790–801. ©2010 AACR. Estrogens play a pivotal role in breast cancer devel- is the most effective endocrine treatment for postmeno- opment (1), and estrogen suppression is an effective pausal women with ER+ tumors (5). therapeutic option among premenopausal as well as post- It is well established that the concentration of E2,the menopausal women harboring estrogen receptor–positive most biologically active estrogen, in breast tumors can (ER+) tumors (2, 3). In postmenopausal women, estrogens be as much as 10-fold higher than plasma levels in post- are synthesized through aromatization of circulating an- menopausal women (6). Several explanations, including drogens (mainly androstenedione and, to a minor degree, local estrogen synthesis (7–9) as well as active uptake testosterone) into estrogens (mainly E1 and, to a minor de- from the circulation (10), have been suggested. Studies at- gree, E2) in peripheral tissues (4). Inhibition of aromatase tempting to directly evaluate the contribution from local synthesis versus circulatory uptake have involved a limited number of patients and have indicated substantial interin- dividual variation (7, 11). Authors' Affiliations: 1Academic Biochemistry and 2Medicine, Royal Marsden Hospital, London, United Kingdom; 3Department of Oncology, A role for aromatase in local estrogen synthesis has Haukeland University Hospital; 4Department of Molecular Biology and been indicated by studies that reported higher levels 5Section of Oncology, Institute of Medicine, University of Bergen, of aromatase activity and mRNA in the adipose tissue Bergen, Norway; 6Institute of Medicine, University of Oslo, Faculty Division at Akershus University Hospital, Lørenskog, Norway; and directly adjacent to breast cancers compared with nor- 7Cancer Research UK Clinical Trials and Statistics Unit, Institute of mal breast tissue distant from the tumor (12, 13). Cancer Research, Sutton, Surrey, United Kingdom However, only a few weak correlations between clinical Note: Supplementary data for this article are available at Clinical Cancer outcome and biochemical measurements of aromatase Research Online (http://clincancerres.aacrjournals.org/). have been forthcoming possibly due to the low abun- Corresponding Author: Ben P. Haynes, Department of Academic Bio- dance of the enzyme (14). Immunohistochemical as- chemistry, Royal Marsden Hospital, Fulham Road, London SW3 6JJ, United Kingdom. Phone: 44-20-7808-2969; Fax: 44-2073763918; E-mail: sessment of aromatase expression has been fraught [email protected]. with difficulties. Notably, in a recent study applying a doi: 10.1158/1078-0432.CCR-09-2481 well-validated antibody, aromatase expression did not ©2010 American Association for Cancer Research. predict clinical response to letrozole in the neoadjuvant 1790 Clin Cancer Res; 16(6) March 15, 2010 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 9, 2010; DOI: 10.1158/1078-0432.CCR-09-2481 Intratumoral Estrogen Disposition in Breast Cancer showing higher E2 in tumors than in serum or nontumor Translational Relevance tissue, the concentrations reported varied widely and some studies showed no difference between premenopausal and Estrogen deprivation is a major means of endocrine postmenopausal women. This may partly be related to the – therapy of estrogen receptor positive breast cancer difficulty of accurately measuring estrogen concentrations and, through aromatase inhibition, is the most effec- in breast tissues (6, 29–32). tive treatment for postmenopausal women. Inhibition In a recent study (33), some of us confirmed elevated E2 of other enzymes involved in estrogen disposition and levels in tumor as compared with normal tissue. This relat- targeted aromatase inhibition or suppression in breast ed to ER+ tumors only. In contrast, intratumoral levels of tumors have been suggested as alternative or addition- E1 were consistently lower than normal tissue levels from al strategies. The rationale for these depends on a clear the same breast. The aim of the current study was to identify understanding of the contributors to intratumoral factors that might explain the altered estrogen disposition estradiol (E2) levels, but data on this are varied. Our between normal and malignant tissues. Thus, we correlated current study revealed significant correlations of intra- estrogen levels with mRNA levels of the estrogen receptor tumoral E2 with plasma estrogens and intratumoral (ESR1)andallthemajorestrogen-metabolizinggenes α β transcript levels of ER and some of the 17 -hydroxys- [aromatase (CYP19), STS, SULT1E1, and six isoforms of teroid dehydrogenase enzymes involved in estrogen HSD17B] by quantitative real-time PCR in these same interconversion, but not with enzymes involved in es- matched normal and malignant breast tissue samples. trogen synthesis. The data provide strongest support These data would provide correlative evidence for, or for targeting systemic, rather than intratumoral, estro- against, a role for (a) uptake due to binding to ERα,(b) gen synthesis, with HSD17B7 being a potential novel intratumoral synthesis by estrogen metabolism genes, secondary target. and/or (c) plasma estrogen concentrations as contributors to intratumoral E2 levels. In addition, the expression of two classic estrogen-dependent (reporter) genes, PGR and TFF1, was measured as markers of the intratumoral levels of E2. setting, although it was found to be a favorable prog- nostic biomarker (15). Materials and Methods Sulfatase (STS) catalyzes the formation of E1 from es- trone sulfate (E1S), which may act as a possible reservoir Patients. Details of the patients included in this study of E1 due to its high circulating concentrations and pro- have been published elsewhere (33). In brief, premeno- longed half-life (16, 17). The much higher activity of pausal and postmenopausal breast cancer patients select- STS compared with aromatase in breast cancer has been ed for mastectomy (at the Department of Surgery, taken as suggesting that it may be more important than Haukeland University Hospital, Bergen, Norway) were aromatase for intratumoral estrogen synthesis (8). In sup- eligible but were excluded if they had taken in the pre- port of this, STS mRNA levels have been shown to be sig- vious 6 months any kind of hormone replacement ther- nificantly higher in malignant breast tissue than in normal apy or any drug known to interfere with estrogen tissue (18), and high levels of expression of STS are asso- disposition. Tissues were obtained from mastectomy ciated with poor prognosis in ER+ breast cancer (19). specimens immediately on removal and snap-frozen in E1 formed as a result of aromatase or STS activity must liquid nitrogen. About 500 mg of tumor tissue and an be reduced to E2 to achieve its full biological potency, and equivalent amount of normal tissue from each of the this interconversion is done by 17β-hydroxysteroid dehy- four breast quadrants were obtained. All tissue samples drogenase (17βHSD). Multiple isoforms of this enzyme were subsequently stored in liquid nitrogen until proces- exist, with types HSD17B1, HSD17B5, HSD17B7, and sing. Blood samples (20 mL, heparinized) for plasma HSD17B12 acting reductively to catalyze the