Differential expression of estrogen receptor α, β1, and β2 in lobular and ductal breast cancer Bo Huanga, Yoko Omotob,c, Hirotaka Iwased, Hiroko Yamashitac, Tatsuya Toyamac, Raoul Charles Coombese, Aleksandra Filipovice, Margaret Warnera, and Jan-Åke Gustafssona,b,1 aCenter for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX 77204; bDepartment of BioSciences and Nutrition, Karolinska University Hospital, Huddinge, Karolinska Institutet, SE-141 86 Huddinge, Sweden; cOncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan; dDepartment of Breast and Endocrine Surgery, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan; and eDivision of Cancer, Imperial College London, London W12 0NN, United Kingdom Contributed by Jan-Åke Gustafsson, December 19, 2013 (sent for review November 1, 2013) The role of estrogen receptor (ER) α as a target in treatment of from one patient and normal tissue from another patient (8–10). breast cancer is clear, but those of ERβ1 and ERβ2 in the breast Samples taken from different patients have intrinsic limitation remain unclear. We have examined expression of all three recep- (i.e., they cannot account for variations between different patients). tors in surgically excised breast samples from two archives: (i): 187 In addition, because tumors are heterogeneous, core biopsies do invasive ductal breast cancer from a Japanese study; and (ii)20 not fully reflect the histological and biological diversity of breast lobular and 24 ductal cancers from the Imperial College. Samples tumors (29). contained normal areas, areas of hyperplasia, and in situ and in- The roles of ERβ1 and its splice variant ERβ2 in breast cancer vasive cancer. In the normal areas, ERα was expressed in not more are still unclear. As reviewed by Murphy and Leygue (30), some than 10% of epithelium, whereas approximately 80% of epithelial studies show a loss of ERβ1 as ductal cancer progresses, but cells expressed ERβ. We found that whereas ductal cancer is a others do not. Some studies show ERβ2asamarkerofbad highly proliferative, ERα-positive, ERβ-negative disease, lobular prognosis (31), and others not (19). Some of these differences cancer expresses both ERα and ERβ but with very few Ki67-posi- may be due to differences in antibody use and differences in tive cells. ERβ2 was expressed in 32% of the ductal cancers, of tissue fixation and handling. which 83% were postmenopausal. In all ERβ2-positive cancers When ERα and ERβ are coexpressed in breast cancer it is the interductal space was filled with dense collagen, and cell nuclei unclear whether tamoxifen treatment will be successful. This is expressed hypoxia-inducible factor 1α.ERβ2 expression was not because tamoxifen acts as an agonist of ERβ at activator protein confined to malignant cells but was strong in stromal, immune, 1 (AP-1) sites (32) and thus should oppose the antiproliferative and endothelial cells. In most of the high-grade invasive ductal effects of the tamoxifen–ERα complex. Yan et al. (33) have found cancers neither ERα nor ERβ was expressed, but in the high-grade that expression of ERβ predicts tamoxifen benefit in patients with lobularcancerERβ was lost and ERα and Ki67 expression were ERα-negative early breast cancer, whereas Esslimani-Sahla et al abundant. The data show a clear difference in ER expression be- (23) have found that low ERβ level is an independent marker, tween lobular and ductal breast cancer and suggest (i) that tamox- better than ERα level, to predict tamoxifen resistance. Although ifen may be more effective in late than in early lobular cancer and apparently saying different things, these two results actually agree (ii) a potential role for ERβ agonists in preventing in situ ductal with each other: in ERα-negative breast cancer, estrogen is not cancers from becoming invasive. driving proliferation, so tamoxifen via ERβ may interfere with another growth signaling pathway. In ERα-positive cancers whose ductal carcinoma in situ | invasive ductal carcinoma | proliferation is driven by E2, tamoxifen with ERβ would oppose invasive lobular carcinoma the antiproliferative effects of the ERα–tamoxifen complex. Investigation of the expression pattern of ERβ in normal tis- espite decades of research, the etiology of breast cancer sue, DCIS, and IDC is important to understand the function of Dremains unclear. It is currently thought that most breast this receptor in the progression of breast cancer. We have a set cancers occur in the normal terminal duct lobular unit and of samples obtained from surgical excision of breast tumors from progress in a stepwise fashion over time (1). Ductal carcinoma women before pharmacological intervention. The cohorts in- in situ (DCIS) means the cancer has not spread beyond the duct clude lobular cancer, which has not yet been thoroughly studied into any normal surrounding breast tissue and is thought by some for ERβ expression. Lobular cancer is an ERα-positive form of to be the direct precursor of invasive ductal carcinoma (IDC). breast cancer characterized by loss of E-cadherin and relatively Estrogens play an important role in normal breast develop- MEDICAL SCIENCES ment as well as breast cancer progression (2). Most of the effects Significance of estrogen are mediated through its two receptors: estrogen α α β β α – receptor (ER ) and (ER ) (3). ER is expressed in 50 80% Whether breast cancer will respond to the antiestrogen ta- of breast tumors, and its presence is the main indicator for anti- moxifen is determined by whether cellular proliferation is es- hormonal therapy (4). ERβ was first discovered in 1996, and its α – trogen receptor (ER) -mediated. As opposed to early ductal role in breast cancer is still being explored (5 7). cancer, which is an ERα-rich, proliferating disease, in early The first step in understanding the role of ERβ in breast α β β lobular cancer both ER and ER are abundantly expressed and cancer was to define the expression pattern of ER in the normal proliferation is rare. In advanced lobular cancer, ERβ is lost, ERα human breast and in various stages of cancer. Since its discovery, β is retained, and proliferation is high. Thus, tamoxifen may be several laboratories have reported ER expression in clinical an effective pharmaceutical in late but not early lobular cancer. samples (8–28). Most of these studies investigated the expression β – – of ER in invasive breast cancer samples (12 15, 17, 19, 21 23). Author contributions: B.H., M.W., and J.-Å.G. designed research; B.H., Y.O., and M.W. Some studies have reported ERβ expression in invasive breast performed research; H.I., H.Y., T.T., R.C.C., and A.F. contributed new reagents/analytic cancer and normal breast tissue (11, 18, 26–28), but few have tools; B.H., M.W., and J.-Å.G. analyzed data; and B.H., M.W., and J.-Å.G. wrote the paper. compared the expression of ERβ in the normal tissue, DCIS, and The authors declare no conflict of interest. IDC within the same sample. Usually tumor samples are taken 1To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1323719111 PNAS | February 4, 2014 | vol. 111 | no. 5 | 1933–1938 Downloaded by guest on September 29, 2021 Fig. 1. Expression of ERα and ERβ in normal tissue, DCIS, and IDC. (A) Representative immunohistochemical staining of ERα and ERβ in normal breast tissue, DCIS, and IDC. (Scale bars, 25 μm.) (B) Box chart of Allred scores of ERα and ERβ (n = 94) staining. Bottoms and tops of the boxes are the 25th and 75th percentiles, respectively; the lines across the boxes are the median values; the ends of the whiskers represent 10th and 90th percentiles; asterisks represent the minimum and maximum of all of the data (*P < 0.0001). (C) Box chart of Allred scores of ERα and ERβ staining (n = 115). Description of box chart is the same as in B (*P < 0.0001, **P = 0.01). (D) Box chart of Allred scores of ERα and ERβ (n = 94) staining. Description of the box chart is the same as in B (*P < 0.0001, **P = 0.03). low proliferation rate. It is accompanied by a resistance to anoikis showed that the changes in expression levels of ERα and ERβ, (34). It accounts for 10–15% of diagnosed breast cancer, and there which occur in the transition between normal epithelium and are still many questions about the optimal therapeutic approach DCIS, were significant (Fig. 1B, Left and Right,*P < 0.0001). to this cancer. We have explored the changes in expression of Progression from in situ to invasive carcinoma is the first step the two ERs using identical protocols and reagents in different in malignancy. As shown in Fig. 1 A, b and e,ERα expression was developmental stages of breastcancerwithineachpatient. Results Comparison of ERα and ERβ in Normal Breast Epithelium, DCIS, and IDC. The ERβ antibody for the immunohistochemistry is ERβ 503 IgY antibody, which has been used previously (35). Represen- tative staining is shown in Fig. 1A. There were 94 patients who had both normal tissue and DCIS, 115 patients in whose sections there was both DCIS and IDC, and 94 patient samples in which there was normal breast tissue, DCIS, and IDC. In normal breast tissue there was robust expression of ERα in approximately 10% of epithelial cells (Fig. 1 A, a), whereas ERβ was expressed in more than 70% of epithelial cells with in- termediate to strong signals (Fig. 1 A, d). In DCIS the percent- α Fig. 2. Correlation of ERα and ERβ expression with histological grades in IDC.