Estrogen Receptor A^ Negative Breast Cancer Tissues Express

Cancer Prevention

Estrogen Receptor A ^ Negative Breast Cancer Tissues Express Significant Levels of Estrogen-Independent Transcription Factors, ERB1andERB5: Potential Molecular Targets for Chemoprevention Indira Poola,1Suzanne A.W. Fuqua,4 Robert L. DeWitty,2 Jessy Abraham,1 Josephine J. Marshallack,3 and Aiyi Liu5

Abstract We have investigated the expression of two estrogen receptor h (ERh) isoforms, ERh1and ERh5, which activate gene transcription independent of estrogen or growth factors, in ERa-negative breast cancer tissues.We report here, for the first time, that ERa-negative tissues express significant levels of ERh1andERh5, and their expression levels are not different fromlevels in ERa - positive tumors. However, significant differences exist between the two racial groups, African American and Caucasian, in that the patients from the former group express higher levels of ERh1and ERh5 but not ERa.These two transcription factors could be potential molecular targets for designing chemopreventive drugs to treat ERa-negative breast cancers.

It is now well accepted that unopposed stimulation of breast isoforms, ERh1 and ERh5, which can activate the same genes as epithelial cells by the natural hormone, estrogen, plays a major the ERa, independent of estrogen (1), are expressed in these role in the advancement of breast cancers. Although the exact tissues. The rational for our study is that once we establish the mechanism(s) by which estrogen causes breast cancer progres- expression of ERh in ERa-negative tissues, a novel line of ERh- sion are not known, several studies have established that targeted drugs could be designed to treat ERa-negative tumors increased gene transcription by estrogen-activated transcription similar to ERa blockers for ERa-positive tumors. We studied the factor, the estrogen receptor a (ERa), leads to genetic/cellular ERh isoform expression at mRNA levels by quantitative real- aberrations and the genesis and progression of breast cancer. time PCR and at protein levels by Western blotting and Because endogenous estrogens directly affect the growth of immunohistochemistry. We also compared the expression of breast cancer cells, estrogen deprivation either by inhibiting its these isoform mRNA levels with ERa-positive tissues. We report biosynthesis or blocking estrogen-mediated gene transcription here for the first time that ERa-negative breast cancer tissues through ERa is the primary line of therapy for all ERa-positive have significant levels of ERh gene expression, and ERh5isthe cancers. Clinical studies have shown that only ERa-positive most abundantly expressed isoform. We also report here that tumors but not ERa-negative tumors respond to the above two African American patient tumors express significantly higher therapies. The ERa-negative patients do not have the benefits of levels of ERh isoforms compared with Caucasian patient relatively safe and effective targeted endocrine therapies, because tumors. We expect that our results on ERh isoform expression their cancers are considered to be estrogen independent. in ERa-negative breast cancers will have clinical implications in In an effort to develop alternate endocrine therapies for ERa- designing a new line of ERh-targeted molecular therapies to negative breast cancer patients, we investigated whether ERh treat these cancers.

1 Authors’ Affiliations: Departments of Biochemistry and Molecular Biology, Materials and Methods 2Surgical Oncology, and 3Pathology, Howard University College of Medicine, Washington, District of Columbia; 4Breast Center, Baylor College of Medicine, 5 HotStartTaq PCR core kits, Omniscript reverse transcriptase, and Houston, Texas; and Biometry and Mathematical Statistics Branch, National Institutes of Child Health and Human Development, NIH, Bethesda, Maryland MinElute gel extraction kits were from Qiagen, Inc. (Valencia, CA). Received 4/4/05; revised 7/8/05; accepted 7/21/05. Taqman Universal PCR Master Mix, RNase inhibitor, and random Grant support: Department of Defense Breast Cancer Research Initiative Idea hexamers were from Applied Biosystems (Foster City, CA). All the award DAMD17-02-1-0409, Susan G. Komen Breast Cancer Foundation grant primers used in the current study were synthesized by Life Technologies BCTR0100473, and National Cancer Institute grant R33 CA88347 (I. Poola). Bethesda Research Laboratories (Carlsbad, CA), and 5VFAM- and The costs of publication of this article were defrayed in part by the payment of page 3VTAMARA-labeled oligonucleotide probes described here were synthe- charges. This article must therefore be hereby marked advertisement in accordance sized at Applied Biosystems. The bp numbering for ERa and ERh with 18 U.S.C. Section 1734 solely to indicate this fact. primers and probes described here were based on the sequences Requests for reprints: Indira Poola, Department of Biochemistry and Molecular published by Green et al. (2) and Ogawa et al. (3), respectively. PCR Biology, Howard University School of Medicine, 520 W Street Northwest, Washington, DC 20059. Phone: 202-806-5554; Fax: 202-806-5553/5784; quality water and Tris-EDTA buffer were from Bio Whittaker (Rockville, E-mail: [email protected]. MD). Polyclonal antibodies against ERh (H-150) were obtained from F 2005 American Association for Cancer Research. Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and monoclonal doi: 10.1158/1078-0432.CCR-05-0728 antibodies against ERh were obtained from Genetex (San Antonio, TX).

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Protease inhibitor cocktail containing AEBSF, EDTA, Bestatin, E-64 leupeptin, and aprotinin was from Sigma (St. Louis, MO). Horseradish peroxidase–conjugated goat anti-rabbit IgG and protein molecular weight standards were from Bio-Rad (Hercules, CA). Enhanced chemi-luminescence reagents were from Amersham (Piscataway, NJ). Breast tumor samples. Breast tumor tissues were obtained from the Breast Center, Baylor College of Medicine Breast Tumor Bank (Houston, TX) and Howard University Hospital. Fresh tumor tissues were collected immediately after surgery and stored at À80jC until use. Fresh tumor tissue samples for research were routinely harvested immediately adjacent to the histologic/diagnostic sections and considered to be representative of the tissue used for diagnosis. All the samples were examined by a pathologist and tissues containing >80% cancer cells were excised and used for research. ERa status in the tissues collected from Howard University Hospital was determined immuno- Fig. 1. Amplification of ERh1and ERh5 transcripts in ERa-negative breast cancer histochemically using monoclonal antibodies against NH2-terminal tissues by RT-PCR.To show the presence of ERh1and ERh5inERa-negative breast portion of the molecule at Oncotech Laboratories. The tumor tissues cancer tissues, cDNAs fromthese tissues were amplified using a sense primer in were considered positive for ERa if >5% of cancer cells showed positive exon 1and isoform-specific antisense primers as described in Materials and h h for nuclear staining. ERa status in tumor tissues collected from Baylor Methods. ER 1- and ER 5-specific primer pairs amplified 1,165-bp and 1,154-bp products respectively.The PCR products were cloned, sequenced, and College of Medicine Breast Center Tumor Bank was determined by identified as coding sequences of ERh1and ERh5. Expression of ERh isoform ligand binding assay (4). The tissues were diagnosed as ERa positive if in ERa-negative cancer tissues. Representative products fromfive tumorcDNAs. the cancer tissue extract showed >3 fmol ER/mg total tissue extract. A PCR products fromfive ER a-positive breast cancer tissues for comparative total of 60 ERa-negative (20 from Caucasian and 40 from African purposes. Primers also amplified several lower molecular products presumably exon deletion variants. American patients) and 74 ERa-positive (34 from Caucasian and 40 from African-American patients) cancer tissues were included in the current study. Tumor collection procedures were approved by the verified by both electrophoresis in 1.5% agarose gels and amplification Institutional Review Boards of both institutions. of the constitutively expressed gene, glyceraldehyde-3-phosphate dehy- RNA extraction and cDNA synthesis. Total RNA was extracted from drogenase (GAPDH). The total RNAs were reverse transcribed using frozen breast tissues using the Trizol reagent (Life Technologies Bethesda Omniscript reverse transcriptase as previously described (5, 6). Research Laboratories) as previously described (5). RNA integrity was Conventional PCR and identification of PCR products. Conventional PCRs were done in an automatic thermal cycler (MJ Research, Waltham, MA) as previously described (7). To amplify ERh1, ERh4, and ERh5 Table 1. Primers and probes for quantitation of various sequences, a sense primer in exon 1, 5V-CGCTAGAACACACCTTACCTG- ERs by real-time PCR 3V (position, exon 1, 335-355 bp; ref. 3) and isoform-specific antisense primers, 5V-AGCACGTGGGCATTCAGC-3V (position, exon 8, 1,481- Wild type (ERh1) 1,499 bp; ref. 3), 5V-GTCTGGGTTTTATATCGTCTGC-3V (position, exon Sense, 5V-TTTGGGTGATTGCCAAGAGC-3V 8, 1,612-1,632 bp; ref. 1), and 5V-CACTTTTCCCAAATCACTTCACCCT- (position, exon 7, 1,411-1,430 bp) 3V(position, exon 8, 1,464-1,489 bp; ref. 1) respectively, were applied. Antisense, 5V-AGCACGTGGGCATTCAGC-3V All the base pair numbering is given with reference to the translational start site. The PCR amplified products (8.0 AL) were separated by (position, exon 8, 1,580-1,597 bp) electrophoresis in 1% Nu Sieve agarose gels in Tris/acetic acid/EDTA Probe, FAM 5V-CCTCCCAGCAGCAATCCATGCG-3V buffer and detected by ethidium bromide staining. PCR products TAMARA (position, exon 7,1,438-1,460 bp) were purified by gel extraction, cloned into pCR2.1-TOPO vector and h ER 5 identified by sequence analysis as previously described (8). Sense, 5V-TTTGGGTGATTGCCAAGAGC-3V Absolute quantification of ERb1, ERb5, and ERa mRNA copy numbers Antisense, 5V-CACTTTTCCCAAATCACTTCACC-3V by quantitative real-time PCR. Absolute quantification of ERh isoform (position, exon 8, 243-265 bp) and ERa mRNA copy numbers was done by quantitative real-time PCR Probe, FAM 5V-CCTCCCAGCAGCAATCCATGCG-3V in ABI Prism GeneAmp 7900HT Sequence Detection System at a TAMARA modified 50% ramp rate as previously described (9, 10). A typical real- ERa time PCR reaction mixture contained cDNA prepared from reverse transcription of 100 ng of tumor total RNA, 0.04 Amol/L sense and Sense, 5V-CAAGCCCGCTCATGATCAA-3V antisense primers, 0.05 Amol/L 5VFAM- and 3VTAMARA-labeled (position, exon 4, 1,110-1,128bp) oligonucleotide probe, and 1Â Taqman Universal PCR mix in a total Antisense, 5V-CTGATCATGGAGGGTCAAATCCAC-3V volume of 25 AL. The PCR conditions were initial hold at 50jC for 2 (position, exon 5, 1,358-1,338 bp) minutes followed by denaturation for 10 minutes at 95jCand Probe, FAM 5V-AGAACAGCCTGGCCTTGTCCCTG-3VTAMARA denaturation for 15 seconds at 95jC in the subsequent cycles and (position, exon 4, 1,140-1,162 bp) annealing and extension for 1.5 minutes at 60jC for 40 cycles. The GAPDH primer pairs and probes for the quantification of ERa,ERh1, and ERh5 Sense, 5V-TTCCAGGAGCGAGATCCCT-3V by real-time PCR are listed in Table 1. Absolute quantification of every (position, 304-322 bp) isoform was achieved compared with a standard graph that was 2 3 4 5 6 7 8 Antisense, 5V-GGCTGTTGTCATACTTCTCATGG-3V simultaneously generated using 10 ,10,10,10,10,10,10, and 9 (position, 483-505 bp) 10 copies of its reverse-transcribed cRNA (9, 10). All the samples were Probe, FAM 5V-TGCTGGCGCTGAGTACGTCGTG-3V amplified in triplicate and real-time PCRs were repeated four times for TAMARA (position, 342-363 bp) every isoform and normalized to the copy numbers of the housekeeping gene, GAPDH, as previously described (9, 10).

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Immunohistochemical staining. The presence of ERh protein was washed and incubated with peroxidase substrate (3,3V-diaminobenzi- also studied in formalin-fixed, paraffin-embedded breast cancer dine liquid chromogen, from DakoCytomation) for 5 minutes. tissues by immunohistochemistry using monoclonal antibodies Finally, the slides were washed and stained with hematoxylin, against ERh protein. Briefly, slides were deparaffinized in two mounted, and visualized under Leica DMRXA microscope. All slides changes of toluene for 5 minutes each and gradually rehydrated and micrographs for the above marker were evaluated for the through five changes of graded ethanol (100%, 90%, 70%, 50%, presence of ERh. A total of 20 tissue samples from each of the ERa- 30%, and distilled water, 2 minutes each). Antigens were unmasked negative and ERa-positive tissues were stained for ERh in duplicate by steam treating the slides in 10 mmol/L citrate buffer (pH 6.0) for by the above procedure. ERh staining was compared between ERa- 25 minutes. Tissue sections were incubated with blocking buffer positive and ERa-negative tissues by scoring nuclear staining intensity (supplied with the antibody) and then with mouse anti-ERh (1:100 and the proportion of positively stained nuclei, as described by dilution) overnight. The slides were rinsed and incubated with Harvey et al. (11). Slides were scored independently by two EnVision peroxidase conjugated secondary antibody (DakoCytoma- pathologists, and mean scores were compared between ERa-positive tion, Mississauga, Ontario, Canada) for 30 minutes. The slides were and ERa-negative tissues.

Table 2. Expression of ERh1, ER h5, and ERa mRNA Table 2. Expression of ERh1, ER h5, and ERa mRNA (copies/1010 copies of GAPDH) in breast cancer (copies/1010 copies of GAPDH) in breast cancer tissues fromAfrican-American patients tissues fromAfrican-American patients (Cont’d)

No ERA by IHC ERB1ERB5ER A No ERA by IHC ERB1ERB5ER A

1 À 1 Â106 2 Â 107 NA 42 + 7 Â 105 3 Â 106 2 Â 107 2 À 4 Â 105 1 Â105 NA 43 + 4 Â 104 4 Â 106 2 Â 108 3 À 6 Â 105 6 Â 105 NA 44 + 3 Â 105 8 Â 105 6 Â 106 4 À 4 Â 105 2 Â 105 NA 45 + 4 Â 105 1 Â106 4 Â 107 5 À 9 Â 104 3 Â 105 NA 46 + 5 Â 106 5 Â 106 4 Â 108 6 À 1 Â107 1 Â107 NA 47 + 8 Â 107 5 Â 1010 5 Â 109 7 À 8 Â 104 2 Â 105 NA 48 + 3 Â 104 3 Â 106 2 Â 107 8 À 7 Â 104 8 Â 105 NA 49 + 5 Â 104 1 Â106 6 Â 107 9 À 1 Â106 5 Â 108 NA 50 + 2 Â 105 3 Â 106 5 Â 106 10 À 2 Â 105 3 Â 105 NA 51 + 3 Â 105 3 Â 106 4 Â 107 11 À 6 Â 105 1 Â106 NA 52 + 2 Â 105 1 Â106 3 Â 107 12 À 7 Â 105 5 Â 106 NA 53 + 7 Â 104 8 Â 105 1 Â106 13 À 4 Â 105 4 Â 106 NA 54 + 4 Â 105 2 Â 106 2 Â 108 14 À 4 Â 105 3 Â 106 NA 55 + 1 Â106 4 Â 106 3 Â 107 15 À 8 Â 104 2 Â 105 NA 56 + 6 Â 105 3 Â 106 6 Â 106 16 À 2 Â 105 2 Â 106 NA 57 + 5 Â 105 2 Â 106 1 Â107 17 À 4 Â 104 6 Â 105 NA 58 + 3 Â 106 2 Â 107 1 Â107 18 À 4 Â 107 3 Â 107 NA 59 + 1 Â106 3 Â 106 6 Â 107 19 À 2 Â 105 3 Â 106 NA 60 + 3 Â 104 6 Â 104 4 Â 107 20 À 4 Â 105 3 Â 106 NA 61 + 3 Â 105 2 Â 106 1 Â108 21 À 1 Â106 9 Â 106 NA 62 + 8 Â 104 1 Â106 4 Â 107 22 À 4 Â 105 1 Â106 NA 63 + 5 Â 105 1 Â106 6 Â 107 23 À 6 Â 105 1 Â106 NA 64 + 5 Â 104 8 Â 105 6 Â 107 24 À 7 Â 104 2 Â 106 NA 65 + 5 Â 107 8 Â 108 4 Â 108 25 À 5 Â 104 4 Â 105 NA 66 + 9 Â 106 5 Â 106 4 Â 107 26 À 1 Â104 3 Â 106 NA 67 + 4 Â 104 1 Â107 8 Â 107 27 À 6 Â 105 9 Â 105 NA 68 + 5 Â 104 3 Â 106 2 Â 107 28 À 3 Â 104 5 Â 106 NA 69 + 5 Â 106 1 Â107 1 Â108 29 À 3 Â 105 1 Â106 NA 70 + 5 Â 105 3 Â 107 3 Â 107 30 À 9 Â 105 7 Â 106 NA 71 + 3 Â 104 2 Â 105 6 Â 106 31 À 9 Â 104 4 Â 106 NA 72 + 7 Â 106 1 Â107 4 Â 107 32 À 8 Â 105 2 Â 106 NA 73 + 4 Â 105 2 Â 106 1 Â106 33 À 3 Â 105 3 Â 107 NA 74 + 3 Â 104 7 Â 105 4 Â 107 34 À 4 Â 106 4 Â 107 NA 75 + 4 Â 105 7 Â 106 2 Â 106 35 À 2 Â 104 5 Â 106 NA 76 + 4 Â 105 2 Â 106 3 Â 107 36 À 3 Â 105 2 Â 105 NA 77 + 7 Â 104 1 Â106 5 Â 107 37 À 3 Â 104 2 Â 105 NA 78 + 2 Â 105 1 Â105 2 Â 107 38 À 2 Â 105 3 Â 105 NA 79 + 5 Â 104 1 Â106 6 Â 107 39 À 5 Â 104 3 Â 105 NA 80 + 4 Â 105 3 Â 106 3 Â 107 40 À 8 Â 105 5 Â 106 NA 41 + 1 Â105 3 Â 106 5 Â 106 Abbreviations: NA, not applicable; IHC, immunohistochemistry.

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Table 3. Expression of ERh1, ER h5, and ERa mRNA Table 3. Expression of ERh1, ER h5, and ERa mRNA (copies/1010 copies of GAPDH) in breast cancer (copies/1010 copies of GAPDH) in breast cancer tissues fromCaucasian patients tissues fromCaucasian patients (Cont’d)

No ERA by IHC ERB1ERB5ER A No ERA by IHC ERB1ERB5ER A

1 À 9 Â 104 2 Â 105 NA 29 + 4 Â 104 2 Â 105 4 Â 108 2 À 2 Â 104 1 Â105 NA 30 + 1 Â104 4 Â 105 2 Â 107 3 À 8 Â 104 2 Â 106 NA 31 + 2 Â 105 1 Â106 2 Â 107 4 À 2 Â 105 1 Â106 NA 32 + 2 Â 105 1 Â106 1 Â107 5 À 4 Â 104 2 Â 106 NA 33 + 1 Â104 5 Â 105 1 Â107 6 À 5 Â 104 3 Â 106 NA 34 + 8 Â 104 6 Â 105 1 Â108 7 À 4 Â 104 6 Â 105 NA 35 + 8 Â 104 8 Â 105 1 Â107 8 À 2 Â 104 5 Â 105 NA 36 + 1 Â104 1 Â105 4 Â 105 9 À 7 Â 104 3 Â 105 NA 37 + 4 Â 104 4 Â 105 4 Â 106 10 À 5 Â 104 2 Â 105 NA 38 + 8 Â 104 5 Â 105 4 Â 107 11 À 6 Â 104 6 Â 105 NA 39 + 7 Â 104 2 Â 105 1 Â106 12 À 3 Â 104 6 Â 105 NA 40 + 8 Â 104 5 Â 105 4 Â 107 13 À 3 Â 104 2 Â 106 NA 41 + 8 Â 104 1 Â106 4 Â 108 14 À 5 Â 104 6 Â 105 NA 42 + 7 Â 104 4 Â 106 6 Â 107 15 À 2 Â 104 5 Â 105 NA 43 + 6 Â 104 9 Â 105 2 Â 108 16 À 7 Â 104 5 Â 104 NA 44 + 3 Â 104 8 Â 105 5 Â 107 17 À 5 Â 104 3 Â 105 NA 45 + 1 Â105 1 Â106 4 Â 107 18 À 8 Â 104 7 Â 105 NA 46 + 1 Â104 3 Â 105 1 Â106 19 À 2 Â 105 2 Â 106 NA 47 + 1 Â104 3 Â 105 3 Â 107 20 À 3 Â 104 3 Â 104 NA 48 + 4 Â 104 1 Â105 3 Â 107 21 + 8 Â 104 1 Â107 2 Â 107 49 + 6 Â 104 2 Â 106 3 Â 106 22 + 2 Â 104 1 Â106 9 Â 105 50 + 8 Â 104 8 Â 105 2 Â 108 23 + 2 Â 104 3 Â 105 5 Â 107 51 + 3 Â 104 9 Â 105 2 Â 107 24 + 9 Â 104 1 Â105 3 Â 107 52 + 4 Â 104 2 Â 106 1 Â108 5 5 8 25 + 1 Â104 1 Â106 4 Â 107 53 + 1 Â10 8 Â 10 3 Â 10 4 5 7 26 + 5 Â 104 6 Â 105 2 Â 107 54 + 4 Â 10 1 Â10 5 Â 10 27 + 1 Â104 2 Â 105 6 Â 105 28 + 7 Â 104 1 Â105 4 Â 106 Abbreviations: NA, not applicable; IHC, immunohistochemistry.

Protein extraction, electrophoresis, Western blotting, and other terone receptor status was also tested using Wilcoxon rank sum test methods. For extracting total proteins from tumor samples, f10 (nonparametric ANOVA). Test results were considered significant if mg of fresh frozen tumor tissues were homogenized for 5 minutes at P V 0.05. 4jC using 100 AL of 10 mmol/L Tris-HCl buffer (pH 7.6) containing 150 mmol/L NaCl, 1% Triton 100-X, and 1% sodium Results deoxycholate using a T line laboratory stirrer. The extracts were centrifuged at 15,000 Â g for 30 minutes at 4jCandthe ERa-negative breast cancer tissues have significant levels of j h supernatant was stored at À80 C. The presence of ER protein(s) ERb gene expression. We first tested the expression of ERh1, in 20 AL (20 ERa negative and 20 ERa positive) extracts were tested ERh4, and ERh5inERa-negative tissues by conventional by Western blotting using a dilution of 1:200 anti-ERh polyclonal reverse transcription-PCR (RT-PCR) using a sense primer in antibodies (H-150). A 20-AL tumor extract was also probed for the h expression of a housekeeping gene, b-actin, using a dilution of 1:100 exon 1 and isoform-specific antisense primers. The ER 1- and h anti-actin polyclonal antibodies from Santa Cruz Biotechnology. ER 5-specific primer pairs generated expected PCR products of Protein gel electrophoresis and Western blotting were done as 1,165 and 1,154 bp, which were identified by sequence described previously (12, 13). SDS-PAGE (15%) was conducted in a analyses as ERh1 and ERh5, respectively (Fig. 1). However, Bio-Rad slab gel apparatus as described by Laemmli (14). Proteins the ERh4-specific primer pair did not generate any product, were transblotted to nitrocellulose membranes as described by indicating the absence of this isoform in breast cancer tissues, Towbin et al. (15). Blocking and antibody treatments were done consistent with previous observations (10). as described (12, 13). The antigen-antibody complexes were detected We next quantitatively determined ERh1andERh5 using a 1:7,500 dilution of the horseradish peroxidase–conjugated expressions at mRNA levels using molecular assays developed goat anti-rabbit IgG and development with the enhanced chemilu- by us (10) based on reverse transcriptase quantitative minescence detection system. Statistical analysis. The expression of ERh isoforms was compared real-time PCR and isoform-specific primers and probes between ERa-positive and ERa-negative tumors and between two (Table 1). Using the quantitative methods, we were able racial groups using Wilcoxon rank sum test (two sided). The to precisely quantify the exact copy numbers of each iso- association between the expression of every ERh isoform with grade, form mRNAs with respect to the mRNA copy numbers of stage, nodal status, histologic type, menopausal status, and proges- the housekeeping gene, GAPDH. For comparative purposes,

Clin Cancer Res 2005;11(20) October 15, 2005 7582 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. ERb Gene Expression in ERa-Negative Breast Cancers we also determined the expression levels of the above receptors and wild-type ERa in ERa-positive tumor tissues by real-time PCR. The expression levels of ERh1 and ERh5 in ERa-negative and ERa-positive tissues are shown in Tables 2 and 3. The mean values and SD are presented in Table 4 and shown as histograms in Fig. 2. ERa-negative tissues expressed significant levels of ERh1 and ERh5, and their expression levels are not statistically different from ERa-positive tissues. In addition to the mRNA levels, we also established the presence of ERh protein(s) in ERa-negative patient tumors, by Western blotting the tumor extracts and immunohistochemistry of formalin-fixed, paraffin-embedded samples. The expression of ERh protein(s) in eight representative ERa-negative and seven ERa-positive tumor tissues by Western blotting is shown in

Fig. 3. Expression of ERh proteins in ERa-negative breast cancer tissues by Western blotting.To show the presence of ERh proteins, the cancer tissues were homogenized and proteins extracted as described in Materials and Methods.The tumor extracts (20 AL) were separated by SDS-PAGE and immunoblotted using polyclonal antibodies against ERh.Two closely spaced bands of Mr 55 to 58 kDa were observed showing the presence of ERh protein(s) in these tissues. ERh protein expression in ERa-positive cancer tissues for comparative purposes. No significant differences were observed in ERh protein expression levels ERa-positive or ERa-negative tumor tissues.

Fig. 3. Two closely spaced bands of Mr 55 to 58 kDa were visualized when the Western blots were probed with polyclonal antibodies specific to ERh protein. All the 20 samples tested from each group were positive and gave similar pattern by this procedure. To determine any differences in the protein levels between ERa-positive and ERa-negative tissues, the ERh protein bands in the Western blots were scanned, normalized to the housekeeping gene, bactin, and the normalized values were compared between ERa-negative and ERa-positive tissues. By this procedure, we did not find any significant differences in the levels of ERh proteins in ERa-positive and ERa-negative samples. By immunohistochemistry, we observed strong nuclear staining when probed with monoclonal antibodies obtained from Genetex (Fig. 4) in ERa-negative tissues. The polyclonal antibodies, although they detected ERh protein(s) in Western blots, were not suitable for immunohistochemistry. All 20 tissues tested from each group were positive for ERh by immunohistochemistry procedure. For comparative purposes, the ERh protein expression in ERa-positive tissues by immunohistochemistry is also shown in Fig. 4. Expression levels of ERb isoforms are different in breast tumors of Caucasian and African-American patient groups. To test whether ERh gene expression in the two racial groups are similar, we compared the data obtained on mRNA levels of ERh isoforms by quantitative real-time PCR (Tables 2 and 3) using statistical procedures described in Materials and Methods. Statistical analyses of data showed that African-American patient tumors expressed significantly higher levels of these receptor mRNAs in both types of tissues compared with Caucasian patient tumors. (ERa-negative tissues, P = 0.0048 for ERh1 and P = 0.0213 for ERh5; ERa-positive tumors, P = 0.0004 for ERh1 and P = 0.0002 for ERh5; all by two-sided Wilcoxon rank sum tests). Interestingly, ERa mRNA levels in ERa-positive tissues were not significantly different in the two Fig. 2. Expressionlevels of ERh1and ERh5transcriptsinERa-negative and positive racial groups (Tables 2 and 3 and Table 4; Fig. 2). However, the tissues fromAfrican-American and Caucasian patient tumors.Columns, mean expression levels of these two receptors in the two racial groups; bars, SD.The expression levels of the above two receptor mRNAs were not expression level of ERa is also shown in the positive tissues. associated with tumor grade, stage of the cancer, histologic

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Fig. 4. ERh protein expression in ERa-negative breast cancer tissues by immunohistochemistry.To show ERh protein expression in the ERa-negative tissues, paraffin sections fromthese tissues were immunostained using monoclonal antibodies against ERh as described in Materials and Methods. Strong staining could be visualized in the nuclei of ductal epithelial cells. Representative of immunohistochemically stained ERa-negative tissues. A representative fromER a-positive tissues for comparative purposes.

type, menopausal status, progesterone receptor, or nodal status and ERh5 could contribute to tumor progression by activating either in the ERa-positive or ERa-negative tumors (data not the transcription of cancer-promoting genes, independent of shown). estrogen or growth factors. However, there were no reports to show whether ERa-negative breast cancer tissues express Discussion significant levels of ERh isoforms. A number of groups investigated the expression of ERh in It is now widely accepted that aberrant expression of breast cancer tissues by RT-PCR and immunohistochemical growth-promoting genes by the transcription factor, the ERa, methods. All the reports to date established that breast cancer signaled through estrogen or growth factors, promotes survival tissues express ERh mRNA and protein, although at levels and progression of breast cancer cells. When the ERa lower than the normal breast tissues (19–25). However, expression is lost, it is assumed that breast cancer cells gain most of the studies conducted thus far focused on ERa- the ability to progress in the absence of estrogen. Although the positive tissues, and there is little information on ERh mechanism(s) by which the cancer-promoting genes are expression in ERa-negative tumors. Jenson et al. (26) studied expressed in the absence of ERa are not known, it is the expression of ERh by immunohistochemistry in 11 ERa- presumably by other transcription factors that have the ability negative tumor tissues and reported its presence in seven to activate their expression independent of estrogen. One tissues. Shaw et al. (27) studied in six ERa-negative tissues group of molecules that can activate the same genes as ERa in by RT PCR and 17 tissues by immunohistochemistry. They the absence of estrogen or growth factors includes ERh reported the presence of ERh mRNA in 3 of 6 and protein in isoforms, ERh1 and ERh5 (1, 16, 17). The isoform ERh5 7 of 17 tissues studied. However, none of the above studies was recently cloned by our group and shown to have thrice distinguished between ERh1 and ERh5 or reported quantita- higher estrogen-independent transcriptional activity than tive differences between ERa-positive and ERa-negative ERh1 (1). tumors. In the cells where both ERa and ERh isoforms are expressed, In the current study, we investigated the expression levels of the primary function of ERhs seems to regulate the degree of ERh1 and ERh5 isoforms in ERa-negative tissues at mRNA estrogen action by negatively modulating ERa,andthe using isoform-specific molecular assays and protein levels by estrogen-independent transcriptional activity of ERh isoforms immunohistochemical and Western blotting methods. The is inhibited by ERa (1, 16–18). In the absence of inhibiting rational for our studies is that once we establish the presence ERa, as in the case of ERa-negative breast cancer tissues, ERh1 of ERh isoforms in ERa-negative tissues, these molecules

Table 4. Expression levels of ERh1and ERh5(meanandSD)inERa-negative and ERa-positive breast cancer tissues (copies/1010 of GAPDH)

Isoform ERA negative ERA positive African-American Caucasian African-American Caucasian ERh15Â 10 5 and 1 Â10 5 6 Â 10 4 and 1 Â10 4 1 Â10 6 and 3 Â 10 5 6 Â 10 4 and 8 Â 10 3 ERh52Â 107 and 1 Â107 9 Â 10 5 and 2 Â10 5 3 Â 10 7 and 2 Â 10 7 1 Â10 6 and 3 Â 10 5 ERa NA NA 6 Â 10 7 and 1 Â10 7 7 Â 10 7 and 2 Â 10 7

Abbreviation: NA, not applicable.

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could be targeted for molecular therapy similar to ERa- due to low levels of ERh1, which could not be detected by blocking drugs for ERa-positive tumors. Inhibiting the immunohistochemistry and lack of interaction of antibodies estrogen-independent gene activation by ERhs could slow or with ERh5. completely block the progression of ERa-negative cancers. The When we compared the levels of ERh isoforms in tumors data presented here established that ERa-negative tissues have from African-American patients with Caucasian patients, the significant levels of ERh gene expression. The data presented tumors from African-American patients showed significantly above also established for the first time that ERa-negative higher levels of these two receptors. This trend is seen both tissues express ERh isoforms at levels similar to ERa-positive in ERa-positive and ERa-negative tissues (Fig. 2; Table 4). tissues, and ERh5, which was recently been characterized by us The higher levels of ERh isoforms, particularly the most (1), is the most abundant isoform. These observations show abundant estrogen-independent transcription factor, ERh5, that the ERh expression is independent of ERa gene ex- may contribute, in part, to poor survival observed in African- pression. Although the expression of ERh1 is far less than ERa American patients (28). Given the success of ERa-blocking levels, ERh5 levels are comparable with ERa levels of the ERa- drugs for inhibiting tumor growth of ERa-positive tumors, positive tissues (Tables 2, 3 and 4). the drugs that can block ERh1orERh5 or both could be Although all tumor tissues analyzed expressed both ERh1 potential targeted therapies for treating ERa-negative tissues. and ERh5 isoforms, a wide variation was observed in The ERh-targeted therapies could particularly benefit African- between tissues as seen in Table 2. However, the variation American patients, because these patients express comparatively in the expression levels of neither ERh1 nor ERh5 signifi- higher levels of ERh isoforms and bear disproportionately cantly correlated with tumor characteristics. Similar observa- higher percentage of ERa-negative tumors (29, 30). tions were made by Fuqua et al. (21). They reported the presence of ERh in 76% of 242 tissues by immunohisto- Acknowledgments chemistry, but the presence did not correlate with tumor grade or S-phase fraction. The negativity observed in 24% of We thank Rakesh Bhatnagar for immunohistochemical staining of breast cancer tumors by Fuqua et al. and others (21, 26, 27) was probably tissue slides for ERh.

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Indira Poola, Suzanne A.W. Fuqua, Robert L. De Witty, et al.

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