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Clinical Cancer Research Vol. 11, 7579-7585, October 15, 2005
© 2005 American Association for Cancer Research

Cancer Prevention

Estrogen Receptor {alpha}–Negative Breast Cancer Tissues Express Significant Levels of Estrogen-Independent Transcription Factors, ERß1 and ERß5: Potential Molecular Targets for Chemoprevention

Indira Poola1, Suzanne A.W. Fuqua4, Robert L. De Witty2, Jessy Abraham1, Josephine J. Marshallack3 and Aiyi Liu5

Authors' Affiliations: Departments of 1 Biochemistry and Molecular Biology, 2 Surgical Oncology, and 3 Pathology, Howard University College of Medicine, Washington, District of Columbia; 4 Breast Center, Baylor College of Medicine, Houston, Texas; and 5 Biometry and Mathematical Statistics Branch, National Institutes of Child Health and Human Development, NIH, Bethesda, Maryland

Requests for reprints: Indira Poola, Department of Biochemistry and Molecular Biology, Howard University School of Medicine, 520 W Street Northwest, Washington, DC 20059. Phone: 202-806-5554; Fax: 202-806-5553/5784; E-mail: ipoola{at}howard.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
We have investigated the expression of two estrogen receptor ß (ERß) isoforms, ERß1 and ERß5, which activate gene transcription independent of estrogen or growth factors, in ER{alpha}-negative breast cancer tissues. We report here, for the first time, that ER{alpha}-negative tissues express significant levels of ERß1 and ERß5, and their expression levels are not different from levels in ER{alpha} 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 ERß1 and ERß5 but not ER{alpha}. These two transcription factors could be potential molecular targets for designing chemopreventive drugs to treat ER{alpha}-negative breast cancers.

It is now well accepted that unopposed stimulation of breast epithelial cells by the natural hormone, estrogen, plays a major role in the advancement of breast cancers. Although the exact mechanism(s) by which estrogen causes breast cancer progression are not known, several studies have established that increased gene transcription by estrogen-activated transcription factor, the estrogen receptor {alpha} (ER{alpha}), leads to genetic/cellular aberrations and the genesis and progression of breast cancer. Because endogenous estrogens directly affect the growth of breast cancer cells, estrogen deprivation either by inhibiting its biosynthesis or blocking estrogen-mediated gene transcription through ER{alpha} is the primary line of therapy for all ER{alpha}-positive cancers. Clinical studies have shown that only ER{alpha}-positive tumors but not ER{alpha}-negative tumors respond to the above two therapies. The ER{alpha}-negative patients do not have the benefits of relatively safe and effective targeted endocrine therapies, because their cancers are considered to be estrogen independent.

In an effort to develop alternate endocrine therapies for ER{alpha}-negative breast cancer patients, we investigated whether ERß isoforms, ERß1 and ERß5, which can activate the same genes as the ER{alpha}, independent of estrogen (1), are expressed in these tissues. The rational for our study is that once we establish the expression of ERß in ER{alpha}-negative tissues, a novel line of ERß-targeted drugs could be designed to treat ER{alpha}-negative tumors similar to ER{alpha} blockers for ER{alpha}-positive tumors. We studied the ERß isoform expression at mRNA levels by quantitative real-time PCR and at protein levels by Western blotting and immunohistochemistry. We also compared the expression of these isoform mRNA levels with ER{alpha}-positive tissues. We report here for the first time that ER{alpha}-negative breast cancer tissues have significant levels of ERß gene expression, and ERß5 is the most abundantly expressed isoform. We also report here that African American patient tumors express significantly higher levels of ERß isoforms compared with Caucasian patient tumors. We expect that our results on ERß isoform expression in ER{alpha}-negative breast cancers will have clinical implications in designing a new line of ERß-targeted molecular therapies to treat these cancers.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
HotStartTaq PCR core kits, Omniscript reverse transcriptase, and MinElute gel extraction kits were from Qiagen, Inc. (Valencia, CA). Taqman Universal PCR Master Mix, RNase inhibitor, and random hexamers were from Applied Biosystems (Foster City, CA). All the primers used in the current study were synthesized by Life Technologies Bethesda Research Laboratories (Carlsbad, CA), and 5'FAM- and 3'TAMARA-labeled oligonucleotide probes described here were synthesized at Applied Biosystems. The bp numbering for ER{alpha} and ERß primers and probes described here were based on the sequences published by Green et al. (2) and Ogawa et al. (3), respectively. PCR quality water and Tris-EDTA buffer were from Bio Whittaker (Rockville, MD). Polyclonal antibodies against ERß (H-150) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and monoclonal antibodies against ERß were obtained from Genetex (San Antonio, TX). 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 chemiluminescence 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 –80°C 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. ER{alpha} status in the tissues collected from Howard University Hospital was determined immunohistochemically using monoclonal antibodies against NH2-terminal portion of the molecule at Oncotech Laboratories. The tumor tissues were considered positive for ER{alpha} if >5% of cancer cells showed positive for nuclear staining. ER{alpha} status in tumor tissues collected from Baylor College of Medicine Breast Center Tumor Bank was determined by ligand binding assay (4). The tissues were diagnosed as ER{alpha} positive if the cancer tissue extract showed >3 fmol ER/mg total tissue extract. A total of 60 ER{alpha}-negative (20 from Caucasian and 40 from African American patients) and 74 ER{alpha}-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 Institutional Review Boards of both institutions.

RNA extraction and cDNA synthesis. Total RNA was extracted from frozen breast tissues using the Trizol reagent (Life Technologies Bethesda Research Laboratories) as previously described (5). RNA integrity was verified by both electrophoresis in 1.5% agarose gels and amplification of the constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The total RNAs were reverse transcribed using Omniscript reverse transcriptase as previously described (5, 6).

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 ERß1, ERß4, and ERß5 sequences, a sense primer in exon 1, 5'-CGCTAGAACACACCTTACCTG-3' (position, exon 1, 335-355 bp; ref. 3) and isoform-specific antisense primers, 5'-AGCACGTGGGCATTCAGC-3' (position, exon 8, 1,481-1,499 bp; ref. 3), 5'-GTCTGGGTTTTATATCGTCTGC-3' (position, exon 8, 1,612-1,632 bp; ref. 1), and 5'-CACTTTTCCCAAATCACTTCACCCT-3'(position, exon 8, 1,464-1,489 bp; ref. 1) respectively, were applied. All the base pair numbering is given with reference to the translational start site. The PCR amplified products (8.0 µL) were separated by electrophoresis in 1% Nu Sieve agarose gels in Tris/acetic acid/EDTA buffer and detected by ethidium bromide staining. PCR products were purified by gel extraction, cloned into pCR2.1-TOPO vector and identified by sequence analysis as previously described (8).

Absolute quantification of ERß1, ERß5, and ER{alpha} mRNA copy numbers by quantitative real-time PCR. Absolute quantification of ERß isoform and ER{alpha} mRNA copy numbers was done by quantitative real-time PCR in ABI Prism GeneAmp 7900HT Sequence Detection System at a modified 50% ramp rate as previously described (9, 10). A typical real-time PCR reaction mixture contained cDNA prepared from reverse transcription of 100 ng of tumor total RNA, 0.04 µmol/L sense and antisense primers, 0.05 µmol/L 5'FAM- and 3'TAMARA-labeled oligonucleotide probe, and 1x Taqman Universal PCR mix in a total volume of 25 µL. The PCR conditions were initial hold at 50°C for 2 minutes followed by denaturation for 10 minutes at 95°C and denaturation for 15 seconds at 95°C in the subsequent cycles and annealing and extension for 1.5 minutes at 60°C for 40 cycles. The primer pairs and probes for the quantification of ER{alpha}, ERß1, and ERß5 by real-time PCR are listed in Table 1. Absolute quantification of every isoform was achieved compared with a standard graph that was simultaneously generated using 102, 103, 104, 105, 106, 107, 108, and 109 copies of its reverse-transcribed cRNA (9, 10). All the samples were amplified in triplicate and real-time PCRs were repeated four times for every isoform and normalized to the copy numbers of the housekeeping gene, GAPDH, as previously described (9, 10).


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  		Table 1. Primers and probes for quantitation of various ERs by real-time PCR

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

Protein extraction, electrophoresis, Western blotting, and other methods. For extracting total proteins from tumor samples, ~10 mg of fresh frozen tumor tissues were homogenized for 5 minutes at 4°C using 100 µL of 10 mmol/L Tris-HCl buffer (pH 7.6) containing 150 mmol/L NaCl, 1% Triton 100-X, and 1% sodium deoxycholate using a T line laboratory stirrer. The extracts were centrifuged at 15,000 x g for 30 minutes at 4°C and the supernatant was stored at –80°C. The presence of ERß protein(s) in 20 µL (20 ER{alpha} negative and 20 ER{alpha} positive) extracts were tested by Western blotting using a dilution of 1:200 anti-ERß polyclonal antibodies (H-150). A 20-µL tumor extract was also probed for the expression of a housekeeping gene, ß-actin, using a dilution of 1:100 anti-actin polyclonal antibodies from Santa Cruz Biotechnology. Protein gel electrophoresis and Western blotting were done as described previously (12, 13). SDS-PAGE (15%) was conducted in a Bio-Rad slab gel apparatus as described by Laemmli (14). Proteins were transblotted to nitrocellulose membranes as described by Towbin et al. (15). Blocking and antibody treatments were done as described (12, 13). The antigen-antibody complexes were detected using a 1:7,500 dilution of the horseradish peroxidase–conjugated goat anti-rabbit IgG and development with the enhanced chemiluminescence detection system.

Statistical analysis. The expression of ERß isoforms was compared between ER{alpha}-positive and ER{alpha}-negative tumors and between two racial groups using Wilcoxon rank sum test (two sided). The association between the expression of every ERß isoform with grade, stage, nodal status, histologic type, menopausal status, and progesterone receptor status was also tested using Wilcoxon rank sum test (nonparametric ANOVA). Test results were considered significant if P ≤ 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
ER{alpha}-negative breast cancer tissues have significant levels of ERß gene expression. We first tested the expression of ERß1, ERß4, and ERß5 in ER{alpha}-negative tissues by conventional reverse transcription-PCR (RT-PCR) using a sense primer in exon 1 and isoform-specific antisense primers. The ERß1- and ERß5-specific primer pairs generated expected PCR products of 1,165 and 1,154 bp, which were identified by sequence analyses as ERß1 and ERß5, respectively (Fig. 1). However, the ERß4-specific primer pair did not generate any product, indicating the absence of this isoform in breast cancer tissues, consistent with previous observations (10).



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  		Fig. 1. Amplification of ERß1 and ERß5 transcripts in ER{alpha}-negative breast cancer tissues by RT-PCR. To show the presence of ERß1 and ERß5 in ER{alpha}-negative breast cancer tissues, cDNAs from these tissues were amplified using a sense primer in exon 1 and isoform-specific antisense primers as described in Materials and Methods. ERß1- and ERß5-specific primer pairs amplified 1,165-bp and 1,154-bp products respectively. The PCR products were cloned, sequenced, and identified as coding sequences of ERß1 and ERß5. Expression of ERß isoform in ER{alpha}-negative cancer tissues. Representative products from five tumor cDNAs. PCR products from five ER{alpha}-positive breast cancer tissues for comparative purposes. Primers also amplified several lower molecular products presumably exon deletion variants.

 
We next quantitatively determined ERß1 and ERß5 expressions at mRNA levels using molecular assays developed by us (10) based on reverse transcriptase quantitative real-time PCR and isoform-specific primers and probes (Table 1). Using the quantitative methods, we were able to precisely quantify the exact copy numbers of each isoform mRNAs with respect to the mRNA copy numbers of the housekeeping gene, GAPDH. For comparative purposes, we also determined the expression levels of the above receptors and wild-type ER{alpha} in ER{alpha}-positive tumor tissues by real-time PCR. The expression levels of ERß1 and ERß5 in ER{alpha}-negative and ER{alpha}-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. ER{alpha}-negative tissues expressed significant levels of ERß1 and ERß5, and their expression levels are not statistically different from ER{alpha}-positive tissues.


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  		Table 2. Expression of ERß1, ERß5, and ER{alpha} mRNA (copies/1010 copies of GAPDH) in breast cancer tissues from African-American patients

 

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  		Table 3. Expression of ERß1, ERß5, and ER{alpha} mRNA (copies/1010 copies of GAPDH) in breast cancer tissues from Caucasian patients

 

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  		Table 4. Expression levels of ERß1 and ERß5 (mean and SD) in ER{alpha}-negative and ER{alpha}-positive breast cancer tissues (copies/1010 of GAPDH)

 


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  		Fig. 2. Expression levels of ERß1 and ERß5 transcripts in ER{alpha}-negative and positive tissues from African-American and Caucasian patient tumors. Columns, mean expression levels of these two receptors in the two racial groups; bars, SD. The expression level of ER{alpha} is also shown in the positive tissues.

 
In addition to the mRNA levels, we also established the presence of ERß protein(s) in ER{alpha}-negative patient tumors, by Western blotting the tumor extracts and immunohistochemistry of formalin-fixed, paraffin-embedded samples. The expression of ERß protein(s) in eight representative ER{alpha}-negative and seven ER{alpha}-positive tumor tissues by Western blotting is shown in 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 ERß 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 ER{alpha}-positive and ER{alpha}-negative tissues, the ERß protein bands in the Western blots were scanned, normalized to the housekeeping gene, ßactin, and the normalized values were compared between ER{alpha}-negative and ER{alpha}-positive tissues. By this procedure, we did not find any significant differences in the levels of ERß proteins in ER{alpha}-positive and ER{alpha}-negative samples.



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

 
By immunohistochemistry, we observed strong nuclear staining when probed with monoclonal antibodies obtained from Genetex (Fig. 4) in ER{alpha}-negative tissues. The polyclonal antibodies, although they detected ERß protein(s) in Western blots, were not suitable for immunohistochemistry. All 20 tissues tested from each group were positive for ERß by immunohistochemistry procedure. For comparative purposes, the ERß protein expression in ER{alpha}-positive tissues by immunohistochemistry is also shown in Fig. 4.



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

 
Expression levels of ERß isoforms are different in breast tumors of Caucasian and African-American patient groups. To test whether ERß gene expression in the two racial groups are similar, we compared the data obtained on mRNA levels of ERß 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. (ER{alpha}-negative tissues, P = 0.0048 for ERß1 and P = 0.0213 for ERß5; ER{alpha}-positive tumors, P = 0.0004 for ERß1 and P = 0.0002 for ERß5; all by two-sided Wilcoxon rank sum tests). Interestingly, ER{alpha} mRNA levels in ER{alpha}-positive tissues were not significantly different in the two racial groups (Tables 2 and 3 and Table 4; Fig. 2). However, the expression levels of the above two receptor mRNAs were not associated with tumor grade, stage of the cancer, histologic type, menopausal status, progesterone receptor, or nodal status either in the ER{alpha}-positive or ER{alpha}-negative tumors (data not shown).


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
It is now widely accepted that aberrant expression of growth-promoting genes by the transcription factor, the ER{alpha}, signaled through estrogen or growth factors, promotes survival and progression of breast cancer cells. When the ER{alpha} expression is lost, it is assumed that breast cancer cells gain the ability to progress in the absence of estrogen. Although the mechanism(s) by which the cancer-promoting genes are expressed in the absence of ER{alpha} are not known, it is presumably by other transcription factors that have the ability to activate their expression independent of estrogen. One group of molecules that can activate the same genes as ER{alpha} in the absence of estrogen or growth factors includes ERß isoforms, ERß1 and ERß5 (1, 16, 17). The isoform ERß5 was recently cloned by our group and shown to have thrice higher estrogen-independent transcriptional activity than ERß1 (1).

In the cells where both ER{alpha} and ERß isoforms are expressed, the primary function of ERßs seems to regulate the degree of estrogen action by negatively modulating ER{alpha}, and the estrogen-independent transcriptional activity of ERß isoforms is inhibited by ER{alpha} (1, 16–18). In the absence of inhibiting ER{alpha}, as in the case of ER{alpha}-negative breast cancer tissues, ERß1 and ERß5 could contribute to tumor progression by activating the transcription of cancer-promoting genes, independent of estrogen or growth factors. However, there were no reports to show whether ER{alpha}-negative breast cancer tissues express significant levels of ERß isoforms.

A number of groups investigated the expression of ERß in breast cancer tissues by RT-PCR and immunohistochemical methods. All the reports to date established that breast cancer tissues express ERß mRNA and protein, although at levels lower than the normal breast tissues (19–25). However, most of the studies conducted thus far focused on ER{alpha}-positive tissues, and there is little information on ERß expression in ER{alpha}-negative tumors. Jenson et al. (26) studied the expression of ERß by immunohistochemistry in 11 ER{alpha}-negative tumor tissues and reported its presence in seven tissues. Shaw et al. (27) studied in six ER{alpha}-negative tissues by RT PCR and 17 tissues by immunohistochemistry. They reported the presence of ERß mRNA in 3 of 6 and protein in 7 of 17 tissues studied. However, none of the above studies distinguished between ERß1 and ERß5 or reported quantitative differences between ER{alpha}-positive and ER{alpha}-negative tumors.

In the current study, we investigated the expression levels of ERß1 and ERß5 isoforms in ER{alpha}-negative tissues at mRNA using isoform-specific molecular assays and protein levels by immunohistochemical and Western blotting methods. The rational for our studies is that once we establish the presence of ERß isoforms in ER{alpha}-negative tissues, these molecules could be targeted for molecular therapy similar to ER{alpha}-blocking drugs for ER{alpha}-positive tumors. Inhibiting the estrogen-independent gene activation by ERßs could slow or completely block the progression of ER{alpha}-negative cancers. The data presented here established that ER{alpha}-negative tissues have significant levels of ERß gene expression. The data presented above also established for the first time that ER{alpha}-negative tissues express ERß isoforms at levels similar to ER{alpha}-positive tissues, and ERß5, which was recently been characterized by us (1), is the most abundant isoform. These observations show that the ERß expression is independent of ER{alpha} gene expression. Although the expression of ERß1 is far less than ER{alpha} levels, ERß5 levels are comparable with ER{alpha} levels of the ER{alpha}-positive tissues (Tables 2, 3 and 4).

Although all tumor tissues analyzed expressed both ERß1 and ERß5 isoforms, a wide variation was observed in between tissues as seen in Table 2. However, the variation in the expression levels of neither ERß1 nor ERß5 significantly correlated with tumor characteristics. Similar observations were made by Fuqua et al. (21). They reported the presence of ERß in 76% of 242 tissues by immunohistochemistry, but the presence did not correlate with tumor grade or S-phase fraction. The negativity observed in 24% of tumors by Fuqua et al. and others (21, 26, 27) was probably due to low levels of ERß1, which could not be detected by immunohistochemistry and lack of interaction of antibodies with ERß5.

When we compared the levels of ERß isoforms in tumors from African-American patients with Caucasian patients, the tumors from African-American patients showed significantly higher levels of these two receptors. This trend is seen both in ER{alpha}-positive and ER{alpha}-negative tissues (Fig. 2; Table 4). The higher levels of ERß isoforms, particularly the most abundant estrogen-independent transcription factor, ERß5, may contribute, in part, to poor survival observed in African-American patients (28). Given the success of ER{alpha}-blocking drugs for inhibiting tumor growth of ER{alpha}-positive tumors, the drugs that can block ERß1 or ERß5 or both could be potential targeted therapies for treating ER{alpha}-negative tissues. The ERß-targeted therapies could particularly benefit African-American patients, because these patients express comparatively higher levels of ERß isoforms and bear disproportionately higher percentage of ER{alpha}-negative tumors (29, 30).


   Acknowledgments
 
We thank Rakesh Bhatnagar for immunohistochemical staining of breast cancer tissue slides for ERß.


   Footnotes
 
Grant support: Department of Defense Breast Cancer Research Initiative Idea award DAMD17-02-1-0409, Susan G. Komen Breast Cancer Foundation grant BCTR0100473, and National Cancer Institute grant R33 CA88347 (I. Poola).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 4/05; revised 7/ 8/05; accepted 7/21/05.


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 Materials and Methods
 Results
 Discussion
 References
 

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A. M. Shaaban, A. R. Green, S. Karthik, Y. Alizadeh, T. A. Hughes, L. Harkins, I. O. Ellis, J. F. Robertson, E. C. Paish, P. T.K. Saunders, et al.
Nuclear and Cytoplasmic Expression of ER{beta}1, ER{beta}2, and ER{beta}5 Identifies Distinct Prognostic Outcome for Breast Cancer Patients
Clin. Cancer Res., August 15, 2008; 14(16): 5228 - 5235.
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I. Poola, J. Abraham, J. J. Marshalleck, Q. Yue, V. B. Lokeshwar, G. Bonney, and R. L. DeWitty
Molecular Risk Assessment for Breast Cancer Development in Patients with Ductal Hyperplasias
Clin. Cancer Res., February 15, 2008; 14(4): 1274 - 1280.
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I. Poola, B. Shokrani, R. Bhatnagar, R. L. DeWitty, Q. Yue, and G. Bonney
Expression of Carcinoembryonic Antigen Cell Adhesion Molecule 6 Oncoprotein in Atypical Ductal Hyperplastic Tissues Is Associated with the Development of Invasive Breast Cancer
Clin. Cancer Res., August 1, 2006; 12(15): 4773 - 4783.
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