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Decreased Expression of E6-Associated Protein in Breast and Prostate Carcinomas

Cancer Center (X.G., G.F., Z.N.), Creighton University, Omaha, Nebraska 68178; Breast Center (S.K.M.), Baylor College of Medicine, Houston, Texas 77030; and Department of Pathology (Z.G., P.S.), Creighton University Medical Center, Creighton University, Omaha, Nebraska 68131

Address all correspondence and requests for reprints to: Zafar Nawaz, Ph.D., Cancer Center, Criss III, Room 352, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178. E-mail: znawaz{at}creighton.edu.

	Abstract

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The E6-associated protein (E6-AP) is a dual function protein. It acts as an E3 ubiquitin-protein ligase as well as a steroid hormone receptor coactivator. Considering the influence of steroid hormone receptors and their coactivators in the normal development and tumorigenesis of reproductive organs of both genders, we examined the roles of E6-AP in the tumorigenesis of breast and prostate tissues. We demonstrated that the expression of E6-AP protein is decreased in human invasive breast and prostate carcinomas compared with their adjacent normal tissues, and this down-regulation of E6-AP is accompanied by the up-regulation of estrogen receptor (ER)- in breast and androgen receptor (AR) in prostate carcinomas. Furthermore, our in vivo data from E6-AP-knockout animals indicated that the expression levels of ER and AR are increased in E6-AP-null mammary and prostate glands, respectively, when compared with that of normal control animals, suggesting that E6-AP modulates the protein levels of ER in breast and AR in prostate glands.

	Introduction

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BREAST AND PROSTATE carcinomas are among the top three cancers in American women and men, respectively. It is well established that the steroid hormones estrogen, progesterone, and androgen are the principal regulators of mammary gland and/or prostate gland development and function (1, 2, 3). These steroids exert their effect on the target tissues via their cognate intracellular receptors, estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR), all of which are members of the nuclear hormone receptor superfamily and act as transcription factors. The steroids estrogen, progesterone, and androgen not only regulate the normal growth and development of their target tissues but also play important roles in tumorigenesis of these tissues (1, 2, 3). The abnormal expressions of ER and AR have been associated with cancers of breast and prostate, respectively. Furthermore, the development of resistance to the antihormone therapies for these cancers is also related with the aberrant expression of the steroid hormone receptors (SHRs) (1, 2, 3). Currently, the detailed molecular mechanisms by which the SHRs regulate the development and progression of breast cancer and prostate cancer remain largely unknown. Theoretically, any factors that can influence the expression or function of these receptors might contribute to the development and progression of breast and prostate cancers.

The SHR coactivators represent a growing class of proteins that interact with receptors in a ligand-specific manner and serve to enhance their transcriptional activity. Coactivators have been shown to possess enzymatic activities such as histone acetyltransferase, histone methyltransferase, ubiquitin conjugation, and ubiquitin-protein ligase, which contribute to their ability to enhance receptor-mediated transcription (4, 5). Ligand-activated receptors are thought to bring these activities to the promoter region of the target genes and presumably manifest part of their in vivo coactivation functions through these enzymatic activities. Because of their ability to enhance receptor-mediated gene expression, coactivators are thought to play an important role in regulating the magnitude of the biological response to hormones. The level of coactivator expression is critical in determining the activity of the receptor in target tissues, and variations in hormone responsiveness seen in the population may be due to differences in coactivator levels.

The E6-associated protein (E6-AP) is a novel SHR coactivator. It enhances the hormone-dependent transcriptional activities of various SHRs including ER and AR (6). E6-AP is also a member of the E3 class of functionally related ubiquitin-protein ligases, which have been proposed to play a major role in defining substrate specificity of the ubiquitin system (7). Protein ubiquitination also involves two other classes of enzymes, namely the E1 ubiquitin-activating enzyme (UBA) and E2 ubiquitin-conjugating enzymes (UBCs). First, the UBA activates ubiquitin in an ATP-dependent manner. The activated ubiquitin then forms a thioester bond between the carboxyl-terminal glycine residue of ubiquitin and a cysteine residue of the UBA. Next, ubiquitin is transferred from the E1 to one of the several E2s (UBCs), preserving the high-energy thioester bond. In some cases, ubiquitin is transferred directly from the E2 to the target protein. In other instances, the transfer of ubiquitin from UBCs to target proteins proceeds through an E3 ubiquitin-protein ligase intermediate such as E6-AP. The ubiquitin-tagged target proteins undergo degradation via the 26S proteasome pathway.

It has been demonstrated that the ubiquitin-proteasome pathway is required for the degradation and proper function of SHRs (4, 5, 6, 7). As an important component of the ubiquitin-proteasome pathway, E6-AP might also be required for the degradation and function of the SHRs and hence participate in the carcinogenesis of their target tissues. In this study, we examined whether changes in the expression pattern of this dual function protein, E6-AP, is involved in the carcinogenesis of breast and prostate by analyzing the expression levels of E6-AP in human breast and prostate cancer samples. Our data indicated that the expression of E6-AP is down-regulated in advanced-stage carcinomas of breast and prostate. Furthermore, this down-regulation is accompanied by up-regulation of ER and AR in breast and prostate tissues, respectively. Furthermore, we also demonstrated that E6-AP modulates the protein levels of ER and AR in these organs presumably by promoting their degradation.

	Materials and Methods

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Human tumor samples
Formalin-fixed, paraffin-embedded samples of 13 cases of invasive breast carcinomas (IBC), 20 cases of ductal carcinoma in situ (DCIS) of breast, and seven cases of prostate carcinomas were examined in this study. The tumor specimens with either internal normal tissue within the same sections (IBC and DCIS) or normal tissues taken from the tumor-adjacent normal area of the same patients (prostate cancer) were sectioned and mounted on the same slide. Use of human tissues for this study was approved by the local institutional review board.

Animal care and processing of mouse mammary/ prostate tissues
All animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the specific protocol used in this study was approved by the Creighton University Institute Animal Care and Use Committee. The E6-AP-knockout mice were bred and kept at Creighton University’s Animal Facility. The inguinal mammary glands and prostate glands were removed from E6-AP-knockout mice (8) and wild-type control littermate mice. For immunohistochemical study, the mammary tissues were fixed in 10% formalin (Fisher Scientific, Pittsburgh, PA) for 18–24 h, embedded in paraffin, and sectioned on a microtome. For Western blot analysis, the mammary gland or the total prostate gland was homogenized and analyzed by Western blot as described below.

Immunohistochemistry
Five-micrometer-thick paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through graded alcohol. Antigen was retrieved by boiling the slides in the antigen unmasking solution (Vector Laboratories, Burlingame, CA) in a microwave oven for 10 min, and endogenous peroxidase activity was quenched by a 30-min incubation of the sections in 1% hydrogen peroxide at room temperature. The sections were incubated overnight with 10% normal goat serum in Tris-buffered saline at 4 C to block the nonspecific immunoreactivity. In the case of human breast and prostate sections, antibodies against E6-AP (a rabbit polyclonal antibody; kindly donated by Dr. Norman J. Maitland, University of York, York, UK), ER (NCL-ER-6F11, a mouse monoclonal antibody; Novocastra, Newcastle, UK), and AR (441, a mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA) at dilutions of 1:200, 1:100, and 1:100, respectively, were added to the sections and incubated for 1–2 h at room temperature in a humidity chamber. For detection of the immunoreactivity, sections were then incubated with biotinylated antirabbit (for E6-AP) or antimouse (for ER and AR) antibodies (Bio-Rad Laboratories, Hercules, CA), washed in Tris-buffered saline, incubated with streptavidin-conjugated peroxidase (Vectastain ABC kit; Vector Laboratories), washed, and developed with 3,3'-diaminobenzidine (DAB substrate kit; Vector Laboratories) according to the manufacturer’s recommendations. In the case of mouse tissue sections, a rabbit anti-ER- antibody (MC-20; Santa Cruz Biotechnology) was used instead at a dilution of 1:200. Finally, the sections were counterstained with hematoxylin and coverslipped for bright-field microscopy after dehydration in graded alcohol and clearance in xylene. Negative controls were included in each experiment by using nonimmune serum instead of primary antibody.

Evaluation of immunostained slides
The levels of cellular expression of E6-AP, ER, and AR were measured on immunohistochemically stained tissue slides using the Automated Cellular Imaging System (ACIS, ChromaVision Medical Systems, Inc., San Juan Capistrano, CA). This system combines color-based imaging technology with automated microscopy to provide quantitative information on intensity of staining. This proprietary imaging technology allows objective quantification of color intensity in a range from 0 (none, black) to 255 (maximum, white).

In this process, each immunohistochemically stained glass slide was entirely scanned and digital tissue image was constructed. The pathologists then reviewed the images and selected subregions (normal ducts and neoplastic glands) for detailed analysis. Once these regions are outlined, ACIS instantly counts the individual pixels of chromagen color, converts pixel count to staining intensity, and presents a score. The processor can distinguish 256 increments of intensity for a particular color.

Expression of E6-AP is presented as the staining intensity because E6-AP is ubiquitously expressed in the breast and prostate epithelial cells. However, expression of ER and AR was further analyzed with ACIS by taking into consideration the percentage of positive nuclei (those expressing the receptors). A so-called histo-score (H score) was calculated for each case and was obtained by multiplying the percentage (P) of positive cells with the average intensity (I), i.e. H = P x I.

In vitro expression of ER
In vitro synthesis of radiolabeled human ER was performed using transcription and translation-coupled rabbit reticulocyte extracts in the presence of [³⁵S]methionine according to the manufacturer’s recommended condition (Promega, Madison, WI).

Protein degradation and ubiquitination assay
The ³⁵S-labeled human ER was incubated either with or without E6-AP purified from Escherichia coli, in a mixture containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM ATP, 10 mM MgCl₂, 0.2 mM dithiothreitol, and 4 µg of ubiquitin (Sigma, St. Louis, MO) for 1 h at 30 C. Rabbit reticulocyte provides E1 and E2 enzymes for the assay. Reactions were terminated by boiling samples in the presence of sodium dodecyl sulfate-loading buffer (100 mM Tris-HCl, pH 8.0; 200 mM dithiothreitol; 4% sodium dodecyl sulfate; 20% glycerol; and 0.2% bromophenol blue). The reaction mixtures were resolved by 10% SDS-PAGE, and radiolabeled bands were visualized by autoradiography.

Western blot analysis
Protein extracts were prepared from total mouse mammary and prostate glands by homogenizing in a buffer containing 50 mM NaCl, 20 mM HEPES, pH 7.5; 5 mM KCl; 10% glycerol; 1 mM phenylmethylsulfonyl fluoride; and protease inhibitors leupeptin (1.25 µg/ml) and pepstatin A (1.0 µg/ml). Protein concentrations were estimated by the Bradford assay (Bio-Rad Laboratories). Twenty-five micrograms of the total proteins were denatured in SDS sample buffer and analyzed by SDS-PAGE. Proteins were transferred to the nitrocellulose membranes and probed either with anti-ER rabbit polyclonal antibody (MC-20; Santa Cruz Biotechnology) and anti-AR rabbit polyclonal antibody (C-19; Santa Cruz Biotechnology) or anti-ß-actin mouse monoclonal antibody (Sigma) at a concentration of 1:800, 1:1,000, and 1:10,000, respectively. Proteins were detected using enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) following the manufacturer’s protocol.

Statistical methods
Differences between tumor samples and their matched normal tissues were tested using the Student’s paired t test. Correlation between the expression of E6-AP and ER in breast and prostate samples was analyzed using Excel software and expressed as a correlation coefficient.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
To investigate the expression profile of E6-AP in human breast cancer, we compared immunohistochemical expression of E6-AP protein in neoplastic cells of IBC and DCIS to the adjacent normal mammary tissue. Figure 1A shows the immunostaining of E6-AP in the normal breast tissue, IBC and DCIS, which was found in the same section of a breast carcinoma patient. In the normal breast tissue, E6-AP is strongly and ubiquitously expressed in epithelial cells, predominantly in the luminal cells. Compared with the normal tissue, the immunostaining intensity of E6-AP is greatly decreased in the IBC, whereas there was no significant change in the expression of E6-AP in DCIS cases. A total of 13 cases of IBC and 20 cases of DCIS were immunohistochemically stained for their expression of E6-AP, each of which had their adjacent normal gland as a control. The immunostaining intensity was evaluated using an ACIS system, and the results are shown as averages in Fig. 1B. All of the 13 IBC samples exhibited decreased immunostaining intensity compared with the normal glands. On average, there was approximately 25% reduction in the intensity of the immunostaining in tumors than in the adjacent normal tissues. The Student’s paired t test indicated that the differences were statistically significant (P = 0.00001). However, in DCIS, which represents a precursor to IBC, decreased expression of E6-AP was not observed, suggesting that the down-regulation of E6-AP protein levels is a relatively late event and is associated with the invasive phenotype.

View larger version (62K): [in this window] [in a new window]   FIG. 1. Immunohistochemical analysis of E6-AP expression in human breast tissues. A, Representative immunostaining of E6-AP in human breast tissues. Nor, Normal breast. Enlarged images are shown in the right panel, respectively. B, The average immunostaining intensity of E6-AP in both Nor and IBC as analyzed by ACIS.

View larger version (36K): [in this window] [in a new window]   FIG. 2. Immunohistochemical analysis of E6-AP and ER in breast carcinomas. A, Representative immunostaining of E6-AP and ER in IBC and DCIS. B, Average immunostaining intensity of ER in IBC and DCIS as analyzed by ACIS. C, Relative expression of E6-AP in DCIS and IBC taking the level of E6-AP in DCIS as 100. D, Relative expression of ER in DCIS and IBC taking the level of ER in DCIS as 100.

The ubiquitin-proteasome pathway accounts for the selective degradation of short-lived regulatory proteins including many nuclear hormone receptors. Furthermore, it has been shown that UBA and UBCs are necessary for the degradation and turnover of ER protein levels (4, 5, 12), which permits continuous responses to changes in the concentration of estrogen. As a rate-limiting factor in the ubiquitin-proteasome pathway, it is possible that E6-AP also plays an important role in regulation of ER protein levels. To test this hypothesis, we performed in vitro protein degradation and ubiquitination assay on ER protein in the absence and presence of E6-AP. ³⁵S-labeled ER protein was synthesized in vitro by using transcription and translation-coupled rabbit reticulocyte extracts in the presence of radiolabeled methionine. The ³⁵S-labeled ER protein was then incubated with ATP and ubiquitin either in the presence or absence of bacterially expressed E6-AP. As expected, E6-AP promoted the degradation of ER (Fig. 3A). To further confirm that E6-AP modulates the levels of ER, we also examined the ER protein levels in the mammary glands of E6-AP-knockout mice by immunohistochemistry and Western blot analysis. E6-AP-knockout mice exhibited an increased expression of ER in the mammary epithelial cells compared with their wild-type litermates (Fig. 3, B and C). These results confirmed that E6-AP modulates the levels of ER protein by promoting its degradation through the ubiquitin-proteasome pathway.

View larger version (72K): [in this window] [in a new window]   FIG. 3. Degradation of ER through the ubiquitin-proteasome pathway. A, In vitro protein degradation and ubiquitination assay. B, ER immunohistochemistry of mammary glands in wild-type and E6-AP-knockout mice. WT, Wild-type mouse mammary gland; O, knockout mouse mammary gland. C, Western blot analysis of ER expression in the mammary glands of E6-AP-knockout mice: +/+, wild-type; –/+, heterozygous; –/–, homozygous knockout. ß-Actin was used as a loading control.

Prostate cancer exhibits many similarities to breast cancer with regard to its AR expression and its switch from hormone responsiveness at the earlier stage to the hormone resistance at the later stage (2). The changes in the expression of certain AR coactivators have also been associated with prostate cancers (21, 22, 23, 24, 25). Therefore, we were interested in exploring whether the expression pattern of E6-AP is also altered in prostate cancers. Similar to that observed in breast cancer, the immunostaining intensity of E6-AP was decreased in prostate cancers when compared with the matched normal tissues from the same patient. In normal prostate tissue, E6-AP was expressed at high level in the luminal epithelial cells, whereas the expression level of E6-AP was decreased in tumors (Fig. 4A). The immunostaining of E6-AP from seven prostate tumor cases along with their normal glands was evaluated using an ACIS system. All of the tumor samples exhibited a decreased expression of E6-AP compared with the adjacent normal tissue, and the averages of the immunostaining intensity are shown in Fig. 4B. Overall, there was approximately 27% decrease in E6-AP expression in the tumors compared with the normal tissue, and this difference was statistically significant (P = 0.000022).

View larger version (57K): [in this window] [in a new window]   FIG. 4. Immunohistochemical analysis of E6-AP in human prostate cancers. A, Representative immunostaining of E6-AP in human prostate tissue. Enlarged images are shown in the right panel. B, Average immunostaining intensity of E6-AP in both the normal areas and tumor areas as analyzed by ACIS. C,Western blot analysis of AR expression in the prostate glands of E6-AP-knockout mice: +/+, wild-type; –/+, heterozygous; –/–, homozygous knockout. ß-Actin was used as a loading control.

In conclusion, we present data herein showing that E6-AP is down-regulated in breast and prostate tumors and the expression of E6-AP is inversely associated with that of ER and AR. Our data also demonstrate that E6-AP plays a role in the carcinogenesis of breast and prostate glands probably through its participation in the degradation of ER and AR proteins via the ubiquitin-proteasome pathway.

	Footnotes

 
This work was supported in part by grants to Z.N. (National Institutes of Health Grants DK 60907, DK56833, Department of Defense (DOD) DAMD 17-02-1-0281, DOD DAMD 17-99-1-9075, LB595, and LB692). X.G. is a recipient of the DOD Breast Cancer Postdoctoral Research Fellowship (DAMD 17-02-1-0281).

First Published Online February 3, 2005

Abbreviations: ACIS, Automated Cellular Imaging System; AR, androgen receptor; DCIS, ductal in situ carcinoma; E6-AP, E6-associated protein; ER, estrogen receptor; IBC, invasive breast carcinomas; PR, progesterone receptor; SHR, steroid hormone receptor; UBA, ubiquitin-activating enzyme; UBC, ubiquitin-conjugating enzyme.

Received September 8, 2004.

Accepted for publication January 20, 2005.

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This Article Abstract Full Text (PDF) All Versions of this Article: 146/4/1707    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gao, X. Articles by Nawaz, Z. Search for Related Content PubMed PubMed Citation Articles by Gao, X. Articles by Nawaz, Z.