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RESEARCH ARTICLE Open Access
Immunohistochemical analysis of ezrin-radixin-moesin-binding phosphoprotein 50 in prostaticadenocarcinomaTanner L Bartholow1*, Michael J Becich2, Uma R Chandran2 and Anil V Parwani3
Background: Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is an adapter protein which has beenshown to play an active role in a wide variety of cellular processes, including interactions with proteins related toboth tumor suppression and oncogenesis. Here we use immunohistochemistry to evaluate EBP50s expression innormal donor prostate (NDP), benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia(HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), andmetastatic prostatic adenocarcinoma (Mets).
Methods: Tissue microarrays were immunohistochemically stained for EBP50, with the staining intensitiesquantified using automated image analysis software. The data were statistically analyzed using one-way ANOVAwith subsequent Tukey tests for multiple comparisons. Eleven cases of NDP, 37 cases of NAC, 15 cases of BPH, 35cases of HGPIN, 103 cases of PCa, and 36 cases of Mets were analyzed in the microarrays.
Results: Specimens of PCa and Mets had the lowest absolute staining for EBP50. Mets staining was significantlylower than NDP (p = 0.027), BPH (p = 0.012), NAC (p < 0.001), HGPIN (p < 0.001), and PCa (p = 0.006). Additionally,HGPIN staining was significantly higher than NAC (p < 0.009) and PCa (p < 0.001).
Conclusions: To our knowledge, this represents the first study comparing the immunohistochemical profiles ofEBP50 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC and suggests that EBP50 expression isdecreased in Mets. Given that PCa also had significantly higher expression than Mets, future studies are warrantedto assess EBP50s potential as a prognostic biomarker for prostate cancer.
BackgroundProstate cancer is currently the second leading cause ofcancer death in males. Despite this, in the era ofprostate specific antigen (PSA) screening, researchershave now estimated that clinically insignificant prostatecancer is actually overdiagnosed at a rate of 29% forwhites and 44% for blacks, the PSA screen resulting inthe detection of cancers that otherwise would only havebeen detected during autopsy in up to 15% and 37% oftumors in whites and blacks, respectively.There is currently a limited amount of information in
the literature on biomarkers with the potential to dis-cern which cases of prostate cancer have the greatest
potential to metastasize versus remain latent. Evaluat-ing the expression of tumor suppressor proteins thathave been previously examined in other cancers mayindicate novel biomarkers for prostate cancer that havethe potential to assess individual patient prognosis andguide therapy selection.One such biomarker is ezrin-radixin-moesin-binding
phosphoprotein 50 (EBP50), which is also known as Na+/H+ exchanger regulatory factor 1, or NHERF1. A 50kDa, 358 amino acid adaptor protein whose gene islocated at 17q25.1, it consists of two PSD-95/DiscsLarge/ZO-1 (PDZ) domains and a carboxyl-terminalregion that is capable of binding members of the ezrin-radixin-moesin (ERM) protein family[4-6]. With its mul-tiple domains, it has been described as a participant inat least 30 unique cellular interactions, including thoseinvolving ion transport, secondarily coupled signaling
* Correspondence: email@example.com Contributed equally1University of Pittsburgh School of Medicine, Pittsburgh, PA, USAFull list of author information is available at the end of the article
Bartholow et al. BMC Urology 2011, 11:12http://www.biomedcentral.com/1471-2490/11/12
2011 Bartholow et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.
receptors, and tyrosine kinase receptors. Among theoncologically relevant functions for the protein thathave been demonstrated are its ability to recruit thetumor suppressor PTEN for inactivation of the phospha-tidylinositol-3-OH kinase (PI3K)/Akt signaling pathwayin glioblastoma multiforme[5,7], as well as an ability toprovide cortical stabilization of b-catenin at cellularjunctions in murine embryonic fibroblast models,both indicative of a tumor suppressor function.Further supporting this notion, additional work has
shown that an allele for EBP50 was deleted in 28 of 48examined breast cancer cell lines. Knocking-out exist-ing EBP50 expression in T47D and MCF7 breast cancercell lines has also been shown to lead to increased cellproliferation. Zheng et al. have additionally notedthat restoring EBP50 expression to a MDA-MB-231breast cancer line, originally deficient in EBP50, inhib-ited cell growth and increased apoptosis. Subsequentto this, the same group prepared a stably transfectedHeLa-EBP50 clone, which also demonstrated decreasedcell growth, suggesting a tumor suppressor role forEBP50 in cervical cancer as well.In spite of this, a universal tumor suppressor function
for EBP50 has not been observed. EBP50 has beenshown to be overexpressed in hepatocellular carcinoma. Cytoplasmic over expression has also been linkedto the progression of colorectal carcinoma. Further-more, in contrast to the previously described work,Song et al. have reported that EBP50 immunoreactivityin breast cancer was positively associated with tumorstage and lymph node involvement, promptingothers to suggest that its role in oncogenesis or tumorsuppression may vary with cellular location, with amembranous or apical distribution supporting a tumorsuppressor function and a cytoplasmic distribution con-ferring oncogenic properties .To our knowledge, the expression of EBP50 has never
been studied in prostate cancer. Here, we compare theimmunohistochemical profiles in a series of 11 cases ofnormal donor prostate (NDP), 37 cases of normal tissueadjacent to prostatic adenocarcinoma (NAC), 15 casesof benign prostatic hyperplasia (BPH), 35 cases of high-grade prostatic intraepithelial neoplasia (HGPIN), 103cases of primary prostatic adenocarcinoma (PCa), and36 cases of metastatic prostatic adenocarcinoma (Mets)in order to examine if either a tumor suppressor oroncogenic function for EBP50 can be suggested in pros-tate cancer, providing further information about itspotential as a diagnostic and/or prognostic biomarker.
MethodsTissue Microarray Block PreparationTissue microarray (TMA) blocks were constructed usingspecimens obtained from the Health Sciences Tissue
Bank at the University of Pittsburgh Medical Center,with the tissue bank rendering the honest broker ser-vices. All specimens were originally obtained withinformed consent. Cores from the appropriate case spe-cific paraffin-embedded tissue blocks were assembledinto TMAs as described in a previous protocol. Thefinal TMAs consisted of 11 cases of NDP, 37 cases ofNAC, 15 cases of BPH, 35 cases of isolated HGPIN (noaccompanying cancer diagnosed), 103 cases of PCa, and36 cases of Mets. No specimens of HGPIN included inthis study were diagnosed at the time as containing PCa.All cases were initially prepared so that each one wouldbe represented at least in triplicate. Due to variations inTMA processing, however, some cases were only able tobe represented in duplicate. This occurred for threecases of the HGPIN, three cases of the Mets, three casesof the NAC, two cases of the BPH, and eight cases ofthe PCa. In such instances, these cases were still scoredand included as a part of the final analysis.
ImmunohistochemistryEach TMA block was deparaffinized and then rehy-drated with incremental ethanol concentrations. Decloa-ker was then used for heat induced epitope retrieval,followed by a 5 minute TBS buffer rinse. A Dako auto-stainer was then used to stain the TMAs with anti-EBP50 (working dilution 1:400), a mouse monoclonalantibody (Catalogue # MA1-19291) from Thermo Scien-tific (Waltham, MA). Immunolabeling was conductedusing Dako Dual Envision + Polymer (Catalogue #K4061) from Dako (Carpinteria, CA). The slides werecounterstained with hematoxylin and coverslipped.
Scoring of SlidesAll slides for this project were scanned as digital wholeslides images (WSI) using ScanScope XT (by Aperio,Vista, CA). The individual tissue cores for each WSIwere viewed using Aperio ImageScope (Version184.108.40.2066) and scored by applying the Positive PixelCount Algorithm to each one. In order to detect theEBP50 staining, a hue value of 0.1 and hue width of 0.5was chosen for the algorithm, corresponding to the sug-gested range for the detection of brown immunostainingusing the software (Aperio Positive Pixel Count Algo-rithm instruction manual). By analyzing the averagepixel intensity with a predetermined hue value andwidth, the stromal tissue and cell nuclei that appearblue and do not feature the immunostain are negated bythe software and excluded from the final analysis thatdetermines the average staining intensity. This, in effect,controls for the glandular to stromal tissue ratio presentin the TMA cores.The validity of using Aperio software for quantitative
immunohistochemistry has been previously documented
Bartholow et al. BMC Urology 2011, 11:12http://www.biomedcentral.com/1471-2490/11/12
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in other studies[16,17]. The average staining intensitywas then determined by the software for each core, uti-lizing a formula that sums the intensities of weak, mod-erate, and strong staining pixels and divides t