EXPRESSION OF EZRIN IN PROSTATIC INTRAEPITHELIAL NEOPLASIA SEE-TONG PANG, XIOALEI FANG, ALEXANDER VALDMAN, GUNNAR NORSTEDT, ÅKE POUSETTE, LARS EGEVAD, AND PETER EKMAN
ABSTRACT Objectives. To study the protein expression and gene copy number of ezrin in a set of high-grade prostatic intraepithelial neoplasia (HGPIN) samples with concomitant prostate cancer. Ezrin is a cytoskeleton linker protein that is actively involved in regulating the growth and metastatic capacity of cancer cells. Methods. Nineteen HGPIN samples obtained from radical prostatectomy specimens were used for the study. Among them, 13 samples also contained invasive prostate cancer. The expression of ezrin was studied by immunohistochemistry. The same samples were also used for fluorescence in situ hybridization to study the gene copy number of ezrin. Results. Immunoreactivity for ezrin was absent or weak in benign prostatic epithelial cells. Weak or moderate immunostaining was detected in 11 of 13 prostate cancer specimens. However, the immunostaining was moderate or strong in all HGPIN samples. In addition, whenever HGPIN and prostate cancer were found in the same sample, the staining was always more intense in the HGPIN cells than in the cancer cells. No alteration was found in the gene copy number detected by fluorescence in situ hybridization. Conclusions. We have shown that ezrin is overexpressed in HGPIN and prostate cancer compared with adjacent benign prostatic epithelium. In addition, HGPIN has a greater expression level of ezrin compared with that of prostate cancer. Our results indicate that aberrant expression of ezrin might be involved in the pathogenesis of prostate cancer, and ezrin expression may be useful for the diagnosis of HGPIN. UROLOGY 63: 609–612, 2004. © 2004 Elsevier Inc.
rostate cancer is the leading cancer among men in the industrialized Western world.1,2 Organconfined prostate cancer can be cured by surgical treatment or radiotherapy. For advanced prostate cancer, endocrine therapy is still the mainstay of treatment. However, most advanced prostate canThis study was supported by a research grant from the Swedish Cancer Foundation, Swedish Medical Research Council (13X08556), Maud and Birger Gustavsson’s Foundation, Johanna Hagstrand and Sigfrid Linne´r Foundation, Percy Falk Foundation, and Chang Gung Memorial Hospital (to S.T.P). From the Departments of Molecular Medicine, Urology, Women and Child Health, Andrology Center, and Pathology and Cytology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden; Division of Urology, Department of Surgery, Chang Gung Hospital, Tao-Yuan, Taiwan; Department of Urology, Qilu Hospital, Shandong University, Jinan, China; and Department of Urology, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia Reprint requests: See-Tong Pang, M.D., Division of Urology, Department of Surgery, Chang Gung Hospital, No. 5 Fushing Street, Kweishan, Tao-Yuan 333, Taiwan Submitted: July 15, 2003, accepted (with revisions): September 29, 2003 © 2004 ELSEVIER INC. ALL RIGHTS RESERVED
cers will transform into hormone-resistant cancer, and those patients often die of their disease. Efficient treatment of hormone-resistant prostate cancer is still lacking.2 Understanding the pathogenesis of prostate cancer and the mechanisms involved in cancer progression could help us to improve the management of the disease. In a previous study, we identified many novel androgen-regulated genes that may be important in the growth regulation of the prostate gland.3 We have shown that ezrin is downregulated in androgen withdrawal-induced apoptosis and upregulated in androgen replacement-stimulated proliferation in rat ventral prostatic epithelial cells. Ezrin is a member of the ezrin-raxidin-moesin family, which share a homology with the amino-terminal membrane-binding domain of erythrocyte band 4.1 and possesses membrane-cytoskeleton linking functions.4 It has been shown that ezrin plays a positive role in maintaining cell shape and cell polarity and participates in membrane-trafficking pathways, cell migration, cell signaling, growth 0090-4295/04/$30.00 doi:10.1016/j.urology.2003.09.068 609
regulation, and differentiation.5 Ezrin can interact with several membrane proteins, including CD44,6 CD43,7 intercellular adhesion molecule-1 and intercellular adhesion molecule-2, and phosphatidylinositol (4,5)-bisphosphate.8 In addition, it can signal cell survival through the phosphatidylinositol 3-kinase/Akt pathway.9 Because of its unique functions, ezrin is also actively involved in tumor biology, especially in regulating the growth and metastatic capacity of cancer. Overexpression of ezrin has been detected in several human epithelial tumors, including brain hemangioblastoma,10 uterine endometrioid adenocarcinoma,11 osteosarcoma,12 and uveal malignant melanoma.13 To date, no study has specifically evaluated the expression of ezrin in prostate cancer or precursor lesions. In this study, our aim was to assess the patterns of ezrin expression in high-grade prostatic epithelial neoplasia (HGPIN), prostate cancer, and benign prostatic tissue. In parallel, we also investigated its gene copy number by fluorescence in situ hybridization (FISH) on the same samples. MATERIAL AND METHODS TUMOR SAMPLES The study included 19 radical prostatectomy specimens collected from January to December 2000 at our department. The mean patient age at surgery was 61.3 years (range 50 to 74). None of the patients had received hormonal treatment or radiotherapy before prostatectomy. The prostatectomy specimens were selected for the study because they contained welldefined areas of HGPIN. From each specimen, one tissue section with HGPIN was selected. In 13 of them, foci of invasive prostate cancer were also present in the same section. In 13 (68%) and 6 (32%) of the cases, the main tumors originated from the peripheral and transitional zone, respectively. Only one specimen showed seminal vesicle invasion, and nine specimens (47%) had capsule penetration. The tumors were obtained with informed consent, and the local ethical committee approved the study.
IMMUNOHISTOCHEMISTRY The prostates were fixed overnight in 10% buffered formalin. The specimens were inked and sliced horizontally at 4-mm intervals. The slices were cut in two to six segments (usually quadrants), and the entire prostate was subsequently blocked in standard cassettes. The specimens were dehydrated, embedded in paraffin, sectioned at 4 m, and stained with hematoxylin-eosin. Cancer and HGPIN were outlined on the slides by one of us (L.E.). Standard biotin-avidin-complex immunohistochemistry was performed using a mouse monoclonal antibody against ezrin (Ab-1; Neomarker, Lab Vision, Calif) at a dilution of 1:300. The specificity of this antibody was confirmed with Western blot analysis; it does not cross-react with other ezrinraxidin-moesin family proteins. Placenta tissue was used for positive and negative controls for the immunostaining. The intensity of the immunohistochemical staining was scored by two observers (L.E. and S.T.P.) in an open discussion. The cytoplasmic immunostaining intensity in the cancer areas was scored as absent, weak, moderate, or strong compared with adjacent normal epithelial cells. The staining in 610
HGPIN was also compared with the staining of the cancer areas in the same samples. Scoring was performed without knowledge of any clinical features.
FLUORESCENCE IN SITU HYBRIDIZATION The sections from the same paraffin blocks were used for FISH analysis. However, three samples were omitted because of technical problems. The specific ezrin gene probe was isolated from the BAC clone RP11-507C10 (obtained from Children’s Hospital, Oakland, Calif), and the centromere probe of chromosome 6 (pEDZ6) was kindly provided by Cytogenetic Unit, University of Bari, Italy using a QIAGEN Plasmid Midi Kit (QIAGEN, Hilden, Germany). Both probes were directly labeled with SpectrumRed or SpectrumGreen (Vysis, Downers Grove, Ill) fluorophore conjugated with dUTP using standard nick translation (Vysis). The labeled probes were then coprecipitated using ethanol/sodium acetate together with Cot-1 (Vysis) and carrier DNA. FISH was performed on 4-m, formalin-fixed, paraffin-embedded tumor sections. After dewaxing and a mild digestion with pepsin, the slides were rinsed in 2 ⫻ SSC, dehydrated in graded ethanol, and air dried. Twenty microliters of hybridization mixture containing 100 ng of each probe, 55% formamide, 10% dextran, and 2 ⫻ SSC was applied to each slide. The slides were sealed with cover slips and denatured with the probe mixture simultaneously at 92°C for 10 minutes. The hybridization was performed at 37°C overnight (14 to 16 hours) in a humidified chamber. The cover slips were removed, and the slides were washed in 2 ⫻ SSC for 10 minutes at 72°C, dehydrated in graded ethanol, air dried, and mounted in antifade solution containing DAPI (Vector Laboratories). The slides were then analyzed by two observers (X.F. and S.T.P.) under an epifluorescent microscope using different band filters for DAPI/FITC/TRITC (Nikon, Tokyo, Japan). HGPIN and invasive prostate cancer areas were identified in immunostained slides, and the corresponding areas on FISH slides were marked with India ink. One hundred nuclei in the marked areas were counted for each hybridization.
RESULTS EZRIN EXPRESSION IN BENIGN PROSTATIC EPITHELIUM, HGPIN, AND CANCER Immunohistochemistry showed that ezrin was present in the cytoplasm of prostatic epithelial cells, and the basal cells occasionally stained positive, especially in reactive basal cell proliferations. Immunoreactivity was absent or weak in benign prostatic epithelial cells. HGPIN was positive for ezrin in all cases. When HGPIN and benign epithelium were found in the same gland, an abrupt transition occurred from atypical cells with positive immunostaining to benign cells with negative staining (Fig. 1A). Weak or moderate immunostaining was detected in 11 of 13 prostate cancers, and the immunostaining was heterogeneous in all cases. However, the immunostaining was moderate or strong in all areas of HGPIN. In addition, when HGPIN and prostate cancer were found in the same sample, the staining in HGPIN was more intense than that in the invasive cancer (Fig. 1B). FISH ANALYSIS Sixteen HGPIN and nine prostate cancer samples with good FISH signals were analyzed. The overall UROLOGY 63 (3), 2004
FIGURE 1. (A) HGPIN with abrupt transition from atypical cells was positive for ezrin (bottom right) compared with negative benign cells (left) within same gland. Original magnification ⫻ 400. (B) Stronger immunostaining for ezrin in HGPIN (asterisks) compared with adjacent invasive prostate cancer (arrows). Basal cells occasionally stained positive, especially in reactive basal cell proliferations. Original magnification ⫻ 200.
ratio of green/red fluorescence (gene copy/chromosome) detected in both lesions was nearly 1. Hence, no ezrin gene amplification or deletion was detected. COMMENT In this study, we have shown that ezrin, a cytoskeleton linker, has a greater expression level in HGPIN and prostate cancer compared with normal prostatic epithelium. Moreover, the expression of ezrin was always stronger in HGPIN than in the concomitant prostate cancer specimens. This aberrant expression of ezrin cannot be explained by a corresponding alteration on the genomic level, as UROLOGY 63 (3), 2004
shown by FISH. In a previous study, we found that ezrin was regulated by androgens in the rat ventral prostate.3 Therefore, the aberrant expression noted in HGPIN and prostate cancer may be related to the androgen responsive status of ezrin. Deregulation of androgen-responsive genes in prostate cancer is not uncommonly seen.14 Additional investigation of the mechanism of ezrin regulation in HGPIN and prostate cancer would be of interest. PIN is an intraductal or intra-acinar epithelial proliferation with significant nuclear atypia in the secretory (luminal) cells.15,16 Similar to dysplasia in the uterine cervix, it has been suggested that PIN is a precursor of invasive cancer.15,17–19 PIN is more common in prostates with cancer than in prostates without cancer.20 –23 The molecular event involved in PIN development is still unclear. Common genetic changes have been detected in PIN and prostate cancer, suggesting a link between these lesions.24 Ezrin is mapped to chromosome 6q25-26. Although deletion of chromosome 6q is frequently seen in prostate cancer,25 we were unable to detect any ezrin copy number change in either HGPIN or prostate cancer specimens. Interestingly, HGPIN is not the only precancerous lesion with overexpression of ezrin. Overexpression has also been found in atypical endometrial hyperplasia, a precursor lesion of endometrial adenocarcinoma.11 Therefore, ezrin expression in cancer precursor lesions may be important for tumorigenesis. Overexpression of ezrin has been correlated to the metastatic potential of several cancers, as previously mentioned. However, it has been shown that ezrin can bind to key adhesion proteins E-cadherin and beta-catenin, and removal of ezrin by antisense treatment increases the invasive capacity of colorectal epithelial tumor cell lines.26 The high expression level of ezrin in HGPIN compared with in prostate cancer is, therefore, intriguing. A possible function of ezrin may instead be to strengthen the cells’ ability to retain their normal growth pattern. Only a limited amount of research is available regarding the functional role of ezrin in prostate cancer. Recently, Harrison et al.27 have shown that ezrin can co-localize with CD44 in DU145 and PC3, and the CD44/ezrin complex is involved in the capture and invasion of endothelial cells by these prostate cancer cells. CD44 is a transmembrane protein family that has been implicated in cell migration and tumor metastasis, including prostate cancer.28,29 Therefore, whether the expression level of ezrin could be a relevant factor for invasiveness of prostate cancer awaits additional studies. CONCLUSIONS We have shown that ezrin is overexpressed in HGPIN and prostate cancer compared with in adjacent benign prostatic epithelium. In addition, a 611
greater expression of ezrin is seen in HGPIN compared with in prostate cancer. However, FISH analysis of the ezrin gene was unremarkable, suggesting a lack of mutations and that an increase in the copy number of the gene cannot account for the increased expression. Our results indicate that the aberrant expression of ezrin might be involved in the pathogenesis of prostate cancer, and ezrin expression may be used as a tool for the diagnosis of HGPIN. Additional studies on a larger series of prostate cancer specimens are warranted to confirm these findings. ACKNOWLEDGMENT. To Margareta Rodensjo¨ for technical assistance with immunohistochemistry. REFERENCES 1. Mettlin C: Recent developments in the epidemiology of prostate cancer. Eur J Cancer 33: 340 –347, 1997. 2. Reid P, Kantoff P, and Oh W: Antiandrogens in prostate cancer. Invest New Drugs 17: 271–284, 1999. 3. Pang ST, Dillner K, Wu X, et al: Gene expression profiling of androgen deficiency predicts a pathway of prostate apoptosis that involves genes related to oxidative stress. Endocrinology 143: 4897–4906, 2002. 4. Sato N, Funayama N, Nagafuchi A, et al: A gene family consisting of ezrin, radixin and moesin: its specific localization at actin filament/plasma membrane association sites. J Cell Sci 103(Pt 1): 131–143, 1992. 5. Tsukita S, and Yonemura S: ERM (ezrin/radixin/moesin) family: from cytoskeleton to signal transduction. Curr Opin Cell Biol 9: 70 –75, 1997. 6. Tsukita S, Oishi K, Sato N, et al: ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 126: 391–401, 1994. 7. Serrador JM, Nieto M, Alonso-Lebrero JL, et al: CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts. Blood 91: 4632–4644, 1998. 8. Heiska L, Alfthan K, Gronholm M, et al: Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2): regulation by phosphatidylinositol 4, 5-bisphosphate. J Biol Chem 273: 21893–21900, 1998. 9. Gautreau A, Poullet P, Louvard D, et al: Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci USA 96: 7300 –7305, 1999. 10. Bohling T, Turunen O, Jaaskelainen J, et al: Ezrin expression in stromal cells of capillary hemangioblastoma: an immunohistochemical survey of brain tumors. Am J Pathol 148: 367–373, 1996. 11. Ohtani K, Sakamoto H, Rutherford T, et al: Ezrin, a membrane-cytoskeletal linking protein, is highly expressed in
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