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l arti opo ros , Mia nd Mil ond Mi 17 Sep Ab P tin and M EPO a car a (PIN 30 R igh-gr BP R scor EPOR or EPO score and Gleason score in PCa. Conclusions: Up-regulation of EPOR has a more important role in comparison with EPO in prostate carcinogenesis. Differential ove bet Key 1. ing tio adv tum mo vas car tiss lev clo [1, (05 653 riginal 107 doi ARTICLE IN PRESS r-expression of EPOR and EPO in benign and malignant prostatic tissue is ascribed to different mechanisms involved in up-regulation ween EPO and EPOR. © 2009 Elsevier Inc. All rights reserved. words: Prostate carcinoma; Erythropoietin; Erythropoietin receptor; Hypoxia Introduction Hypoxia is a common phenomenon in solid tumors, result- from inadequate blood supply with impaired vascular func- n. Hypoxia stress has not always been considered to be erse to the survival and growth of tumors. Actually, once or cells adapt to the hypoxia environment, they become re aggressive as hypoxia directs the remodeling of tumor culature or phenotypic changing of tumor cells. Hypoxia regions has been demonstrated in human prostate cinoma (PCa) by analyzing the extent of hypoxia in tumor ues with Eppendorf pO2 microelectrode, and increasing els of hypoxia in prostate carcinomas were reported to be sely associated with increasing clinical stage and patient age 2]. Moreover, over-expression of hypoxia-inducible fac- tor-1� (HIF-1�), the surrogate marker of hypoxia, was shown in human PCa, and its expression was considered as an early event for prostate carcinogenesis [3]. Once the transcription factor HIF-1� is activated, more than 60 putative target genes will actively participate in tumor cells’ response to hypoxia stress. Of them, erythro- poietin (EPO) with its receptor (EPOR), a member of the type I cytokine receptor superfamily, has recently been reported to be expressed in human PCa [4]. Previously, we have demonstrated up-regulation of EPOR in human PCa and high-grade prostate intraepithelial neoplasia (high- grade PIN) [5]. In the present study, we further investigated the differential up-regulation of EPO and EPOR in normal, benign and malignant human prostatic tissue. 2. Materials and methods 2.1. Patients and samples � This project was funded by a grant from Shanghai PuJiang Program PJ14005). * Corresponding author. Tel.: �86-21-25070586; fax: �86-21- 38288. Origina Differential up-regulation of erythr and malignant p Chuanliang Xu, M.D.a,1, Tie Zhou, M.D.a,1 a Department of Urology, Shanghai Hospital, The Seco b Department of Pathology, Shanghai Hospital, The Sec Received 22 June 2008; received in revised form stract urpose: To investigate differential up-regulation of erythropoie aterials and methods: An immunohistochemical analysis of cinoma (PCa) with 16 high-grade prostate intraepithelial neoplasi normal prostatic tissue samples as control. esults: Over-expression of EPOR was only shown in PCa and h H tissue. There was significant relationship between EPO and EPO Urologic Oncology: Seminars and O pro E-mail address: sunyh@medmail.com.cn (Y. Sun). 1 These authors contributed equally to this work. 8-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. :10.1016/j.urolonc.2008.09.023 cle ietin and its receptor in benign tatic tissue� oxia He, M.D.b, Yinghao Sun, M.D.a,* itary Medical University, Shanghai, P. R. China litary Medical University, Shanghai, P. R. China tember 2008; accepted 22 September 2008 its receptor in benign and malignant prostatic tissue. nd EPOR expression was performed on 30 cases of prostate ), 50 cases of benign prostatic hyperplasia (BPH) lesions, and ade PIN tissue but over-expression of EPO was also shown in e in BPH, high-grade PIN, and PCa but no association between Investigations xx (2009) xxx Study protocols involving human materials were ap- ved by the institutional ethics committee at Changhai Ho the tw fro (av pro fro ye ng tat ob ye an the sco Gl So som tox rev pa 2.2 for sec no ma scr po no sli gra in bu an 4°C de En 30 bu DA zid aft sta sam (ne sec pa bra ba 2.3 po tat wi sta ma for pe sys als sta tita pre int str be inc an wi im DU we wh sam sta 2.4 EP wa EP sco for NC be 3. H ch fou mu gla Tab Ch PSA Me 15. P 2 C. Xu et al. / Urologic Oncology: Seminars and Original Investigations xx (2009) xxx ARTICLE IN PRESS spital. All the samples were from specimens archived in Department of Pathology at the Changhai Hospital be- een 2003 and 2004. PCa tissue samples were obtained m 30 patients with an age range from 60 to 77 years erage 70 years), who had undergone radical retropubic statectomy in the hospital. BPH tissue samples were m 50 patients (aged from 62 to 79 years, average 72 ars) with prostate-specific antigen (PSA) level less than 4 /ml, who underwent transurethral resection of the pros- e. A total of 30 normal prostate tissue samples were tained by autopsy from the patients aged from 20 to 30 ars (average 24 years). Patients with PCa did not receive y treatment such as radiation, chemotherapy, or hormonal rapy before the removal of the prostate. Tumors were red with the Gleason system. PSA, tumor stage, and eason score of patients with PCa are shown in Table 1. me PCa samples had adjacent high-grade PIN lesions and e BPH samples contained inflammation lesions. Hema- ylin and eosin (H & E) stained slides of all cases were iewed, and the diagnosis was confirmed by two senior thologists. . Immunohistochemical staining Four-�m-thick paraffin sections were serially cut from malin-fixed, paraffin-embedded tissues. One of these tions was stained with H & E for histopathologic diag- sis. Immunohistochemical assays were performed on for- lin-fixed, paraffin-embedded sections as previously de- ibed [6]. The primary antibodies used were rabbit lyclonal antibodies purchased from Santa Cruz Biotech- logy, Inc. (Santa Cruz, CA). Sections of 4 �m on glass des were deparaffinized in Hemo-D and rehydrated in ded alcohols, followed by endogenous peroxidase block 3% H2O2 and antigen retrieval in boiling 10% citrate ffer. Then, slides were incubated with the polyclonal tibody against EPO, EPOR (1:200 dilution) overnight at , and subsequently with horseradish peroxidase labeled xtran polymer coupled to anti-rabbit antibody (DAKO vision� System HRP, DAKO Inc., Carpinteria, CA) for minutes at room temperature after 3 washes with tris ffered saline containing Tween 20 (TBST, pH 7.6, KO). Finally, slides were developed with diaminoben- ine for 10 minutes and counterstained with hematoxylin er washing three times with TBST. The specificity of ining was confirmed by processing sections from the le 1 aracteristics of patients with PCa, n � 30, 2003–2004 (ng/ml) Tumor stages Gleason score dian range pT2 pT3a pT3b 2–4 5–7 8 9 6 4.1–23.6 16 8 6 7 16 6 1 SA � prostate-specific antigen; PCa � prostate carcinoma. e paraffin block with omission of the primary antibody of gative control). As a positive control, reactions with tions of breast cancer archived in the pathological de- rtment of our hospital were used. Cytoplasmic or mem- ne staining that was clearly distinguishable from the ckground was considered positive. . Semiquantitative analysis and interpretation of staining At least 500 epithelial cells within each area showing sitive immunoreactivity were evaluated in normal pros- e, BPH, high-grade PIN, and PCa. The percentage of cells th no staining (0) or weak (1), moderate (2), or intense ining (3) was analyzed by visual inspection under �100 gnification and a staining score was calculated using the mula: weighted mean of stain intensity � (�intensity � rcentage of cells)/total percentage of cells. The scoring tem took into account not only the staining intensity but o the percentage of the cells that exhibit EPO, EPOR ining. EPO and EPOR expression was graded semiquan- tively according to the results of staining score. In the sent study, EPO and EPOR staining were also classified o overexpression, which was defined as moderate or ong staining shown in any prostatic epithelium within nign or malignant tissues, and normal expression, which luded weak or negative staining of EPO or EPOR. These alyses were performed using a Nikon E-400 microscope th computer-aided image analysis system, and digital ages were captured using a digital camera (Nikon 100; Tokyo, Japan) at �200 magnification [7]. Slides re evaluated twice at different times by 3 investigators o were unaware of the pathologic characteristics of the ples, and the resulting mean levels were used for the tistical analyses. . Statistics Kruskal-Wallis test was used to compare differences in O, EPOR staining scores among groups. Spearman test s used to analyze the relationship between EPO and OR score for each group, and to relate EPO or EPOR re to Gleason score in PCa. Computations were per- med using SAS 9.1.3 software (SAS Institute Inc., Cary, ). A two-sided P value less than 0.05 was considered to statistically significant. Results PCa, high-grade PIN, and BPH lesions were identified on & E staining sections according to morphological anges (Fig. 1). A total of 16 high-grade PIN lesions were nd in 16 PCa samples, and no PCa sample showed ltiple high-grade PIN lesions. EPO and EPOR expression were observed mainly in the ndular epithelium. A predominantly cytoplasmic pattern staining for EPO and EPOR was observed, but membrane im an tis ma ov PIN sta pro EP fur gra tre bu an EP score 4 Tab EP nor Imm EP � � � � EP EP H pro ma ove a b 3C. Xu et al. / Urologic Oncology: Seminars and Original Investigations xx (2009) xxx ARTICLE IN PRESS munoreactivity was also noted for EPOR. Staining level d median score of EPO and EPOR in different prostate sues are shown in Table 2. Over-expression of EPOR was inly shown in high-grade PIN and PCa (Fig. 2), whereas er-expression of EPO was shown not only in high-grade and PCa but also in BPH tissue (Fig. 3). No positive ining for EPO and EPOR was found for all the normal static tissues tested. PCa and high-grade PIN had significantly higher median OR score than BPH (P � 0.0001; P � 0.0096) and thermore, PCa had higher median EPOR score than high- de PIN (P � 0.0072). With regard to EPO, an increased Fig. 1. Morphology of BPH (A), high-grade PIN (B), PCa (Gleason le 2 O and EPOR immunohistochemistry in different prostate lesions and mal prostatic epithelia unoreactivity PCa High-grade PIN BPHa Normalb O EPOR � 22 10 0 0 � 0 2 22 0 � 8 4 0 0 � 0 0 28 30 O Score 2.38 1.75 0.98 — OR Score 2.50 1.65 0.73 — igh-grade PIN � high-grade prostatic intraepithelial neoplasia; PCa � state carcinoma; BPH � benign prostatic hyperplasia; Normal � nor- l prostate; � � normal expression (negative or weak expression); � � rexpression (moderate or strong expression). 10 BPH samples contained inflammation lesions. whNegative expression of EPO and EPOR in normal prostate. nd was observed among BPH, high-grade PIN, and PCa, t only the difference of median EPO score between PCa d BPH was statistically significant (P � 0.0064). There was a significant correlation between EPO and OR score in BPH, high-grade PIN, and PCa (P � 0.031, � 0.014, P � 0.003, respectively). The relationship be- een Gleason score and EPO or EPOR score in PCa was t observed. Statistic analysis showed that there was no ociation between EPOR/EPO score and Gleason score in a (P � 0.085, P � 0.616). Comment EPO, a 30.4 kDa glycoprotein, is the principal regulator of blood cell production in mammals by binding EPOR [8], produced primarily in the adult kidney under the control of oxygen-sensing mechanism. Although there are several tors, such as hypoglycemia, increased intracellular calcium, ulin release, estrogen, and androgenic steroids, and various okines can modulate EPO production, low tissue oxygen sion is still the main driver that induces EPO gene expres- n through both transcriptional activation and mRNA stabi- ation [9,10]. The hypoxia dependent up-regulation of EPO ectly results from the activation of HIF-1�, which binds a poxia responsive element in the 3= flanking region of the O gene as a transcription factor. Recently, expression of EPOR and EPO in tumor cell es as well as primary cancers has been reported [11–18], ) (C), and PCa (Gleason score 7) (D). H & E staining (�200). P tw no ass PC 4. red and an fac ins cyt ten sio liz dir hy EP lin ich suggests the potential for generation of an autocrine or cel ate inc ch EP Fig mo leason Fig in h 4 C. Xu et al. / Urologic Oncology: Seminars and Original Investigations xx (2009) xxx ARTICLE IN PRESS paracrine growth-stimulatory EPO-EPOR loop in cancer ls. Thus, the activation of EPO-EPOR loop may be associ- d with modulation of various aspects of tumor biology, . 2. Immunohistochemical staining of EPOR in high-grade PIN and PCa. derate in high-grade PIN (�400); (C) PCa (Gleason score 4); (D) PCa (G . 3. Immunohistochemical staining of EPO in BPH, high-grade PIN, and igh-grade PIN (�400); (C) PCa (Gleason score 4); (D) PCa (Gleason score 7) e luding cellular proliferation, apoptosis, and sensitivity to emoradiation therapy. Furthermore, the expression of OR in vascular endothelium in tumors has suggested gative staining for EPOR in BPH (�200); (B) expression of EPOR is score 8) exhibiting strong EPOR immunoreactivity (�400). ) Moderate EPO staining in BPH (�400); (B) moderate EPO staining PCa. (A (A) Ne xhibiting strong EPO immunoreactivity (�400). po ge an [19 of ten inv sio en ma cli bre fou hy EP can ex sie Th tor filt au na can sig les log EP via EP pre cri tho rel hy reg ma fou wi ov hy an mi on of eri sug BP Fu wa BP po EP wa eff firm fin 5. ha co EP asc be Re [1 [2 [3 [4 [5 [6 [7 [8 [9 [10 [11 [12 [13 [14 5C. Xu et al. / Urologic Oncology: Seminars and Original Investigations xx (2009) xxx ARTICLE IN PRESS tential effects of EPO on the stimulation of tumor angio- nesis. EPOR expression in human prostate has been reported, d the expression of EPOR in prostate was functional ,20]. In the previous study, we also found overexpression EPOR in PCa and high-grade PIN. Considering the po- tial existence of EPO-EPOR loop in PCa, we further estigated, in this study, whether there was overexpres- n of EPO in PCa and explored the significance of differ- tial up-regulation of EPO and its receptor in benign and lignant prostate tissue. Acs et al. reported high levels of EPO and EPOR in 50 nical specimens of breast carcinoma but not in normal ast, benign papilloma, or fibrocystic tissue [21]. They nd the highest levels of EPO and EPOR expression in poxic tumor regions and thought that enhanced EPO- OR signaling contributed to the promotion of human cer by tissue hypoxia. Mohyeldin et al. investigated the pression and function of EPO and EPOR in tumor biop- s and cell lines from human head and neck cancer [16]. ey reported the highest expression of EPO and its recep- in malignant cells, particularly within hypoxic and in- rating tumor regions. Their data supported a role for tocrine or paracrine EPO-EPOR signaling in the malig- nt progression and local invasiveness of head and neck cer. More importantly, Yasuda et al. found EPO-EPOR naling pathway in 24 malignant human cell lines regard- s of their origins, types, genetic characteristics, and bio- ical properties [13]. Their findings suggested that EPO- OR signaling was indispensable for the growth and bility of malignant tumor. Similar to previous studies, up-regulation of EPO and OR was also found in PCa and high-grade PIN in the sent study, which suggested a potential role of the auto- ne EPO-EPOR signaling in prostate tumorigenesis. Al- ugh the mechanism involved in EPO-EPOR signaling- ated prostate tumorigenesis is still unknown, undoubtedly poxia plays an important and indispensable role. Up to now, to our best knowledge, the differential up- ulation of EPO and its receptor (EPOR) in benign and lignant prostatic tissue has not been investigated. We nd an over-expression of EPOR in all PCa tissue tested, th the majority of them over-expressing EPO. Although er-expressed EPO and EPOR have been ascribed to be poxia-related, the differential over-expression of EPO d EPOR in PCa tissue suggested non-hypoxia mechanism ght be also involved in up-regulation of EPOR. Actually, cogenic mechanism has been stressed in the up-regulation EPOR [22]. Over-expressed EPO was also shown in BPH. Consid- ng that EPO was one of the target genes of hypoxia, we gest that hypoxia may play a role in development of H, even malignant transition of benign prostatic tissue. rthermore, given the fact that over-expression of EPOR s observed only in high-grade PIN and PCa but not in H tissue, up-regulation of EPOR may have a more im- rtant role in prostate carcinogenesis. In this study, we did not find any relationship between O/EPOR score and Gleason score. Gleason score is al- ys related to prognosis of PCa, so EPO or EPOR as an ective prognostic factor for PCa has not yet been con- ed. Considering that the current sample is small, the al conclusion needs a further large-sample study. Conclusions Our data have demonstrated that up-regulation of EPOR s a more important role for prostate carcinogenesis in mparison with EPO. Differential over-expression of OR and EPO in benign and malignant prostatic tissue is ribed to different mechanisms involved in up-regulation tween EPO and EPOR. ferences ] Movsas B, Chapman JD, Horwitz EM, et al. Hypoxic regions exist in human prostate carcinoma. Urology 1999;53:11–8. ] Movsas B, Chapman JD, Greenberg RE, et al. Increasing levels of hypoxia in prostate carcinoma correlate significantly with increasing clinical stage and patient age. Cancer 2000;89:2018–24. ] Zhong H, Semenza GL, Simons JW, et al. Up-regulation of hypoxia- inducible factor 1alpha is an early event in prostate carcinogenesis. 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Blood 1999;94:1864–77. ] Batra S, Perelman N, Luck LR, et al. Pediatric tumor cells express erythropoietin and a functional erythropoietin receptor that promotes angiogenesis and tumor cell survival. Lab Invest 2003;83:1477–87. ] Arcasoy MO, Jiang X, Haroon ZA. Expression of erythropoietin receptor splice variants in human cancer. Biochem Biophys Res Commun 2003;307:999–1007. ] Yasuda Y, Fujita Y, Matsuo T, et al. Erythropoietin regulates tumor growth of human malignancies. Carcinogenesis 2003;24:1021–9. ] Kumar SM, Acs G, Fang D, et al. Functional erythropoietin autocrine loop in melanoma. Am J Pathol 2005;166:823–30. [15] Lai SY, Childs EE, Xi S, et al. Erythropoietin mediated activation of JAK-STAT signaling contributes to cellular invasion in head and neck squamous cell carcinoma. Oncogene 2005;24:4442–9. [16] Mohyeldin A, Lu H, Dalgard C, et al. Erythropoietin signaling pro- motes invasiveness of human head and neck squamous cell carci- noma. Neoplasia 2005;7:537–43. 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