Journal of Pathology J Pathol 2011; 224: 110–120 ORIGINAL PAPER Published online 7 March 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.2846 Down-regulation of oestrogen receptor-β associates with transcriptional co-regulator PATZ1 delocalization in human testicular seminomas Francesco Esposito,1,2# Francesca Boscia,3# Renato Franco,4 Mara Tornincasa,2 Alfredo Fusco,2 Sohei Kitazawa,5 Leendert H Looijenga6 and Paolo Chieffi1,2* 1 Dipartimento di Medicina Sperimentale, II Universita` di Napoli, Naples, Italy 2 IEOS and Dipartimento di Biologia e Patologia, Universita` di Napoli ‘Federico II’, Naples, Italy 3 Dipartimento di Neuroscienze, Universita` di Napoli ‘Federico II’, Naples, Italy 4 Istituto Nazionale dei Tumori ‘Fondazione G. Pascale’, Naples, Italy 5 Department of Molecular Pathology, Kobe University, Japan 6 Department of Pathology, Daniel den Hoed Cancer Centre, JosephineNefkens Institute, Erasmus MC University Medical Centre Rotterdam, The Netherlands *Correspondence to: Paolo Chieffi, Dipartimento di Medicina Sperimentale, Via Costantinopoli 16, 80138 Naples, Italy. e-mail: Paolo.Chieffi@unina2.it #These authors equally contributed to this study. Abstract Oestrogen exposure has been linked to a risk for the development of testicular germ cell cancers. The effects of oestrogen are now known to be mediated by oestrogen receptor-α (ERα)andERβ subtypes, but only ERβ has been found in human germ cells of normal testis. However, its expression was markedly diminished in seminomas, embryonal cell carcinomas and mixed germ cell tumours, but remains high in teratomas. PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. We have previously described that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mislocalization could be associated with the development of testicular germ cell tumours. Here we show that ERβ interacts with PATZ1 in normal germ cells, while down-regulation of ERβ associates with transcriptional co-regulator PATZ1 delocalization in human testicular seminomas. In addition, we show that the translocation of PATZ1 from the cytoplasm into the nucleus is regulated by cAMP, which also induces increased expression and nuclear localization of ERβ, while this effect is counteracted by using the anti-oestrogen ICI 182-780. Copyright 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: ZNF278; PATZ1; MAZR; spermatogenesis; testicular cancer; tumour suppressor; oestrogen receptor-β Received 12 October 2010; Revised 26 November 2010; Accepted 18 December 2010 No conflicts of interest were declared. Introduction certain hormones (eg oestrogen) at the time of testicular differentiation in utero has long been implicated as a risk factor for developing these neoplasmas [4]. Testicular germ cell tumours (TGCTs), the most Recently, it has been suggested that the carcinogenic common malignancy in males aged 15–34 years, rep- resent a major cause of death attributable to can- effects of oestrogen on testicular cells may involve cer in this age group [1–3]. TGCTs can be subdi- oestrogen receptor-mediated oxidative DNA damage vided into seminoma and non-seminoma germ cell [5]. tumours (NSGCTs), including embryonal cell carci- Oestrogen signalling is mediated by two nuclear α α β β noma, choriocarcinoma, yolk sac tumour and teratoma. receptors, oestrogen receptor- (ER )and (ER ), Neoplasms containing more than one tumour cell com- which are oestrogen-dependent transcription factors. ponents, eg seminoma and embryonal cell carcinoma, ERα is expressed at high levels in human epididymis are referred to as mixed germ cell tumours. Semino- and efferent ductules but not in the testis, whereas mas and NSGCTs not only present distinctive clinical ERβ is expressed in spermatogonia, spermatocytes and features but also show significant differences as far as early round spermatids in human testis [6,7]. The ERβ therapy and prognosis are concerned [1,2]. Whereas subtype is the principal mediator of oestrogen action in the aetiology of TGCTs remain undefined, exposure to promoting germ cell survival and development [6–9]. Copyright 2011 Pathological Society of Great Britain and Ireland. J Pathol 2011; 224: 110–120 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com PATZ1 and ERβ in human testicular seminomas 111 After activation, these receptors, in association with a approval was given in all instances. The GC-1 cell line myriad of co-activators and repressors, act as nuclear was cultured in Dulbecco’s modified Eagle’s medium transcription factors for targeted genes [10,11]. It has (D-MEM) supplemented with 10% fetal bovine serum ◦ been well documented in literature that ERβ,whichis (FBS; Gibco BRL, Italy) and grown in a 37 C humid- expressed in normal testicular cells, is instead down- ified atmosphere of 5% CO2 [24]. TCam-2 cells were ◦ regulated in seminomas and embryonal cell carcinomas grown at 37 Cina5%CO2 atmosphere in RPMI [12,13]. 1640 (Lonza) supplemented with 10% FBS [25,26]. PATZ1, also named ZNF278 or MAZR, is a recently Spermatogonia were prepared from 7dpp testes, as pre- discovered ubiquitously expressed transcriptional reg- viously described [27]. ulatory factor gene whose product binds to the RING TCam-2 cells were also treated with 8-bromoadeno- finger protein RNF4 that, in turn, associates with a sine-3,5-cyclic monophosphate (8Br-cAMP, 100 µM; variety of transcription regulators [14–17]. By virtue Sigma, Milan, Italy) and with ICI 182–780 (100 µM; of the POZ domain, PATZ1 acts as a transcriptional Zeneca, London, UK). Cells were transfected with repressor on different promoters [18], although it has plasmids contained human PATZ1 and ERβ full-length been also shown to function as a strong activator cDNA using the lipofectamineplus reagent (Invitrogen, of the c-myc promoter [19]. Indeed, PATZ1 is able Milan, Italy), as suggested by the manufacturer. to bind proteins involved in chromatin remodelling, such as HMGA1, that it has been shown to be over- Antibodies expressed in human testicular seminomas [20] and the Antibodies were purchased from the following sources: above mentioned RNF4 [14]. Interestingly, it has been (a) polyclonal mouse antibody anti-PATZ1 (no. found rearranged through a paracentric inversion of ab68646; Abcam, Cambridge, UK); (b) polyclonal rab- 22q12 with the EWS gene, in small round cell sar- bit antibody anti-PATZ1 (no. sc-86 776; Santa Cruz coma, suggesting a potential tumour suppressor role Biotechnology, CA, USA); (c) polyclonal rabbit anti- [17]. Four alternatively spliced transcript variants have body anti-oestrogen receptor-β (ERβ; no. sc-8974, been described for the PATZ1 gene. Our previous pub- Santa Cruz); (d) mouse monoclonal anti-oestrogen lished data demonstrated that only variant 3 of PATZ1 receptor β (clone PPG5/10; code no. M7292, Dako (537 amino acids, about 60 kDa) is expressed in the Cytomation, Denmark); (e) mouse monoclonal anti-β- testis and TGCTs [21]. −/− tubulin (no. T-4026, Sigma, St. Louis, MO, USA); Recently, we have shown that male PATZ1 mice (f) mouse monoclonal anti γ-tubulin (no. T-5326, are unfertile and, although PATZ1 protein expression Sigma); (g) mouse monoclonal anti-Golgin-97 (code was up-regulated in testicular germ cell tumours when no. A21270, Molecular Probes); (h) polyclonal rabbit compared to normal human testis, it was delocalized anti-Hemagglutinin Tag Anitibody (HA) (no. sc-805, in the cytoplasm, thus suggesting an impaired function Santa Cruz) that recognized PATZ1 proteins conju- [21]. In addition, it has been documented, using the gated to HA; (i) polyclonal rabbit anti-SP1 (no. sc- null mice model, that the zinc-finger protein MAZR 14 027, Santa Cruz); (j) mouse monoclonal anti-PKA- is part of the transcription factor network that controls regulatory subunit (PKA-R; code no. 612 242, BD the CD4 versus CD8 lineage fate of double-positive Transduction Laboratories, Franklin Lakes, NJ, USA); thymocytes [22,23]. (k) rabbit polyclonal anti PKA-catalytic subunit (PKA- β Here we show that PATZ1 interacts with ER in C; code no. sc-903, Santa Cruz). normal germ cells. Moreover, in human testicular seminomas, PATZ1 protein delocalization associates Histological analysis and immunohistochemistry with ERβ down-regulation. In addition, we show that the translocation of PATZ1 from the cytoplasm into For light microscopy, tissues were fixed in 10% for- the nucleus is mediated by cAMP, which is also malin and embedded in paraffin by standard proce- responsible for an increased expression and nuclear dures. Sections (4 µm) were stained with haematoxylin localization of ERβ. Interestingly, both PATZ1 and and eosin (H&E) or processed for immunohistochem- ERβ nuclear translocation mediated by cAMP was istry. The classical avidin–biotin peroxidase complex counteracted by using the anti-oestrogen ICI 182–780. (ABC) procedure was used for immunohistochem- Taken together, these results suggest that the failed istry, as described [20,21]. Sections were incubated association between the two proteins might have a role overnight with antibodies against: (a) PATZ1 (diluted in the genesis of the testicular neoplasia. 1 : 200; polyclonal rabbit antibody anti-PATZ1, no. sc- 86 776, Santa Cruz); (b) oestrogen receptor-β (diluted 1 : 200; mouse monoclonal anti-oestrogen receptor-β, β Materials and methods code no.