Prostate Cancer and Prostatic Diseases (2011) 14, 38–45 & 2011 Macmillan Publishers Limited All rights reserved 1365-7852/11 www.nature.com/pcan ORIGINAL ARTICLE Transcription alterations of members of the ubiquitin–proteasome network in prostate carcinoma OJC Hellwinkel1,2,5, LE Asong3,5, J-P Rogmann3,HSu¨ ltmann4, C Wagner2, T Schlomm2 and C Eichelberg3 1Department of Legal Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 2Martini-Klinik, Prostate Cancer Center, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 3Department of Urology, University Hospital Hamburg-Eppendorf, Hamburg, Germany and 4Working Group Cancer Genome Research, Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany The purpose of this work was to investigate the role of the ubiquitin–proteasome network (UPN) in prostate cancer (PCA) and to elicit potential markers for this disease. The UPN represents a key factor in the maintenance of cellular homoeostasis as a result of its fundamental function in the regulation of intracellular protein degradation. Members of this network have a role in the biology of haematological and solid tumours. Tumour cells and normal epithelial cells from 22 prostatectomy specimens were isolated by laser microdissection. Prostate biopsy samples from healthy individuals served for technical calibration and as controls. Transcript levels of eight selected genes with E3 ubiquitin ligase activity (labelling target proteins for proteasome degradation) and two genes belonging to the proteasome–multienzyme complex itself were analysed by quantitative real-time RT-PCR. The proteasome genes PSMC4 and PSMB5 and the E3 ubiquitin ligase NEDD4L were significantly and coherently upregulated in PCA cells compared with the corresponding adjacent normal prostate tissue. Transcription of the E3 ubiquitin ligase SMURF2 was significantly higher in organ-confined tumours (pT2) compared with non-organ- confined cancers (pT3). The results indicate a role for PSMC4 and PSMB5 and the E3 ubiquitin ligase NEDD4L in prostate tumourigenesis, whereas SMURF2 downregulation could be associated with clinical progression. NEDD4L and SMURF2 both target transforming growth factor (TGF)-b for degradation. This reflects the pleiotropic role of the TGF-b signalling pathway acting as a tumour suppressor in normal and pre-cancerous cells, but having oncogenic properties in progressing cancer. Further studies have to elucidate whether these alterations could represent clinically relevant PCA-diagnostic and progression markers. Prostate Cancer and Prostatic Diseases (2011) 14, 38–45; doi:10.1038/pcan.2010.48; published online 23 November 2010 Keywords: proteasome; E3 ubiquitin ligase; transcription; prostate carcinoma; tumour marker Introduction cycle progression, signal transduction, proliferation, apoptosis, modulation of surface receptors and regula- Knowledge about expression patterns of genes involved tion of tumour suppression proteins2–4 (Figure 1). Pro- in the control of cellular homoeostasis is important to teins for proteasome degradation are labelled by understand malign transformation and cancer progres- conjugation of multiple ubiquitin moieties (ubiquitina- sion, and could improve cancer diagnosis and staging. tion). Ubiquitin is an abundant small highly conserved, Cellular disposal of molecules is essential for normal composed of 76 amino acids, protein found only in cellular homoeostasis. The ubiquitin–proteasome net- eukaryotes, and is transferred by a cascade of three work (UPN) is accountable for the selective cellular enzyme types: protein E1 activates the C-terminus of the removal of mis-folded, damaged, and unassembled ubiquitin, whereas E2 transfers the activated ubiquitin to polypeptide chains and regulatory proteins.1–2 It reg- one of the multiple E3 ubiquitin ligase proteins. E3 ulates numerous basic cellular processes such as cell ubiquitin ligases are responsible for the specificity of protein recognition and ubiquitination, thus ensuring the fidelity of selective proteolysis. The proteasome is a Correspondence: Dr OJC Hellwinkel, Department of Legal Medicine, 2.2 MDa barrel-shaped multi-catalytic proteinase com- Transplantation Tissue Bank (Building W40, 2nd floor), University plex, the 26S proteasome, composed of two complexes, a Hospital Eppendorf, Martinistrasse 52, Hamburg 20246, Germany. 20S core particle and a 19S regulator cap. The 20S core E-mail: [email protected] 5These authors contributed equally to this work. contains the protease subunits and is composed of four Received 22 July 2010; revised 27 September 2010; accepted 17 rings (two identical peripheral alpha rings and two October 2010; published online 23 November 2010 identical central beta rings), each ring containing seven Transcription alterations in prostate carcinoma OJC Hellwinkel et al 39 p53 TH1 Mcm7 7 3 2 SMAD1 Degradation products TGF UBE3A * 1R SMURF2 * SMAD2 NEDD4L * p53 Poly- Ubiquitin PSMC4 * PSMB5 * label IGF-IR MDM2 * Ub Ub Ub PTEN NEDD4 * 19S 20S 19S IGF-IR regulator core regulator proteasome HECW1 * DVL1 SIAH1 * SOD1 CBL * DCC -Cat EDF-R PDGF IGF-IR -R Figure 1 The ubiquitin–proteasome network. The proteasome alongside some E3 ubiquitin ligases (light blue ellipses) are demonstrated. Asterisks illustrate the investigated transcripts. Some tumour-relevant degradation targets are depicted as small green circles. The color reproduction of this figure is available on the html full text version of the manuscript. non-identical subunits (resulting in 2 Â 14 non-identical board was obtained according to the directions of the subunits). The 19S cap (alternative name PA700) is made German authorities. of two sub-complexes, the lid and the base and a total of A total of 22 PCA patients undergoing radical 17 peptide molecules. It contains 11 non-ATPase subunits prostatectomy were included in this study (Table 2; the and 6 ATPases that unfold the substrates and translocate identical collective has been investigated in an earlier them into the core for proteolysis (substrate specificity; for study (see Hellwinkel et al.20097). None of the patients further information see http://www.atlasgeneticsoncology. had been treated with radio-, chemotherapy or ablative org/Deep/UbiquitininCancerID20083.html). The protea- hormonal therapy. Tumour and normal tissue samples some is not only found attached to the endoplasmatic were taken with a 6-mm punch biopsy instrument from reticulum and to the nucleus but is also found free in the tumourous and non-tumourous areas as described cytosol.1–2 Alterations of this critical degradation machin- before.8 The specimens were incubated in RNAlater ery have an important role in tumourigenesis and cancer (Qiagen, Hilden, Germany) at 4 1C for 2 h and then stored progression.4–6 Its hierarchical character further provides a for a long term at –80 1C. To select epithelial (tumour and platform for a novel approach in innovative anti-cancer adjacent normal) cells for gene expression analyses, therapy.5 we performed laser microdissection as previously des- The aim of this pilot study is a compact analysis cribed.7–8 of the transcription patterns of selected important First, the specimens were briefly thawed on ice and ‘representatives’ of those arms of the UPN that control then cryosections of up to 15 mm thickness were prepared (prostate) cancer-relevant pathways (eight E3 ubiquitin and transferred to RNAse-free membrane slides (PALM ligases and two integral members of the proteasome, Microlaser Technologies AG, Bernried, Germany). The Table 1 and Figure 1). Such findings could provide cryosections were air-dried and stained with cresyl violet valuable information on prostate cancer (PCA) biology following standard procedures. Additionally, represen- and further indicate potential diagnostic and prognostic tative sections were hematoxylin and eosin stained for markers. pathological examination and archival storing. To produce samples of highest uniformity, tissue areas containing either only tumour (always both Gleason Materials and methods components at proportions corresponding to the histo- pathological data given in Table 2) or normal prostate Tissue samples, cryosection and laser microdissection duct cells (1000–3000 mm2) were microdissected and Informed consent from the patients involved in this collected employing an ultraviolet laser-based microdis- project and the healthy volunteers (calibrator, see below), section system (PALM Microlaser Technologies) accord- as well as the approval from the institutional review ing to manufacturer’s protocols. Microdissected samples Prostate Cancer and Prostatic Diseases Prostate Cancer and Prostatic Diseases 40 Table 1 Information on the analysed transcripts (catalogue numbers of the used TaqMan real-time RT-PCR assays from Applied Biosystems are given between brackets) Gene symbol Name Aliases Chromosome Function/role in cancer (TaqMan assay) location UBE3A Ubiquitin protein ligase E6-AP; Human papilloma 15q11–q13 E3 ubiquitin-protein ligase. Targets p53, Blk, TH1and itself for (Hs00963673_ml) E3A virus E6-associated protein; degradation. Mediates Mcm7, HHR23A ubiquitination. Overexpressed in FLJ26981 breast cancer compared with adjacent normal tissue. Downregulated in invasive breast and prostate carcinomas compared with adjacent normal tissue. Transcription alterations in prostate carcinoma SMURF2 SMAD-specific E3 E3 ubiquitin-ligase SMURF2; 17q22-q23 E3 ubiquitin-protein ligase. Targets Smad1, Smad2, Smad3, Smad7 and (Hs00909283_ml)