Heterogeneous nuclear ribonucleoprotein K: altered pattern of expression associated with diagnosis and prognosis of prostate cancer

British Journal of Cancer (2009) 100, 1608 – 1616 & 2009 Cancer Research UK All rights reserved 0007 – 0920/09 $32.00 www.bjcancer.com Heterogeneous nuclear ribonucleoprotein K: altered pattern of expression associated with diagnosis and prognosis of prostate cancer P Barboro1, E Repaci1, A Rubagotti1,2, S Salvi1, S Boccardo1, B Spina1, M Truini1, C Introini1, P Puppo1, N Ferrari1, G Carmignani3, F Boccardo1,2 and C Balbi*,1 1 Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10-16132 Genova, Italy; 2Dipartimento di Oncologia, Biologia e Genetica, Università di Genova, Largo Rosanna Benzi, 10 – 16132 Genova, Italy; 3Dipartimento di Urologia Università di Genova, Largo Rosanna Benzi, 10 – 16132 Genova, Italy Molecular Diagnostics Using proteomic analysis of the nuclear matrix (NM), we found that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the hnRNP family with pleiotropic functions, was differentially expressed in prostate cancer (PCa) tissues. This study aimed to characterise the expression of hnRNP K and its subcellular localisation in PCa, utilising immunohistochemical and quantitative western blot techniques. Furthermore, the hnRNP K expression was studied in human PCa cell lines in order to determine its modulation by bicalutamide, the anti-androgen widely used in PCa therapy. Immunohistochemical staining of paraffinembedded tissues showed that hnRNP K was overexpressed in PCa, where it was localised both in the cytoplasm and in the nucleus. Staining of non-tumour tissues showed exclusively nuclear localisation and a less intense or absent signal. Immunoblot analysis demonstrated that the hnRNP K level within the NM was higher in PCa compared with non-tumour tissues and closely correlated with Gleason score (P ¼ 0.008). Higher expression within the NM was significantly (P ¼ 0.032) associated with poor prognosis. In two-dimensional western blot analysis hnRNP K presented several isoforms; the one with pI 5.1 was the most differently expressed between non-tumour and PCa tissues. Preliminary results indicate that hnRNP K can be modulated in vitro by a non-steroidal antiandrogen. Taken together, our findings suggest that hnRNP K has potential implications at the diagnostic, prognostic and therapeutic levels in PCa. British Journal of Cancer (2009) 100, 1608 – 1616. doi:10.1038/sj.bjc.6605057 www.bjcancer.com Published online 28 April 2009 & 2009 Cancer Research UK Keywords: heterogeneous nuclear ribonucleoprotein K; prostate cancer; nuclear matrix; immunohistochemistry; western blot; biological markers Prostate cancer (PCa) continues to represent a major health concern. As the introduction of serum prostate-specific antigen (PSA) into clinical practice in the late 1980s, the incidence of this tumour has increased whereas the impact on mortality rates is less clear-cut. PSA has poor specificity and cannot distinguish indolent tumours from the aggressive ones that need immediate treatment. New investigation into more accurate diagnostic and prognostic biomarkers is needed to improve risk stratification and identify new targets for therapy of PCa (Damber and Aus, 2008). As the nuclear matrix (NM) is considered to be a good source of cancer-specific biomarkers (Leman and Getzenberg, 2008), we utilised a proteomic approach to compare the NM proteins of PCa with those isolated from non-tumour (NT) tissues. In PCa, we observed an increase in the complexity of the protein pattern, which correlated with the level of tumour differentiation (Alberti et al, 1996, 2000); moreover, a few proteins (called NM-6, 7 and 8) *Correspondence: Dr C Balbi, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10-16132 Genova, Italy; E-mail address: Received 28 January 2009; revised 20 March 2009; accepted 30 March 2009; published online 28 April 2009 were significantly correlated with the risk of biochemical progression (Boccardo et al, 2003). To characterise these potential biomarkers, protein spots were selected from high-resolution two-dimensional gels and analysed by mass spectrometry. NM-6 was identified as heterogeneous nuclear ribonucleoprotein K (hnRNP K) (Barboro et al, 2005). HnRNP K is a member of the large hnRNP family. It is a ubiquitous protein present primarily in the nucleus, but it has also been found in the cytoplasm and mitochondria; it is involved in the transcription, splicing and translation processes (Bomsztyk et al, 2004). It is active at the chromatin level, where it is present in a higher density at transcribed genes with respect to silent ones (Ostrowski et al, 2003). Moreover, hnRNP K binds directly to the promoter region of the human c-myc gene (Michelotti et al, 1996) and promotes neoplastic transformation in an eIF4E-dependent manner (Lynch et al, 2005). In breast cancer cells, overexpression of hnRNP K enhances cell proliferation and anchorage-independent growth (Mandal et al, 2001), and in several states of enhanced cell proliferation, increased expression of this protein has also been found (Ostrowski and Bomsztyk, 2003). Overexpression of hnRNP K has been shown in many human tumours too, including lung cancer (Pino et al, 2003), esophageal cancer (Hatakeyama et al, 2006), oral squamous cell carcinoma (Roychoudhury and Heterogeneous nuclear ribonucleoprotein K in prostate cancer P Barboro et al 1609 MATERIALS AND METHODS Patients and tissue samples Studies were performed on PCa specimens obtained from 49 patients undergoing radical retropubic prostatectomy for clinically localised PCa between 1996 and 2003. NT tissue was obtained from contralateral lobe to the cancer zone and four normal human prostates (NHP) were collected from patients undergoing cystectomy for bladder cancer. The project was approved by the local Ethics Committee. Fresh tissues were immediately frozen in liquid nitrogen until sample preparation. All tissues were histologically confirmed by haematoxylin and eosin staining of frozen sections and only the specimens containing more than 80% of tumour cells were processed to isolate the NM. The patients’ characteristics are summarised in Table 1 and the tumours were classified according to the TNM system. Out of 49 patients included in the present analysis five patients received postoperative irradiation, five were treated with adjuvant hormone therapy and one with both Table 1 Patient demographics and tumour characteristics N ¼ 49 Median preoperative age, years (range) Median preoperative PSA, ng ml – 1 (range) (%) 64.0 (48.0 – 77.0) 11.0 (5.0 – 120.0) Tumour stage pT2 pT3 pT4a 24 24 1 (49.0) (49.0) (2.0) Pelvic nodes involved pN0 pN1-2 pNx 26 10 13 (53.1) (20.4) (26.5) Surgical margins involved Seminal vesicles involved 20 12 (40.8) (24.5) Gleason score p6 ¼7 47 16 12 21 (32.7) (24.5) (42.9) & 2009 Cancer Research UK treatments. Patients were followed at regular intervals and PSA determined. A PSA level of at least 0.4 ng ml – 1, which was confirmed by anoth (...truncated)