Clusterin is epigenetically regulated in prostate cancer
Hanna E. Rauhala
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
The first two authors contributed equally to this work.
Search for more papers by this authorKati P. Porkka
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
The first two authors contributed equally to this work.
Search for more papers by this authorOuti R. Saramäki
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
Search for more papers by this authorTeuvo L.J. Tammela
Department of Urology, University of Tampere, and Tampere University Hospital, Tampere, Finland
Search for more papers by this authorCorresponding Author
Tapio Visakorpi
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
Fax: +358-3-3551-8597
Institute of Medical Technology, FIN-33014 University of Tampere, Tampere, FinlandSearch for more papers by this authorHanna E. Rauhala
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
The first two authors contributed equally to this work.
Search for more papers by this authorKati P. Porkka
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
The first two authors contributed equally to this work.
Search for more papers by this authorOuti R. Saramäki
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
Search for more papers by this authorTeuvo L.J. Tammela
Department of Urology, University of Tampere, and Tampere University Hospital, Tampere, Finland
Search for more papers by this authorCorresponding Author
Tapio Visakorpi
Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland
Fax: +358-3-3551-8597
Institute of Medical Technology, FIN-33014 University of Tampere, Tampere, FinlandSearch for more papers by this authorAbstract
Lack of good models has complicated investigations on the mechanisms of prostate cancer. By far, the most commonly used transgenic mouse model of prostate cancer is TRAMP, which, however, has not been fully characterized for genetic and epigenetic aberrations. Here, we screened TRAMP-derived C2 cell line for the alterations using different microarray approaches, and compared it to human prostate cancer. TRAMP-C2 had relatively few genomic copy number alterations according to array comparative genomic hybridization (aCGH). However, the gene copy number and expression were significantly correlated (p < 0.001). Screening genes for promoter hypermethylation using demethylation treatment with 5-aza-2′-deoxycytidine and subsequent expression profiling indicated 43 putatively epigenetically silenced genes. Further studies revealed that clusterin is methylated in the TRAMP-C2 cell line, as well as in the human prostate cancer cell line LNCaP. Its expression was found to be significantly reduced (p < 0.01) in untreated and hormone-refractory human prostate carcinomas. Together with known function of clusterin, the data suggest an epigenetic component in the regulation of clusterin in prostate cancer. © 2008 Wiley-Liss, Inc.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
| Filename | Description |
|---|---|
| IJC_23658_sm_supptable1.doc44.5 KB | Supplementary Table S1. Primer sequences, annealing temperatures and CpG island positions relative to transcription start site (TSS). |
| IJC_23658_sm_suppfig1.tif11.6 MB | Supplementary Fig. S1. Human homologous segments of the mouse chromosomal regions that showed losses or gains in TRAMP-C2 by the aCGH analysis. The vertical lines on the left sides of the chromosome ideograms indicate losses of the corresponding regions in TRAMP-C2, whereas the lines on the right side indicate gains. |
| IJC_23658_sm_suppfig2.tif330 KB | Supplementary Fig. S2. FISH analysis of gained and lost chromosomal regions in TRAMP-C2 cell line. A) The gain of 16qC4 is shown by 5 locus-specific signals (green) as compared to 3 reference signals (red). B) The loss of 7qD1 is shown by 2 locus-specific signals (green) as compared to 4 reference signals (red). |
| IJC_23658_sm_suppfig3.tif10.9 MB | Supplementary Fig. S3. Expression and methylation analyses of decorin gene. A) Decorin expression in the TRAMP-C2 cell line after treating the cells with 5azadC and TSA as indicated. Relative expression is represented as fold induction, where the control experiment (untreated) is designated as 1. For microarrays (white bars), the fold induction is calculated from the Cy5/Cy3 ratios (treated/untreated) and for real-time Q-RT-PCR (black bars), from the TBP-normalized expression values. B) Methylation analysis of the decorin gene in TRAMP-C2. The mouse decorin gene promoter/5'UTR CpGs and bisulphite sequencing primer positions. Dashed vertical lines represent single CpG dinucleotides. Horizontal solid lines represent the amplified and sequenced genomic regions and the arrowheads the used primers. TSS indicates the transcription start site. CpGs are represented by open dots if unmethylated and by black dots if methylated. Dashed line dots represent CpGs where sequencing results were not obtained. Each row represents a single PCR clone sequenced. The percentage of CpG methylation is indicated. |
| IJC_23658_sm_suppfig4.tif146.8 KB | Supplementary Fig. S4. Clusterin protein expression by Western blotting. No significant difference in clusterin protein levels between the prostate cancer cell lines was observed. Protein samples from human prostate cancer cell lines (20 μg) were run on 10 % SDS-polyacrylamide gel. Proteins were semi-dry transferred to PVDF membrane (Millipore, Billerica, MA, USA). Clusterin was probed using a mouse monoclonal antibody (clone 41D, Millipore, 1:500). After incubation with the secondary antibody (anti-mouse-IgG/HRP; DakoCytomation, Glostrup, Denmark), the proteins were visualized with ECL reagent (Santa Cruz Biotech Inc., Santa Cruz, CA, USA). |
| IJC_23658_sm_suppfig5.tif9 MB | Supplementary Fig. S5. Clusterin protein expression in the epithelial cells of normal prostate by immunohistochemistry. Clusterin protein expression in prostate tissue was analyzed using a standard immunohistochemical technique with mouse monoclonal antibody (clone 41D, Millipore). The slides were autoclaved in pretreatment buffer (5 mM Tris-HCl/1mM EDTA, pH 9) at 121°C for 2 min, followed by incubation with the primary antibody in pre-block solution (1:200) over night. The bound antibody was visualized with a conjugate of a secondary antibody and horseradish peroxidase with diaminobenzidine as a chromogen (PowerVision+ Detection System, ImmunoVision Inc., Springdale, AR, USA). |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1 Porkka KP,Visakorpi T. Molecular mechanisms of prostate cancer. Eur Urol 2004; 45: 683–91.
- 2 Saramäki OR,Porkka KP,Vessella RL,Visakorpi T. Genetic aberrations in prostate cancer by microarray analysis. Int J Cancer 2006; 119: 1322–9.
- 3 van Bokhoven A,Varella-Garcia M,Korch C,Johannes WU,Smith EE,Miller HL,Nordeen SK,Miller GJ,Lucia MS. Molecular characterization of human prostate carcinoma cell lines. Prostate 2003; 57: 205–25.
- 4 Greenberg NM,DeMayo F,Finegold MJ,Medina D,Tilley WD,Aspinall JO,Cunha GR,Donjacour AA,Matusik RJ,Rosen JM. Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA 1995; 92: 3439–43.
- 5 Foster BA,Gingrich JR,Kwon ED,Madias C,Greenberg NM. Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res 1997; 57: 3325–30.
- 6 Rauhala HE,Porkka KP,Tolonen TT,Martikainen PM,Tammela TL,Visakorpi T. Dual-specificity phosphatase 1 and serum/glucocorticoid-regulated kinase are downregulated in prostate cancer. Int J Cancer 2005; 117: 738–45.
- 7
Pollack JR,Perou CM,Alizadeh AA,Eisen MB,Pergamenschikov A,Williams CF,Jeffrey SS,Botstein D,Brown PO.
Genome-wide analysis of DNA copy-number changes using cDNA microarrays.
Nat Genet
1999;
1:
41–6.
10.1038/12640 Google Scholar
- 8 Hyytinen E,Visakorpi T,Kallioniemi A,Kallioniemi OP,Isola JJ. Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization. Cytometry 1994; 16: 93–9.
- 9 Linja MJ,Savinainen KJ,Saramaki OR,Tammela TL,Vessella RL,Visakorpi T. Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res 2001; 61: 3550–5.
- 10 Aapola U,Maenpaa K,Kaipia A,Peterson P. Epigenetic modifications affect Dnmt3L expression. Biochem J 2004; 380: 705–13.
- 11 Li LC,Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002; 18: 1427–31.
- 12 Kim H,Lapointe J,Kaygusuz G,Ong DE,Li C,van de Rijn M,Brooks JD,Pollack JR. The retinoic acid synthesis gene ALDH1a2 is a candidate tumor suppressor in prostate cancer. Cancer Res 2005; 65: 8118–24.
- 13 Lodygin D,Epanchintsev A,Menssen A,Diebold J,Hermeking H. Functional epigenomics identifies genes frequently silenced in prostate cancer. Cancer Res 2005; 65: 4218–27.
- 14 Li LC,Chui R,Nakajima K,Oh BR,Au HC,Dahiya R. Frequent methylation of estrogen receptor in prostate cancer: correlation with tumor progression. Cancer Res 2000; 60: 702–6.
- 15 Leiblich A,Cross SS,Catto JW,Phillips JT,Leung HY,Hamdy FC,Rehman I. Lactate dehydrogenase-B is silenced by promoter hypermethylation in human prostate cancer. Oncogene 2006; 25: 2953–60.
- 16 Banerjee AG,Liu J,Yuan Y,Gopalakrishnan VK,Johansson SL,Dinda AK,Gupta NP,Trevino L,Vishwanatha JK. Expression of biomarkers modulating prostate cancer angiogenesis: differential expression of annexin II in prostate carcinomas from India and USA. Mol Cancer 2003; 2: 34.
- 17 Phillips JL,Hayward SW,Wang Y,Vasselli J,Pavlovich C,Padilla-Nash H,Pezullo JR,Ghadimi BM,Grossfeld GD,Rivera A,Linehan WM,Cunha GR, et al. The consequences of chromosomal aneuploidy on gene expression profiles in a cell line model for prostate carcinogenesis. Cancer Res 2001; 61: 8143–9.
- 18 Gao C,Furge K,Koeman J,Dykema K,Su Y,Cutler ML,Werts A,Haak P,Vande Woude GF. Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations. Proc Natl Acad Sci USA 2007; 104: 8995–9000.
- 19 Sato K,Qian J,Slezak JM,Lieber MM,Bostwick DG,Bergstralh EJ,Jenkins RB. Clinical significance of alterations of chromosome 8 in high-grade, advanced, nonmetastatic prostate carcinoma. J Natl Cancer Inst 1999; 91: 1574–80.
- 20 Porkka KP,Tammela TL,Vessella RL,Visakorpi T. RAD21 and KIAA0196 at 8q24 are amplified and overexpressed in prostate cancer. Genes Chromosomes Cancer 2004; 39: 1–10.
- 21 Nupponen NN,Porkka K,Kakkola L,Tanner M,Persson K,Borg A,Isola J,Visakorpi T. Amplification and overexpression of p40 subunit of eukaryotic translation initiation factor 3 in breast and prostate cancer. Am J Pathol 1999; 154: 1777–83.
- 22 Ribeiro FR,Henrique R,Hektoen M,Berg M,Jeronimo C,Teixeira MR,Lothe RA. Comparison of chromosomal and array-based comparative genomic hybridization for the detection of genomic imbalances in primary prostate carcinomas. Mol Cancer 2006; 5: 33.
- 23 Morey SR,Smiraglia DJ,James SR,Yu J,Moser MT,Foster BA,Karpf AR. DNA methylation pathway alterations in an autochthonous murine model of prostate cancer. Cancer Res 2006; 66: 11659–67.
- 24 Caporali A,Davalli P,Astancolle S,D'Arca D,Brausi M,Bettuzzi S,Corti A. The chemopreventive action of catechins in the TRAMP mouse model of prostate carcinogenesis is accompanied by clusterin over-expression. Carcinogenesis 2004; 25: 2217–24.
- 25 Lund P,Weisshaupt K,Mikeska T,Jammas D,Chen X,Kuban RJ,Ungethüm U,Krapfenbauer U,Herzel HP,Schäfer R,Walter J,Sers C. Oncogenic HRAS suppresses clusterin expression through promoter hypermethylation. Oncogene 2006; 25: 4890–903.
- 26 Jones SE,Jomary C. Clusterin. Int J Biochem Cell Biol 2002; 34: 427–31.
- 27 Nizard P,Tetley S,Le Dréan Y,Watrin T,Le Goff P,Wilson MR,Michel D. Stress-induced retrotranslocation of clusterin/ApoJ into the cytosol. Traffic 2007; 8: 554–65.
- 28 Leskov KS,Klokov DY,Li J,Kinsella TJ,Boothman DA. Synthesis and functional analyses of nuclear clusterin, a cell death protein. J Biol Chem 2003; 278: 11590–600.
- 29 Moretti RM,Marelli MM,Mai S,Cariboni A,Scaltriti M,Bettuzzi S,Limonta P. Clusterin isoforms differentially affect growth and motility of prostate cells: possible implications in prostate tumorigenesis. Cancer Res 2007; 67: 10325–33.
- 30 Yang CR,Leskov K,Hosley-Eberlein K,Criswell T,Pink JJ,Kinsella TJ,Boothman DA. Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein that signals cell death. Proc Natl Acad Sci USA 2000; 97: 5907–12.
- 31 Zhang H,Kim JK,Edwards CA,Xu Z,Taichman R,Wang CY. Clusterin inhibits apoptosis by interacting with activated Bax. Nat Cell Biol 2005; 7: 909–15.
- 32 Zhang Q,Zhou W,Kundu S,Jang TL,Yang X,Pins M,Smith N,Jovanovic B,Xin D,Liang L,Guo Y,Lee C. The leader sequence triggers and enhances several functions of clusterin and is instrumental in the progression of human prostate cancer in vivo and in vitro. Br J Urol 2006; 98: 452–60.
- 33 Bettuzzi S,Davalli P,Astancolle S,Carani C,Madeo B,Tampieri A,Corti A. Tumor progression is accompanied by significant changes in the levels of expression of polyamine metabolism regulatory genes and clusterin (sulfated glycoprotein 2) in human prostate cancer specimens. Cancer Res 2000; 60: 28–34.
- 34 Miyake H,Nelson C,Rennie PS,Gleave ME. Testosterone-repressed prostate message-2 is an antiapoptotic gene involved in progression to androgen independence in prostate cancer. Cancer Res 2000; 60: 170–6.
- 35 July LV,Akbari M,Zellweger T,Jones EC,Goldenberg SL,Gleave ME. Clusterin expression is significantly enhanced in prostate cancer cells following androgen withdrawal therapy. Prostate 2002; 50: 179–88.
- 36 Scaltriti M,Brausi M,Amorosi A,Caporali A,D'Arca D,Astancolle S,Corti A,Bettuzzi S. (SGP-2. Apo. J) expression is downregulated in low- and high-grade human prostate cancer. Int J Cancer 2004; 108: 23–30.
- 37 Gleave M,Miyake H. Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer. World J Urol 2005; 23: 38–46.
- 38 Miyake H,Muramaki M,Kurahashi T,Yamanaka K,Hara I,Gleave M,Fujisawa M. Expression of clusterin in prostate cancer correlates with Gleason score but not with prognosis in patients undergoing radical prostatectomy without neoadjuvant hormonal therapy. Urology 2006; 68: 609–14.
Citing Literature
