Novel Biomarkers for Prostate Cancer Including Noncoding Transcripts
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080868
ISSN1525-2191
AutoresTammy L. Romanuik, Takeshi Ueda, Nhu Le, Simon Haile, Theresa M.K. Yong, Thomas A. Thomson, Robert L. Vessella, Marianne D. Sadar,
Tópico(s)Cancer-related gene regulation
ResumoLevels of 27 transcripts were investigated as potential novel markers for prostate cancer, including genes encoding plasma membrane proteins (ADAM2, ELOVL5, MARCKSL1, RAMP1, TMEM30A, and TMEM66); secreted proteins (SPON2, TMEM30A, TMEM66, and truncated TMEFF2 (called POP4)); intracellular proteins (CAMK2N1, DHCR24, GLO1, NGFRAP1, PGK1, PSMA7, SBDS, and YWHAQ); and noncoding transcripts (POP1 (100 kb) from mRNA AK000023), POP2 (4 kb from mRNA AL832227), POP3 (50 kb from EST CFI40309), POP5 (intron of NCAM2, accession DO668384), POP6 (intron of FHIT), POP7 (intron of TNFAIP8), POP8 (intron of EFNA5), POP9 (intron of DSTN), POP10 (intron of ADAM2, accession DO668396), POP11 (87kb from EST BG194644), and POP12 (intron of EST BQ226050)). Expression of POP3 was prostate specific, whereas ADAM2, POP1, POP4, POP10, ELOVL5, RAMP1, and SPON2 had limited tissue expression. ELOVL5, MARCKSL1, NGFRAP1, PGK1, POP2, POP5, POP8, PSMA7, RAMP1, and SPON2 were significantly differentially expressed between laser microdissected malignant versus benign clinical samples of prostate tissue. PGK1, POP2, and POP12 correlated to clinical parameters. Levels of CAMK2N1, GLO1, SDBS, and TMEM30A transcripts tended to be increased in primary prostate cancer from patients who later had biochemical failure. Expression of GLO1, DHCR24, NGFRAP1, KLK3, and RAMP1 were significantly decreased in metastatic castration-recurrent disease compared with androgen-dependent primary prostate cancer. These novel potential biomarkers may therefore be useful in the diagnosis/prognosis of prostate cancer. Levels of 27 transcripts were investigated as potential novel markers for prostate cancer, including genes encoding plasma membrane proteins (ADAM2, ELOVL5, MARCKSL1, RAMP1, TMEM30A, and TMEM66); secreted proteins (SPON2, TMEM30A, TMEM66, and truncated TMEFF2 (called POP4)); intracellular proteins (CAMK2N1, DHCR24, GLO1, NGFRAP1, PGK1, PSMA7, SBDS, and YWHAQ); and noncoding transcripts (POP1 (100 kb) from mRNA AK000023), POP2 (4 kb from mRNA AL832227), POP3 (50 kb from EST CFI40309), POP5 (intron of NCAM2, accession DO668384), POP6 (intron of FHIT), POP7 (intron of TNFAIP8), POP8 (intron of EFNA5), POP9 (intron of DSTN), POP10 (intron of ADAM2, accession DO668396), POP11 (87kb from EST BG194644), and POP12 (intron of EST BQ226050)). Expression of POP3 was prostate specific, whereas ADAM2, POP1, POP4, POP10, ELOVL5, RAMP1, and SPON2 had limited tissue expression. ELOVL5, MARCKSL1, NGFRAP1, PGK1, POP2, POP5, POP8, PSMA7, RAMP1, and SPON2 were significantly differentially expressed between laser microdissected malignant versus benign clinical samples of prostate tissue. PGK1, POP2, and POP12 correlated to clinical parameters. Levels of CAMK2N1, GLO1, SDBS, and TMEM30A transcripts tended to be increased in primary prostate cancer from patients who later had biochemical failure. Expression of GLO1, DHCR24, NGFRAP1, KLK3, and RAMP1 were significantly decreased in metastatic castration-recurrent disease compared with androgen-dependent primary prostate cancer. These novel potential biomarkers may therefore be useful in the diagnosis/prognosis of prostate cancer. Prostate-specific antigen (PSA) has been used as a serum biomarker to monitor and screen for prostate cancer since 1986 and 1994, respectively.1Lilja H Ulmert D Vickers AJ Prostate-specific antigen and prostate cancer: prediction, detection and monitoring.Nat Rev Cancer. 2008; 8: 268-278Crossref PubMed Scopus (667) Google Scholar A recommendation for biopsy is set at an arbitrary serum PSA level of 4 ng/ml. At this threshold, PSA displays 93% specificity and a poor sensitivity of 24% for the detection of prostate cancer.2Thompson IM Ankerst DP Chi C Goodman PJ Tangen CM Lucia MS Feng Z Parnes HL Coltman Jr, CA Assessing prostate cancer risk: results from the prostate cancer prevention trial.J Natl Cancer Inst. 2006; 98: 529-534Crossref PubMed Scopus (770) Google Scholar In addition to carcinoma of the prostate, PSA is expressed in normal prostate tissue, prostatitis, and benign prostatic hyperplasia.3Bostwick DG Burke HB Djakiew D Euling S Ho SM Landolph J Morrison H Sonawane B Shifflett T Waters DJ Timms B Human prostate cancer risk factors.Cancer. 2004; 101: 2371-2490Crossref PubMed Scopus (468) Google Scholar Furthermore, 27% of men with borderline serum PSA levels (3.1 to 4 ng/ml) have detectable prostate cancer by biopsy.4Thompson IM Pauler DK Goodman PJ Tangen CM Lucia MS Parnes HL Minasian LM Ford LG Lippman SM Crawford ED Crowley JJ Coltman Jr, CA Prevalence of prostate cancer among men with a prostate-specific antigen level < or = 4.0 Ng per milliliter.N Engl J Med. 2004; 350: 2239-2246Crossref PubMed Scopus (1983) Google Scholar Serum PSA levels correlate with the degree of dissemination5Stamey TA Yang N Hay AR Mcneal JE Freiha FS Redwine E Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate.N Engl J Med. 1987; 317: 909-916Crossref PubMed Scopus (2084) Google Scholar, 6Pinsky PF Andriole G Crawford ED Chia D Kramer BS Grubb R Greenlee R Gohagan JK Prostate-specific antigen velocity and prostate cancer Gleason grade and stage.Cancer. 2007; 109: 1689-1695Crossref PubMed Scopus (51) Google Scholar and aggressiveness6Pinsky PF Andriole G Crawford ED Chia D Kramer BS Grubb R Greenlee R Gohagan JK Prostate-specific antigen velocity and prostate cancer Gleason grade and stage.Cancer. 2007; 109: 1689-1695Crossref PubMed Scopus (51) Google Scholar of prostate cancer. For example, serum PSA levels > 10 ng/ml are associated with a high pathological stage (odds ratio (OR) 1.7) and high Gleason sum (i.e., 7 to 10; OR 1.9), respectively, compared with PSA levels < 4 ng/ml.6Pinsky PF Andriole G Crawford ED Chia D Kramer BS Grubb R Greenlee R Gohagan JK Prostate-specific antigen velocity and prostate cancer Gleason grade and stage.Cancer. 2007; 109: 1689-1695Crossref PubMed Scopus (51) Google Scholar Following radical prostatectomy or brachytherapy, 7 to 15% of prostate cancers will exhibit biochemical recurrence at 8 years of follow-up as defined by rising PSA levels.7Pound CR Partin AW Eisenberger MA Chan DW Pearson JD Walsh PC Natural history of progression after PSA elevation following radical prostatectomy.J Am Med Assoc. 1999; 281: 1591-1597Crossref PubMed Scopus (2772) Google Scholar, 8Zelefsky MJ Kuban DA Levy LB Potters L Beyer DC Blasko JC Moran BJ Ciezki JP Zietman AL Pisansky TM Elshaikh M Horwitz EM Multi-institutional analysis of long-term outcome for stages T1–T2 prostate cancer treated with permanent seed implantation.Int J Radiat Oncol Biol Phys. 2007; 67: 327-333Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar Approximately 1% of prostate cancer patients will develop metastases following first-line therapy concomitant with serum PSA levels ≤ 2 ng/ml.9Leibovici D Spiess PE Agarwal PK Tu SM Pettaway CA Hitzhusen K Millikan RE Pisters LL Prostate cancer progression in the presence of undetectable or low serum prostate-specific antigen level.Cancer. 2007; 109: 198-204Crossref PubMed Scopus (90) Google Scholar Thus, measurement of serum PSA levels is inadequate for monitoring progression for a small subset of patients. Patients receiving androgen-deprivation therapy for disseminated disease will relapse and their disease will progress to the terminal, castration-recurrent prostate cancer for which there is no effective treatment.10Crawford ED Eisenberger MA Mcleod DG Spaulding JT Benson R Dorr FA Blumenstein BA Davis MA Goodman PJ A controlled trial of leuprolide with and without flutamide in prostatic carcinoma.N Engl J Med. 1989; 321: 419-424Crossref PubMed Scopus (1367) Google Scholar, 11Petrylak DP Tangen CM Hussain MH Lara Jr, PN Jones JA Taplin ME Burch PA Berry D Moinpour C Kohli M Benson MC Small EJ Raghavan D Crawford ED Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer.N Engl J Med. 2004; 351: 1513-1520Crossref PubMed Scopus (3340) Google Scholar, 12Tannock IF De Wit R Berry WR Horti J Pluzanska A Chi KN Oudard S Theodore C James ND Turesson I Rosenthal MA Eisenberger MA Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer.N Engl J Med. 2004; 351: 1502-1512Crossref PubMed Scopus (5048) Google Scholar Initial response to androgen-deprivation therapy is measured by PSA nadir. PSA nadir is prognostic of the time it takes to reach castration-recurrent prostate cancer and death.13Kwak C Jeong SJ Park MS Lee E Lee SE Prognostic significance of the nadir prostate specific antigen level after hormone therapy for prostate cancer.J Urol. 2002; 168: 995-1000Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar However, it is unknown whether pretreatment serum PSA levels can predict response to androgen-deprivation therapy. These limitations of PSA emphasize the need for new biomarkers to accurately detect, monitor, and predict the aggressiveness of prostate cancer. In particular, biomarkers that are prognostic and/or signify the propensity to rapidly develop advanced disease are required. Such biomarkers may stem from gene expression studies using in vivo models of advanced prostate cancer. Here, we characterize the expression of genes and novel non-coding transcripts that were previously identified by Long Serial Analysis of Gene Expression (LongSAGE) (unpublished data) and Subtractive Hybridization14Quayle SN Hare H Delaney AD Hirst M Hwang D Schein JE Jones SJ Marra MA Sadar MD Novel expressed sequences identified in a model of androgen independent prostate cancer.BMC Genomics. 2007; 8: 32Crossref PubMed Scopus (5) Google Scholar technologies using samples from the in vivo LNCaP Hollow Fiber model.15Sadar MD Akopian VA Beraldi E Characterization of a new in vivo hollow fiber model for the study of progression of prostate cancer to androgen independence.Mol Cancer Ther. 2002; 1: 629-637PubMed Google Scholar Both technologies can be used to discover unannotated transcripts, and Subtractive Hybridization is particularly well suited for the identification of differentially expressed low abundance transcripts.16Diatchenko L Lau YF Campbell AP Chenchik A Moqadam F Huang B Lukyanov S Lukyanov K Gurskaya N Sverdlov ED Siebert PD Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific CDNA probes and libraries.Proc Natl Acad Sci USA. 1996; 93: 6025-6030Crossref PubMed Scopus (2748) Google Scholar Novel transcripts identified by Subtractive Hybridization are referred to as POP 1 through 12: POP1, transcript 100 kb from mRNA AK000023; POP2, transcript 4 kb from mRNA AL832227; POP3, transcript 50 kb from EST CFI40309; POP4, transcript from the intron of transmembrane protein with epidermal growth factor-like and two follistatin-like domains 2 (TMEFF2); POP5, transcript from the intron of neural cell adhesion molecule 2 (NCAM2; accession DO668384); POP6, transcript from the intron of fragile histidine triad gene (FHIT); POP7, transcript from the intron of tumor necrosis factor, α-induced protein 8 (TNFAIP8); POP8, transcript from the intron of ephrin-A5 (EFNA5); POP9, transcript from the intron of actin depolymerizing factor destrin (DSTN); POP10, transcript from the intron of ADAM2 (accession DO668396); POP11, transcript 87 kb from EST BG194644; and POP12, transcript from the intron of EST BQ226050.14Quayle SN Hare H Delaney AD Hirst M Hwang D Schein JE Jones SJ Marra MA Sadar MD Novel expressed sequences identified in a model of androgen independent prostate cancer.BMC Genomics. 2007; 8: 32Crossref PubMed Scopus (5) Google Scholar Genes previously identified by LongSAGE and examined here were the known genes ADAM2, CAMK2N1, ELOVL5, GLO1, MARCKSL1, NGFRAP1, PGK1, PSMA7, RAMP1, SBDS, SPON2, TMEM30A, TMEM66, and YWHAQ. DHCR24 is correlated with a higher incidence of metastases17Lapointe J Li C Higgins JP Van De Rijn M Bair E Montgomery K Ferrari M Egevad L Rayford W Bergerheim U Ekman P Demarzo AM Tibshirani R Botstein D Brown PO Brooks JD Pollack JR Gene expression profiling identifies clinically relevant subtypes of prostate cancer.Proc Natl Acad Sci USA. 2004; 101: 811-816Crossref PubMed Scopus (1072) Google Scholar, 18Henshall SM Afar DE Hiller J Horvath LG Quinn DI Rasiah KK Gish K Willhite D Kench JG Gardiner-Garden M Stricker PD Scher HI Grygiel JJ Agus DB Mack DH Sutherland RL Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse.Cancer Res. 2003; 63: 4196-4203PubMed Google Scholar, 19Hendriksen PJ Dits NF Kokame K Veldhoven A Van Weerden WM Bangma CH Trapman J Jenster G Evolution of the androgen receptor pathway during progression of prostate cancer.Cancer Res. 2006; 66: 5012-5020Crossref PubMed Scopus (186) Google Scholar and was included here as a reference gene for comparison. The expression of these transcripts was measured in a variety of cell types and tissues, including clinical samples of androgen-dependent primary prostate cancer and metastasis of castration-recurrent prostate cancer. Here, we examine tissue specificity, androgen regulation, and feasibility of these transcripts in the prognosis of prostate cancer. Cell lines were maintained in RPMI 1640 media (LNCaP, 22Rv1, and COS1), Dulbecco's modified Eagle's medium (PC-3, DU145, and RKO), BRFF-HPC1 medium (MDA PCa 2b), or minimal essential medium (MG63, CV1, HEPG, and MCF7). All media (Stem Cell Technologies, Vancouver, BC, Canada) was supplemented with 100 U/ml penicillin and 100 units/ml streptomycin (Invitrogen, Burlington, ON, Canada) and fetal bovine serum (HyClone, Logan, UT). For androgen treatments, LNCaP cells were serum-starved for 48 hours and then treated for 16 hours in serum-free medium with 10 nmol/L synthetic androgen R1881 (PerkinElmer, Woodbridge, ON, Canada) or vehicle control (ethanol, final concentration 2.85 × 10−4%). Total RNA from cell lines was harvested using TRIzol Reagent (Invitrogen), and RNA from benign human tissue was obtained commercially (BD Clontech, Mountain View, CA). Informed consent was obtained from each patient participating in the study according to guidelines set forth by the University of British Columbia/British Columbia Cancer Agency Research Ethics Board. Frozen primary androgen-dependent prostate specimens from patients in Japan who had undergone radical prostatectomies were obtained through coauthor T. Ueda and were embedded in Optimal Cutting Temperature™ (OCT) compound (Tissue-Tek, Torrance, CA). Prostatectomy specimens were accompanied by information, including the age of the patient, prior treatment history, serum PSA levels before surgery, and Tumor-Node-Metastasis (TNM) clinical and pathological stage (Table 1). Any patient who had received presurgical hormone ablation treatment was excluded. Malignant or benign epithelial cells were stringently obtained by laser microdissection using the μCut MMI AG Microscope (MMI Molecular Machines & Industries, Glattbrugg, Switzerland) (Figure 1, A–F). Total RNA was isolated and purified with the RNA Easy Micro kit (Qiagen, Mississauga, ON, Canada). Contaminating genomic DNA was removed from RNA samples by TURBO DNA-free (Ambion, Austin, TX) or DNase I from the RNA Easy Micro Kit. RNA quality and quantity were assessed using the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada) with RNA 6000 Nano LabChip kit (Caliper Technologies, Hopkinton, MA). RNA of poor quality (RNA integrity number < 2.8) and insufficient quantity (<531 ng) was not used in this study. SuperScript III First-Strand Synthesis System (Invitrogen) with Oligo(dT) was used to reverse transcribe an input of 2.5 ng of RNA per quantitative (q)PCR.Table 1Demographics, PSA, Stage, Gleason grades, and Sum of the Prostate Cancer Patients Used in This Study (Laser Capture-Microdissected Samples)Sample no.*Sample no., sample number labeled "1" to "38".Patient ID†Patient ID, patient identification labeled "A" to "AC".Age (years)PSA‡PSA serum levels upon diagnosis. (ng/ml)Stage§Stage, tumor node metastasis (TNM) staging system.Normal or tumor¶N (normal), normal/benign prostate tissue; T (Tumor), malignant prostate tissue.Gleason grade∥Gleason grade, grading system to describe degree of differentiation of tumor tissue cells. Gleason grading was applied to the slide of tissue used for laser microdissection by a trained pathologist.Gleason sum**Gleason sum, cumulative score of the two most prominent Gleason grades present on the slide of tissue.ClinicalPathological1A599.7NA2bNNANA2A599.7NA2bT3 + 473B6419.01c3bNNANA4B6419.01c3bT4 + 485C7124.22b2bNNANA6C7124.22b2bT3 + 477D689.52a3bNNANA8D689.52a3bT5 + 499E6419.12a3bT3 + 4710F715.52b2bNNANA11G6925.11cNANNANA12G6925.11cNANNANA13G6925.11cNAT4 + 3714H676.42a2aNNANA15H676.42a2aT4 + 4816I647.72a2aNNANA17J7029.92b3bT3 + 3618K6210.02a2bNNANA19L6315.62a2bT3 + 4720M745.22b2bNNANA21N7014.12b3bT4 + 3722ONANANANAT3 + 4723ONANANANANNANA24ONANANANAT3 + 5825P745.72a2aNNANA26Q698.02b3bNNANA27R6822.22a3aNNANA28S7320.33a3bT4 + 4829T6330.83a3aT4 + 3730U758.41c3aT3 + 3631V7310.22b2bT5 + 3832W6212.12b3aT3 + 4733X6221.93a3bT4 + 4834Y7125.93a3bT3 + 3635Z6711.02a3bT4 + 4836AA6413.73a2bT4 + 4837AB68109.02b2bT3 + 4738AC6718.42a2bT5 + 49NA, not applicable or not available.* Sample no., sample number labeled "1" to "38".† Patient ID, patient identification labeled "A" to "AC".‡ PSA serum levels upon diagnosis.§ Stage, tumor node metastasis (TNM) staging system.¶ N (normal), normal/benign prostate tissue; T (Tumor), malignant prostate tissue.∥ Gleason grade, grading system to describe degree of differentiation of tumor tissue cells. Gleason grading was applied to the slide of tissue used for laser microdissection by a trained pathologist.** Gleason sum, cumulative score of the two most prominent Gleason grades present on the slide of tissue. Open table in a new tab NA, not applicable or not available. Clinical samples of primary prostate cancer from men who later did, and did not, develop biochemical failure within 5 years after radical prostatectomy as well as lymph node metastasis from men with castration-recurrent metastatic disease (Prostate Cancer Rapid Autopsy Program) were obtained from the University of Washington through coauthor R. L. Vessella. Total RNA was extracted from tissue with RNA STAT-60 kit (Teltest, Friendswood, TX), and cDNA was synthesized with Oligo dT using Advantage RT for PCR kit (BD Clontech). Input RNA was reverse transcribed with SuperScript III First Strand Synthesis kit (Invitrogen). For most RNA samples, a quantity of 0.5 μg was used in the RT reaction, but for limited sample quantities, such as those from the laser microdissected prostate tissue, 0.1 or 0.05 μg of RNA was used. A 10-μl quantitative (q)RT-PCR consisted of 1 μl of template cDNA, 1× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), and 0.9 μmol/L each of forward and reverse primers and 0.25 μmol/L of TaqMan probe (FAM-BHQ-1 or TET-BHQ-1; Integrated DNA Technologies, San Diego, CA) that produce specific PCR products ranging in size between 85 and 235 bp (see Table 2 for primer and probe sequences). qRT-PCR reactions were cycled as follows in a 7900HT Sequence Detection System (Applied Biosystems): 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 0.25 minutes, followed by 60°C for 1 minute. All qRT-PCRs were performed using technical triplicates. cDNAs (from different conditions/patients) and genes (target and reference) to be directly compared were assayed in the same instrument run. Glyceraldehyde-3-phosphate (GAPDH) was used as a reference gene for all experiments, except the androgen regulation experiment using LNCaP cells in which succinate dehydrogenase complex, subunit A, flavoprotein was used due to its consistent levels of expression despite androgen stimulation. These reference genes were carefully assessed for consistent levels of expression across all conditions. Reactions without template were run for each gene to ensure that DNA had not contaminated the qRT-PCR reactions. Efficiency checks were performed for each primer pair. Relative expression was determined using the Pfaffl method, which takes into consideration the efficiency of the reaction for both the target and reference genes.Table 2Primer and Probe Sequences for qRT-PCR of Candidate TranscriptsGeneForward primerProbeReverse primerADAM25′-TGGTGAAAGTTAATTTCCAAAGG-3′5′-ATTCAAGCGATGAGCAACCT-3′5′-TCATGGCATCTCTGTTGTCC-3′CAMK2N15′-TGCAGGACACCAACAACTTC-3′5′-AGCAAGCGGGTTGTTATTGA-3′5′-GCACGTCATCAATCCTATCATC-3′DHCR245′-GAGGCAGCTGGAGAAGTTTG-3′5′-TGCTGTATGCCGACTGCTAC-3′5′-CTTGTGGTACAAGGAGCCATC-3′ELOVL55′-GTTTGTCGTCAGTCCCTTCC-3′5′-CGTCCATACCTCTGGTGGAA-3′5′-TGGTCTGGATGATTGTCAGC-3′GAPDH5′-CTGACTTCAACAGCGACACC-3′5′-CGACCACTTTGTCAAGCTCA-3′5′-TGCTGTAGCCAAATTCGTTG-3′GLO15′-AAAGGTTTGAAGAACTGGGAGTC-3′5′-AAGGCCTGGCATTTATTCAA-3′5′-TTCAATCCAGTAGCCATCAGG-3′MARCKSL15′-GCAGCCAGAGCTCCAAGG-3′5′-CCAACGGCCAGGAGAATG-3′5′-AAGTCTCCATTGCTTTTCACG-3′NGFRAP15′-GTCACTCGCGTCTGGCTAC-3′5′-AAAGCGGAGCAGGTCTGC-3′5′-GCCGCGGAGACACTTAGC-3′PGK15′-GAAGGGAAGGGAAAAGATGC-3′5′-CGAGCCAGCCAAAATAGAAG-3′5′-GACATCCCCTAGCTTGGAAAG-3′POP1*Genes were represented by Human Genome Organisation Gene Nonenclature Committee-approved nomenclature when available. Non-Human Genome Organisation Gene Nonenclature Committee gene names were not italicized.5′-AAGCTCTTGCTAGGCATGTAGG-3′5′-CCTGGACAGCCCATTCTTTA-3′5′-TTTGGGTAGACATTTCCCC-3′POP25′-GGAGGATCAACAGCAGCATT-3′5′-CAACTGTGCTCCATTGACGT-3′5′-GGTATCATTGAGGCTGGGTG-3′POP35′-TATGGTGTGCCATTTCTGGA-3′5′-CCGTTTGCATCTCTGAGTGA-3′5′-GTGGAACAAAATCCCCTCCT-3′POP45′-CCCTTGTGCAAATGGGTTA-3′5′-TCATTTATGATAGCCACACATGA-3′5′-TTGTTCCCTTCACTCTTTTGTTC-3′POP55′-TTTGGAAAGGTGAGCCTCTG-3′5′-CATTGTTTGGGCAGGAGAGT-3′5′-AAAGAAGTGGACGTGGCAC-3′POP65′-TTTAAGTGGTTCAGCACACAAAAC-3′5′-CAAAAGGATGACCTTGGGAA-3′5′-TGATGACTTCCTTGTGTTTAACAAA-3′POP75′-TTGGTTTCTGGACCCTTTTG-3′5′-AAAGCTTGAGGGTGGTGATG-3′5′-CAGAAGAGCAGGGTGGGTAG-3′POP85′-TTTCGGTTCCTTTCCTCTTC-3′5′-CCCACATTCCATTTCAAACA-3′5′-ATTCCTTTATGGCTTGAAGGGT-3′POP95′-CCTGTTTCCCAGTCACACCT-3′5′-TTAACAATTCCCAAGCACCC-3′5′-ATTTGTCTTCCACCACAGGC-3′POP105′-TTGCTAGGGAAAAGCAGCAT-3′5′-TTCTTCACCAAACTCTCTAAAACAGA-3′5′-GAATCATAAGGCAGCCTCCTT-3′POP115′-GTTCGCTCTTGGCTTTGAAC-3′5′-TTCCCTGTCCCCTAACTCCT-3′5′-TTTGCCTTTTGCAGAATGTG-3′POP125′-TGTGACAAAATGGGAGGACA-3′5′-GCTTGTTTGAGTTGCAAGCA-3′5′-CAGAAAAGTGTATGGCAGGGA-3′PSMA75′-CGTCAAGAAGGGCTCGAC-3′5′-AAGAAGTCAGTGGCCAAACTG-3′5′-CGCACTGTTCTTTCATCCTG-3′RAMP15′-CCTCACCCAGTTCCAGGTAG-3′5′-CAGGACCATCAGGAGCTACA-3′5′-CATGTGCCAGGTGCAGTC-3′SBDS5′-CGCCTGCTACAAAAACAAGG-3′5′-CGTGGAAAAAGACCTCGAT-3′5′-CAAACACTGAGTGGGTCTGC-3′SDHA5′-ACCAGGTCACACACTGTTGC-3′5′-ACATGGAGGAGGACAACTGG-3′5′-C CCTGTGGTGTCGTAGAAATGC-3′SPON25′-CCCAGCAGGGACAATGAG-3′5′-TGTAGACAGCGCCTCAGTTC-3′5′-CACAGTCCCCAGGACGAC-3′TMEM30A5′-GGATGTGACACCTTGCTTTTG-3′5′-CCATTAACTTCACACTGGAAAAG-3′5′-ACGTAACGACGATGGTTTTG-3′TMEM665′-GGGCAGCTATTCGGTATGTTC-3′5′-CGAAAACCAGAACTGCATCA-3′5′-TGCATCCAGTGTTTGACTCC-3′YWHAQ5′-CTGAGATCCATCTGCACCAC-3′5′-AGCCAATGCAACTAATCCAGA-3′5′-ACCGGAAGTAATCACCCTTC-3′* Genes were represented by Human Genome Organisation Gene Nonenclature Committee-approved nomenclature when available. Non-Human Genome Organisation Gene Nonenclature Committee gene names were not italicized. Open table in a new tab LNCaP cells (2 × 106) were cultured in 15-cm plates in RPMI 1640 medium containing 5% fetal bovine serum for 24 hours. Medium was changed to serum-free and phenol red-free RPMI 1640 medium. After at least 24 hours of serum starvation, cells were treated with the indicated concentrations of R1881 or ethanol (vehicle) and were further incubated for 24 or 48 hours. Cells were harvested, and total cell lysates were analyzed by Western blot analysis with the respective antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). To identify significant changes in gene expression in response to androgen, we used the two-sample Student's t-test for unequal variance. Nonparametric methods were used with data that were sampled from nonnormal distributions. For gene expression analysis on RNA from laser microdissected prostatic tissue, the Spearman's correlation test was used to identify associations to patient age or PSA levels, and the Kruskal-Wallis test was used to identify significant differences between mean gene expression in normal (benign) and tumor tissue, TNM stages of cancer, Gleason scores, or biochemical recurrence and terminal castration-recurrent metastases. Tissue-specific expression of transcripts in cell lines were measured by qRT-PCR using RNA isolated from five human prostate cancer cells (LNCaP, MDA-PCa-2B, 22Rv1, PC-3, and DU145) and nonprostate human cancer cell lines that included the following: MG63, osteosarcoma cells; RKO, colon carcinoma cells; HEPG, hepatocellular carcinoma cells; MCF7, mammary adenocarcinoma cells; and large T-antigen transformed and normal monkey kidney cells (COS1 and CV1, respectively). Expression of genes ADAM2 and POP11 were relatively specific for LNCaP cells (Figure 2A). ADAM2 and POP10 (intron of ADAM2) showed differences in expression in HEPG cells suggesting tissue-specific expression of splice variants of ADAM2. MARCKSL1, POP1, POP2, POP3, POP4, POP5, POP12, and SPON2 were enriched in human prostate cancer cell lines compared with all other human cancer cell lines tested. To address tissue-specific expression in benign tissues, levels of transcripts in 20 human tissue samples were measured. Gene expression was displayed relative to the levels in benign human prostate tissue. POP3 was the only transcript to exhibit exclusive expression in benign prostate tissue versus other benign human tissues tested (Figure 2B), suggesting it is prostate specific. This was consistent with expression of POP3 predominantly in LNCaP, MDA-PCa-2B, and 22RV1 cells that express androgen receptor and low expression in all other cell lines examined (Figure 2A). Some genes were expressed at a level on par with that of the benign prostate in the adrenal gland (ELOVL5) and testis (ELOVL5 and POP1; Figure 2B). Both adrenal glands and testes produce androgens that are essential for regulating the growth of the prostate.20Geller J Rationale for blockade of adrenal as well as testicular androgens in the treatment of advanced prostate cancer.Semin Oncol. 1985; 12: 28-35PubMed Google Scholar POP1 had relatively specific expression in prostate cancer cell lines with similar expression patterns to POP3, whereas ELOVL5 had broad expression across most cell lines (Figure 2A). ADAM2 and POP10 (intron of ADAM2) showed similar expression patterns in prostate, placenta, and testis with the exception of expression of only ADAM2 in thymus tissue (Figure 2B). These data support the tissue-specific expression of splice variants of ADAM2. POP4 (splice variant of TMEFF2) was expressed in the prostate, brain, and prostate cancer cells only expressing the androgen receptor. Both RAMP1 and SPON2 had relatively restricted expression in prostate and uterine tissues (Figure 2B). SPON2 had expression generally specific for prostate cancer cell lines (LNCaP and MDA-PCa-2B), while RAMP1 was also highly expressed in MG63 osteosarcoma cells (Figure 2A). Taken together, these data suggest that ADAM2, ELOVL5, POP1, POP3, POP4, POP10, RAMP1, and SPON2 have relatively restricted expression patterns in the prostate when comparing benign tissues (Figure 2B). The androgen signaling axis plays an important role in the growth, survival, and differentiation of the prostate.21Balk SP Knudsen KE AR. The cell cycle, and prostate cancer.Nucl Recept Signal. 2008; 6: e001Google Scholar, 22Huggins C Hodges C Studies on prostatic cancer: the effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate.Cancer Res. 1941; : 293-297Google Scholar, 23Cunha GR Ricke W Thomson A Marker PC Risbridger G Hayward SW Wang YZ Donjacour AA Kurita T Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.J Steroid Biochem Mol Biol. 2004; 92: 221-236Crossref PubMed Scopus (248) Google Scholar Treatment for locally advanced and metastatic prostate cancer includes androgen-deprivation therapy. Thus, it is essential to determine whether levels of expression of any of the 27 transcripts were altered by androgen. To do this, levels of expression of these genes were assessed in prostate cancer cells with androgen receptor (LNCaP, MDA-PCa-2B, and 22Rv1) and without a functional androgen receptor (PC-3 and DU145).24Horoszewicz JS Leong SS Kawinski E Karr JP Rosenthal H Chu TM Mirand EA Murphy GP LNCaP model of human prostatic carcinoma.Cancer Res. 1983; 43: 1809-1818PubMed Google Scholar, 25Kaighn ME Narayan KS Ohnuki Y Lechner JF Jones LW Establishment and characterization of a human prostatic carcinoma cell line (PC-3).Invest Urol. 1979; 17: 16-23PubMed Google Scholar, 26Stone KR Mickey DD Wunderli H Mickey GH Paulson DF Isolation of a human prostate carcinoma cell line (DU 145).Int J Cancer. 1978; 21: 274-281Crossref PubMed Scopus (1038) Google Scholar, 27Sramkoski RM Pretlow 2nd, TG Giaconia JM Pretlow TP Schwartz S Sy MS Marengo SR Rhim JS Zhang D Jacobberger JW A new human prostate carcinoma cell line, 22Rv1.In Vitro Cell Dev Biol Anim. 1999; 35: 403-409Crossref PubMed Scopus (448) Google Scholar, 28Navone NM Olive M Ozen M Davis R Troncoso P Tu SM Johnston D Pollack A Pathak S Von Eschenbach AC Logothetis CJ Establishment of two human prostate cancer cell lines derived from a single bone metastasis.Clin Cancer Res. 1997; 3: 2493-2500PubMed Google Scholar Expression of ADAM2, CAMK2N1, DHCR24, MARCKSL1, NGFRAP1, POP1, POP3, POP4, POP5, POP7, POP8, POP10, POP11, SPON2, and TMEM66 transcripts were enriched in prosta
Referência(s)