MicroRNAs (miRNA) are small, endogenously expressed noncoding RNAs that negatively regulate expression of proteincoding genes at the translational level. Accumulating evidence, such as aberrant expression of miRNAs, suggests that they are involved in the development of cancer. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. To identify the miRNA signature specific for prostate cancer, miRNA expression profiling of 6 prostate cancer cell lines, 9 prostate cancer xenografts samples, 4 benign prostatic hyperplasia (BPH), and 9 prostate carcinoma samples was carried out by using an oligonucleotide array hybridization method. Differential expression of 51 individual miRNAs between benign tumors and carcinoma tumors was detected, 37 of them showing down-regulation and 14 up-regulation in carcinoma samples, thus identifying those miRNAs that could be significant in prostate cancer development and/or growth. There was a significant trend (P = 0.029) between the expression of miRNAs and miRNA locus copy number determined by array comparative genomic hybridization, indicating that genetic aberrations may target miRNAs. Hierarchical clustering of the tumor samples by their miRNA expression accurately separated the carcinomas from the BPH samples and also further classified the carcinoma tumors according to their androgen dependence (hormone naive versus hormone refractory), indicating the potential of miRNAs as a novel diagnostic and prognostic tool for prostate cancer. [Cancer Res 2007;67(13):6130-5]

BACKGROUND Androgens play a critical role in the growth of both androgen dependent and castration‐resistant prostate cancer (CRPC). Only a few micro‐RNAs (miRNAs) have been suggested to be androgen regulated. We aim to identify androgen regulated miRNAs. METHODS We utilized LNCaP derived model, we have established, and which overexpresses the androgen receptor (AR), the VCaP cell line, and 13 intact‐castrated prostate cancer (PC) xenograft pairs, as well as clinical specimens of untreated (PC) and CRPC. The expression of miRNAs was analyzed by microarrays and quantitative RT‐PCR (Q‐RT‐PCR). Transfection of pre‐miR‐141 and anti‐miR‐141 was also used. RESULTS Seventeen miRNAs were >1.5‐fold up‐ or downregulated upon dihydrotestosterone (DHT) treatment in the cell lines, and 42 after castration in the AR‐positive xenografts. Only four miRNAs (miR‐10a, miR‐141, miR‐150*, and miR‐1225‐5p) showed similar androgen regulation in both cell lines and xenografts. Of those, miR‐141 was found to be expressed more in PC and CRPC compared to benign prostate hyperplasia. Additionally, the overexpression of miR‐141 enhanced growth of parental LNCaP cells while inhibition of miR‐141 by anti‐miR‐141 suppressed the growth of the LNCaP subline overexpressing AR. CONCLUSIONS Only a few miRNAs were found to be androgen‐regulated in both cell lines and xenografts models. Of those, the expression of miR‐141 was upregulated in cancer. The ectopic overexpression of miR‐141 increased growth of LNCaP cell suggesting it may contribute to the progression of PC. Prostate 71:604–614, 2011. © 2010 Wiley‐Liss, Inc.

Amplification at the long arm of chromosome 8 occurs in a large fraction of breast and prostate cancers. To clone the target genes for this amplification, we used suppression subtraction hybridization to identify overexpressed genes in the breast cancer cell line SK-Br-3, which harbors amplification at 8q (8q21 and 8q23-q24). A differentially expressed gene identified by SSH, the p40 subunit of eukaryotic translation initiation factor 3 (eIF3), was localized to 8q23 and found to be highly amplified and overexpressed in the breast and prostate cancer cell lines studied. High-level amplification of eIF3-p40 was found in 30% of hormone-refractory prostate tumors and in 18% of untreated primary breast tumors. In the vast majority of the cases, p40 and c-myc were amplified with equal copy numbers. Tumors with higher copy numbers of p40 than c-myc were also found. Expression of p40 mRNA was analyzed with in situ hybridization. The amplification of eIF3-p40 gene was associated with overexpression of its mRNA, as expected for a functional target gene of the amplification. These results imply that genomic aberrations of translation initiation factors, such as eIF3-p40, may contribute to the pathogenesis of breast and prostate cancer.

Purpose: The androgen receptor (AR)-mediated signaling pathway seems to be essentially involved in the development and progression of prostate cancer. In vitro studies have shown that altered expression of AR coregulators may significantly modify transcriptional activity of AR, suggesting that these coregulators could also contribute to the progression of prostate cancer. Here, our goal was to assess alterations in the expression of the AR coregulators in prostate cancer in vivo.Experimental Design: The expression of 16 AR coactivators and corepressors (SRC1, ␤-catenin, TIF2, PIAS1, PIASx, ARIP4, BRCA1, AIB1, AIB3, CBP, STAT1, NCoR1, AES, cyclin D1, p300, and ARA24) was measured in prostate cancer cell lines, xenografts, and clinical prostate tumor specimens by using real-time quantitative reverse transcription-PCR. In addition, gene copy number of SRC1 was analyzed by fluorescence in situ hybridization.Results: Both AR-positive and AR-negative cell lines and xenografts expressed the coregulators. Most of the coregulators studied were expressed at equal levels in benign prostatic hyperplasia and untreated and hormone-refractory carcinomas. However, the expression of PIAS1 and SRC1 was significantly (P ‫؍‬ 0.048 and 0.017, respectively) lower in hormone-refractory prostate tumors than in untreated prostate tumors. No overexpression of the coregulators was found in the clinical material. Paradoxically, the SRC1 gene was found to be amplified and highly expressed in a LuCaP 70 prostate cancer xenograft. Conclusions:These findings suggest that the decreased expression of PIAS1 and SRC1 could be involved in the progression of prostate cancer. In addition, gene amplification of SRC1 in one of the xenografts implies that, in some tumors, genetic alteration of SRC1 may provide a growth advantage.

The aim of this study was to screen genetic as well as expression alterations in prostate cancer. Array comparative genomic hybridization (aCGH) to a 16K cDNA microarray was performed to analyze DNA sequence copy number alterations in 5 prostate cancer cell lines and 13 xenografts. The aCGH confirmed the previously implicated common gains and losses, such as gains at 1q, 7, 8q, 16p and 17q and losses at 2q, 4p/q, 6q, 8p, 13q, 16q, 17p and 18q, which have previously been identified by chromosomal CGH (cCGH). Because of the higher resolution of aCGH, the minimal commonly altered regions were significantly narrowed-down. For example, the gain of 8q was mapped to three independent regions, 8q13.3-q21.11, 8q22.2 and 8q24.13-q24.3. In addition, a novel recurrent gain at 9p13-q21 was identified. The concomitant expression analysis indicated that genome-wide DNA sequence copy number (gene dosage) was significantly associated with the expression level (p < 0.0001). The analyses indicated several individual genes whose expression was associated with the gene copy number. For example, gains of PTK2 and FZD6, were associated with the increased expression, whereas losses of TNFRSF10B (alias DR5) and ITGA4 with decreased expression. In conclusion, the aCGH mapping data will aid in the identification of genes altered in prostate cancer. The combined expression and copy number analysis suggested that even a low-level copy number change may have significant effect on gene expression, and thus on the development of prostate cancer. ' 2006 Wiley-Liss, Inc.Key words: prostatic carcinoma; neoplasia; amplification; deletion; expression; cDNA microarray; molecular cytogenetics Genetic alterations underlying the development and progression of prostate cancer are incompletely known. Chromosomal comparative genomic hybridization (cCGH) as well as analysis of loss of heterozygosity (LOH) have been used to screen prostate tumors for chromosomal aberrations. Several chromosomal regions have been implicated in these studies (reviewed in Ref. 1). The chromosomal arms containing losses most often are 6q, 8p, 10q, 13q, 16q and 18q, whereas the most common gains are found in chromosomes 7, 8q and Xq. The losses at 6q, 8p and 13q seem to be early events, found also in prostate intraepithelial neoplasia (PIN). On the other hand, the gains of chromosome 7 and 8q are late events and are associated with aggressive phenotype. The gain of Xq is found especially in hormone-refractory prostate carcinomas. The actual genes affected in these chromosomal regions are often not known. Moreover, only a few genes that are commonly altered, either genetically or epigenetically, in prostate cancer have been identified (reviewed in Ref. 1).Microarray technologies are now commonly being used for expression analysis of large numbers of genes. Several studies, using either oligo or cDNA microarrays, on expression profiling in prostate cancer have already been published (reviewed in Ref. 2). These have indicated that a number of genes, such as EZH2, 3 AMACR4 hepsin, ...

SUMMARY:By using subtraction and cDNA array hybridizations, we recently identified an anonymous transcript that was differentially expressed in benign prostate hyperplasia and prostate cancer cell line PC-3. Here, we report the cloning of the full-length cDNA of the gene, designated STEAP2 (six-transmembrane epithelial antigen of the prostate 2). The gene is located at the chromosomal region 7q21 and encodes for a 490 -amino acid protein with six predicted transmembrane domains and is predominantly expressed in prostate epithelial cells. Green fluorescent protein fusion construct indicated that the STEAP2 protein is localized mainly in the plasma membrane. Real-time quantitative RT-PCR showed that the gene is expressed at levels more than 10 times higher in normal prostate than in other tissues studied. Of the prostate cancer cell lines, STEAP2 was expressed in significant levels only in androgen-responsive LNCaP. The expression of STEAP2 was significantly higher (p ϭ 0.002) in both untreated primary and hormone-refractory prostate carcinomas than in benign prostate hyperplasias, suggesting that it may be involved in the development of prostate cancer. As a cell-surface antigen, STEAP2 is a potential diagnostic or therapeutic target in prostate cancer. (Lab Invest 2002, 82:1573-1582.

Inactivation of tumor suppressor genes through deletion, mutation and epigenetic silencing has been shown to occur in cancer. In our study, we combined DNA demethylation and histone deacetylation inhibition treatments with suppression subtraction hybridization (SSH) and cDNA microarrays to identify potentially epigenetically downregulated genes in PC-3 prostate cancer cell line. We found 11 genes whose expression was upregulated after relieving epigenetic regulation. Expression of 3 genes [dual-specificity phosphatase 1 (DUSP1), serum/glucocorticoid regulated kinase (SGK) and spermidine/spermine N1-acetyltransferase (SAT)] was subsequently studied in clinical sample material using real-time quantitative RT-PCR and immunohistochemistry. The DUSP1 and SGK mRNA expression was lower in hormone-refractory prostate carcinomas compared to benign prostate hyperplasia (BPH) or untreated prostate carcinomas. BPH, normal prostate and highgrade prostate intraepithelial neoplasia (PIN) expressed high levels of DUSP1 and SGK proteins. Ninety-two percent and 48% of the prostate carcinomas showed almost complete lack of DUSP1 and SGK proteins, respectively, indicating common downregulation of these genes. The genomic bisulphite sequencing did not reveal dense hypermethylation in the promoter regions of either DUSP1 or SGK. In conclusion, the data suggest that downregulation of DUSP1 and SGK is an early event and could be important in the tumorigenesis of prostate cancer. ' 2005 Wiley-Liss, Inc.

To detect genes that are overexpressed in prostate cancer, a subtracted cDNA library was first constructed from the PC-3 cell line and subsequently screened by using cDNA microarray hybridization. Sixty-eight genes were found to be overexpressed (ratio>3) in PC-3. Half of these genes were in chromosomal regions, which, using comparative genomic hybridization, we previously showed to be gained in PC-3. Subsequently, the expression and copy number of selected genes were studied by quantitative RT-PCR and fluorescence in situ hybridization in prostate cancer cell lines, xenografts, and clinical tumor specimens of benign prostate hyperplasia and untreated as well as hormone-refractory prostate carcinomas. Two genes from chromosomal region 8q24-RAD21 and KIAA0196-showed increased expression in clinical prostate carcinomas and were also amplified in 30-40% of xenografts and hormone-refractory tumors. In addition, the expression of KIAA0196 was significantly (P=0.0051) higher in tumors with the gene amplification than in those without it. The data suggest that KIAA0196 and possibly RAD21 are putative target genes for the common amplification of 8q23-24 in prostate cancer.