Kati P. Porkka

MicroRNA Expression Profiling in Prostate Cancer

MicroRNAs (miRNA) are small, endogenously expressed noncoding RNAs that negatively regulate expression of protein-coding 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.

Amplification and Overexpression of p40 Subunit of Eukaryotic Translation Initiation Factor 3 in Breast and Prostate Cancer

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.

Androgen regulation of micro-RNAs in prostate cancer

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.

Genetic aberrations in prostate cancer by microarray analysis.

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.

Dual-specificity phosphatase 1 and serum/glucocorticoid-regulated kinase are downregulated in prostate cancer

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 high‐grade 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.

RAD21 and KIAA0196 at 8q24 are amplified and overexpressed in prostate cancer.

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.

Amplification and overexpression of elongin C gene discovered in prostate cancer by cDNA microarrays

Functional significance of several oncogenes is mediated by overexpression. To identify overexpressed genes in prostate cancer, we analyzed expression of 1081 transcripts in three prostate cancer cell lines (PC-3, DU145, and LNCaP) using cDNA microarray hybridization. The cDNA microarray analyses were validated by quantitative real-time RT-PCR. On average, 64% of the genes were expressed at detectable levels in the cell lines. Next, the expression profiles were combined with the data on DNA sequence copy number alterations in the cell lines obtained by comparative genomic hybridization. The genes for Elongin C and urokinase type plasminogen-activator, both located in the regions of amplification in the PC-3 cell line (8q21 and 10q22, respectively), were found to be overexpressed in the PC-3. Amplification and overexpression of urokinase type plasminogen-activator in prostate cancer has previously been reported. Here, fluorescence in situ hybridization on tissue microarray showed high-level amplification of the Elongin C gene in 8 (23%) of 35 hormone-refractory carcinomas but in none of the untreated prostate carcinomas (n = 35). Finally, it was shown that the Elongin C gene was overexpressed and amplified also in breast cancer cell line SK-Br-3. The results indicate that Elongin C is a putative target gene for 8q amplification.

Clusterin is epigenetically regulated in prostate cancer

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.

Cloning and Characterization of a Novel Six-Transmembrane Protein STEAP2 , Expressed in Normal and Malignant Prostate

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 = 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.

Detection of differentially expressed genes in prostate cancer by combining suppression subtractive hybridization and cDNA library array

The molecular mechanisms underlying the development and progression of prostate cancer have remained poorly understood. The identification of differentially expressed genes has been used as a tool to recognize genes that are involved in disease processes. In this study we combined suppression subtractive hybridization (SSH) and cDNA array hybridization to identify genes whose expression is decreased in prostate cancer. cDNA from benign prostatic hyperplasia (BPH) was subtracted with cDNA from the prostate cancer cell line PC‐3 and 386 of the subtracted clones were arrayed onto a nylon filter membrane. The differential gene expression was then verified by hybridizing the filter with radioactively labelled first‐strand cDNA preparations from BPH, PC‐3, four other cancer cell lines, and a normal prostate epithelial cell line (PrEC). In order to validate SSH and cDNA array hybridization, the enrichment of clones in the subtraction, as well as the sensitivity and linearity of array hybridization, was first evaluated. The array hydridization results were confirmed by northern analysis and selected clones were sequenced. Altogether, several known genes, such as prostate‐specific antigen (PSA), human glandular kallikrein 2 (hK2), phosphatidic acid phosphatase type 2a (PAP2a), α‐tropomyosin, and insulin‐like growth factor binding protein 7 (IGFBP‐7), as well as an anonymous transcript (EST), were found to be expressed less in PC‐3 than in BPH. Further studies on the significance of these genes in the development of prostate cancer are now warranted. Copyright