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From the Department of Pathology,* University of Pittsburgh Medical Center, Pittsburgh; Department of Pharmacology, University of Pittsburgh School of Medicine; and University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, Pennsylvania
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Production and function of miR requires a set of proteins collectively referred to as the miR machinery. MiRs encoded in the genome are transcribed by RNA polymerase II into primary transcripts, pri-miRNAs.8
The next step of miR maturation is the nuclear cleavage of pri-miRNA and release of a 60- to 70-nt stem-loop intermediate, an miRNA precursor (pre-miRNA). This step requires a 
650-kd microprocessor complex that comprises Drosha, an RNase III endonuclease, and its cofactor, DGCR8.2
Pre-miRs are actively transported from the nucleus to the cytoplasm by the Exportin 5/RanGTP complex.9
In the cytoplasm, Dicer and the human immunodeficiency virus (HIV)-1 transactivating response RNA-binding protein cut both strands of the pre-miRNA duplex, generating a mature 21-nucleotide miR duplex.10,11
Chendrimada et al isolated a 500-kd protein complex containing transactivating response RNA-binding protein, Dicer, and EIF2C212
. This complex also includes Gemin 3 and Gemin 4 and links miRNA processing with the assembly of the RNA-induced silencing complexes.12,13
Dicer has also been found to stably associate with HSPCA and EIF2C1.14,15
In addition, MOV10, TNRC6B, and protein arginine methyltransferase 5 specifically associate with EIF2C proteins.14
PACT (protein kinase, interferon-inducible double-stranded RNA-dependent activator, PRKRA) has been shown to interact with Dicer without facilitating its pre-miRNA cleavage activity.16
Finally, fragile X mental retardation protein interacts with the mammalian EIF2C2 and is associated with Dicer activity.17,18
Maturation of miR is summarized in Figure 1A
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In addition to being an important RNA interference (RNAi) effector, it has been shown that Dicer mutations are associated with defects independent of global errors induced by dysfunction of the miR machinery. In yeast, independently of the RNAi pathway, Dicer has been shown to regulate G1 arrest in response to nitrogen-limiting conditions and initiate the Cdc2-dependent DNA replication and DNA damage checkpoints.24,25 Dicer is crucial for proper structuring of centromeric heterochromatin, a process important for accurate sister chromosome segregation.26 In a chicken-human hybrid cell line, loss of Dicer leads to chromosome mis-segregation and to the accumulation of cells in the G2/M phase of the cell cycle.27 These findings point to the role of Dicer in controlling checkpoints in response to mutagenic stress. In conclusion, it is important to consider the aberrant expression of Dicer and other components of the miR machinery in human malignancies.
In an effort to elucidate the mechanism of miR up-regulation in PCa, we characterized the alterations in expression of genes encoding proteins of miR machinery. Our gene array analysis demonstrated an increased transcription of Dicer and most of its partners in metastatic PCa. The up-regulation of Dicer with PCa progression was confirmed by immunohistochemical analysis of clinical samples and immunocytochemical and Western blot analysis of prostate cell lines.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The tissue microarray set consists of 232 prostate tissue specimens from 166 different patients arrayed onto slides in quadruplicate. The demographic and clinicopathologic features of the patients in this study are listed in Table 1
. Regional lymph nodes were not assessed in two cases (NX). Two specimens were obtained from patients of Asian race, 10 from African Americans, and 200 from Caucasians. The race of 20 patients was not available. Samples were procured as previously described.28
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For immunostaining, 4-µm sections of tissue array were cut and mounted on glass slides. The sections were heated at 60°C for 12 hours and deparaffinized in xylene and ethanol. Antigen retrieval was performed using 25 mmol/L sodium citrate buffer (pH 6.0) at 90°C for 15 minutes, followed by treatment of 3% H2O2 to block endogenous peroxidase. The slides were incubated at 4°C overnight with anti-Dicer antibodies at 1:600 dilution. The sections were then incubated with biotinylated anti-mouse IgG (Vector Elite Kit; Vector Laboratories, Burlingame, CA) for 30 minutes. This was followed by incubating the section with Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine solution (DAKO, Carpinteria, CA) to develop the stain. Hematoxylin was used for counterstaining. Only staining of the luminal epithelial cells was scored. The staining of the basal cells was used as a positive internal control. Strongly positive ("3") staining of Dicer was defined as homogenous cytoplasm staining more intense than in basal cells. Moderately positive ("2") score was assigned to a granular cytoplasm staining with apical cytoplasmic or perinuclear concentration. This level of intensity was equal to that normally seen in basal cells. Weakly positive ("1") staining was less intense than in basal cells and limited to the basal cytoplasm. "0" reflected the lack of Dicer immunoreactivity and was the most common pattern seen in benign luminal cells. Three pathologists (R.D., T.M., and S.C.) scored stained slides. Scores for all cores from one case were averaged. Cases with all cores missing on all examined tissue microarray sections were excluded from the analysis. All statistical analysis of immunohistochemical studies of the tissue microarray set was performed with Sigma Stat (SYSTAT, Point Richmond, CA). Two sample comparisons were performed with the Mann-Whitney rank sum test. Multiple samples were compared using analysis of variance on ranks or one-way analysis of variance.
Immunoblot Detection of Dicer
The cells were washed with phosphate-buffered saline and lysed using a cell disruption buffer (mirVANA PARIS; Ambion, Austin, TX). The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide gel) and blotted onto a nitrocellulose membrane. The membrane was blocked with 5% powdered nonfat milk in Tris-Tween 20 buffer, pH 7.4, overnight at 4°C, followed by 2 hours of incubation with a 1:600 dilution of anti-Dicer antibody. The membrane was then washed three times with Tris-Tween 20 buffer and incubated with anti-mouse IgG for 1 hour at room temperature. Dicer expression was detected with the Super SignalWest Femto Maximum Sensitivity substrate (Pierce, Rockford, IL) according to the manufacturer’s protocol.
Gene Array: cRNA Preparation, Affymetrix Chip Hybridization, Data Preprocessing, and Statistical Analysis
cRNA was prepared and hybridized to Affymetrix HGU95Av2, B, and C chips as previously described.29 The raw scanned array images were first processed through the Affymetrix Microarray Analysis Suite 5.0 (MAS; Affymetrix Corporation, Santa Clara, CA) to generate probe cel intensity (*.cel) files. The *.cel files were then analyzed using MAS 5.0 to generate gene expression signal values for each probe set. Data normalization to remove variation in overall chip intensities was performed by global scaling to a chip mean target intensity of 200 (MAS 5.0). We analyzed 16 normal prostate samples, organ-confined prostate cancer tissue samples from 64 prostate cancer patients, and 24 metastatic tissues from four patients. To identify differentially regulated genes, Affymetrix U95Av2, B, and C chip data were combined, and organ-confined PCa were compared with donors and to metastatic samples by significance analysis of microarrays.30 Before analysis, genes that show low variation across all samples were removed by using the filtering option in the Avadis (Strand Genomics, Bangalore, India) data analysis tool.
Antibodies, Cell Lines, and Cell Culture
Dicer antibody was from Clonegene (Hartford, CT) and was used at a 1:600 dilution. Tubulin antibody was from Oncogene Research Products (San Diego, CA). DU145 and RWPE-1 cell lines were from American Type Culture Collection (Manassas, VA). RWPE-1 was maintained in complete keratinocyte serum-free medium containing 50 µg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor, and 1% antibiotic/antimycotic mixture. Medium was changed every 48 hours. Cells were passaged on confluence and seeded at 1 to 2 x 106 cells/T-25 flask. DU145 were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Gibco, Sydney, Australia). The primary prostate cancer cells, passage 0, were derived from prostatectomy specimen.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Using Affymetrix HGU95 chips we analyzed gene expression in 64 patients with organ-confined PCa and 24 metastatic PCa samples from four patients. The gene expression analysis of the organ-confined PCa and normal prostate samples was previously described by our group.28
All of the genes encoding miR machinery components showed a similar level of expression in normal prostate and in the organ-confined PCa. However, expression of several genes was up-regulated in metastatic PCa (summarized in Figure 1
). POLR2A, polymerase RNA II, was up-regulated 5.1-fold in metastatic PCa. Drosha (RNASE3L) and DGCR8 did not show differential expression in normal prostate versus organ-confined PCa versus metastatic PCa.
Dicer and its closest partner, EIF2C2, were up-regulated 2.5- and threefold, respectively, in metastatic PCa compared with organ-confined PCa. Several major functional partners of Dicer were up-regulated in metastatic PCa as well. In our analysis, XPO5 was up-regulated 1.6-fold. A significantly increased Exportin 5 level in metastatic PCa has been previously shown.31 EIF2C1 and HSPCA were up-regulated 1.8- and 2.7-fold, respectively, in metastatic samples compared with organ-confined PCa.
Immunohistochemical Analysis of Dicer Expression in Benign Human Prostate Tissue
The normal human prostatic epithelium consists of basal, intermediate, secretory luminal, and neuroendocrine cells.32
Within the layer of the basal cells there are stem cells.32
The expression of Dicer was examined in normal prostate tissue, normal tissue adjacent to the PCa (normal adjacent to tumor), and benign prostatic hypertrophy specimens. In normal prostate tissue, Dicer immunoreactivity was limited to the cytoplasm of the basal cells (Figure 2, A and B)
. No nuclear staining was identified. Weak Dicer immunoreactivity was rarely detected in fibromuscular stroma. Dicer was negative in the luminal cells of the glands involved with basal cell hyperplasia, whereas the foci of basal cell hyperplasia were distinctly positive (Figure 2C)
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0.5, and the mean Dicer immunoreactivity in all benign samples (n = 64) was 0.2. Although luminal cells in the normal adjacent to tumor samples tended to show higher Dicer immunoreactivity, no statistically significant difference was observed between normal prostates, normal adjacent to tumor, and benign prostatic hypertrophy specimens. Of note, urethral urothelium and peripheral ganglion cells within the prostate demonstrated strong cytoplasmic Dicer immunoreactivity. The results of the immunohistochemical analysis of Dicer expression in normal prostate tissue are summarized in Table 2
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In prostatic intraepithelial neoplasia (PIN) and PCa, Dicer was observed not only in basal cells but also in the cytoplasm of the luminal cells (Figure 3)
. In PIN specimens, the mean Dicer immunoreactivity was 0.6, a twofold increase over normal prostate tissue. Of the PIN specimens, 53% (19 of 36) showed immunoreactivity of 
0.5. The majority of prostate cancers showed overexpression of Dicer: 81% (89 of 107) of PCa cases demonstrated Dicer intensity of 0.5 with mean immunoreactivity of 1.2 for N0 PCa (no positive regional lymph nodes, n = 83) and mean of 1.7 for N1 PCa (metastases in regional nodes, n = 22) (P = 0.006) (Table 3)
. The difference in mean Dicer immunoreactivity in benign prostate samples, PIN, and PCa was statistically significant (P < 0.001). Dicer expression increased with the clinical stage of the PCa (Table 4)
(P = 0.001) and pathological T stage (P < 0.009). Higher Dicer levels were seen in PCa with Gleason score >7. There was a significant difference in mean immunoreactivity between tumors with Gleason score >7 and those with Gleason score 7 (P < 0.025) (Table 5)
. Mean Dicer immunoreactivity in benign and malignant prostate tissue is summarized in Figure 4
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Western Blot and Immunocytochemical Analysis of Dicer Expression in Prostate Cell Lines
Our findings in clinical PCa samples raised the question as to whether Dicer is up-regulated in neoplastic prostate cell lines. We compared Dicer levels in RWPE-1 and DU145 prostate cell lines. Of note, RWPE-1 is not tumorigenic.33
The DU145 cell line was derived from a human prostate adenocarcinoma metastatic to the brain.34
Western blot analysis of these human prostate cell lines incubated under routine cell culture conditions showed dramatically higher Dicer expression in DU145 compared with RWPE-1 (Figure 5A)
. Likewise, Dicer was detected at a higher level in primary prostate cancer cell lines when compared with primary benign prostate cells (data not shown).
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and three other human PCa cell lines: PPC-1, LNCAP, and PC3 (data not shown). Increased level of Dicer detected in the androgen-receptor-negative cell line DU145 further suggested that it might be a useful marker of aggressive androgen-independent tumors. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Human Dicer is mapped to the subtelomeric region of chromosome 14 (14q32.13). An extensive search of publicly available databases for neoplasms associated with genetic abnormalities at 14q32.13 locus yielded a number of human malignancies.35-37 As for the underlying mechanisms of increased Dicer expression in PCa, it is possible that Dicer up-regulation is induced by a genomic instability at 14q32 (eg, amplification). The up-regulation of other miR machinery components might be secondary to the increased Dicer level. In lung adenocarcinoma, methylation of the Dicer promoter region does not appear to control the level of Dicer.22
A Global Up-Regulation of miRs and Up-Regulated miR Machinery in Prostate Cancer
miR arrays of several solid cancers revealed an almost global up-regulation of miRs as a common feature of oncogenesis in many tissue types.4 Specifically in prostate adenocarcinoma, 39 of 45 differentially expressed miRs are up-regulated.4 Because Dicer is essential for the generation of mature miR, it is likely that higher Dicer level may increase expression of multiple miR species in prostate cancer cells. The opposite effect of reduced Dicer on miR was reported in lung adenocarcinoma, where decreased Dicer levels corresponded to a decrease in miR let-7.38
Up-regulation of the central components of the miR machinery may account for the general up-regulation of miRs in prostate cancer. This hypothesis relies on the assumption that the up-regulated miR machinery is functional. The functionality of the miR machinery is supported by fact that both Dicer protein and its mRNA are up-regulated. Furthermore, many research groups demonstrated a functional RNAi pathway in most prostate adenocarcinoma cell lines by using small interfering RNA (siRNA)/short hairpin RNA constructs targeting mRNAs.39,40
Immunohistochemical Data Explain Gene Array Findings
Our gene array analysis detected no difference in Dicer expression between normal prostate tissue and organ-confined PCa. The immunohistochemical analysis of PCa samples offered an explanation for this finding. In normal prostate tissue, Dicer immunoreactivity was detected in basal cells only. Neoplastic epithelial cells exhibited increased Dicer expression. The hallmark of the invasive PCa is the disappearance of basal cells. Therefore, even though the absolute level of Dicer is probably unchanged when normal prostate tissue is compared with organ-confined PCa, the redistribution of Dicer from vanishing basal cells to proliferating neoplastic cells appears to be biologically significant. As PCa progresses and metastasizes to lymph nodes and visceral organs, the Dicer level continued to rise. Hence, Dicer was up-regulated 2.5-fold in metastatic PCa compared with the organ-confined PCa, as determined by gene array transcriptional profiling.
In cancer, the increasing number of proliferating neoplastic glands and the decrease of intervening stroma may account for a seemingly higher expression of some proteins. Proteins predominantly localized to epithelial glandular compartment of tissues are particularly susceptible to this bias, especially when quantified in protein extracts (by Western blot). In contrast, immunohistochemical analysis allows localization of the antigen of interest in tissue. It allowed us to evaluate Dicer expression only in the luminal cells of the individual glands, benign or malignant. The level of Dicer demonstrated by the most luminal cells within the glands in a particular tissue core was recorded. Our measurements (scoring scheme) were independent of the number of glands in a given microscopic field.
The Pattern of Dicer Expression
Luminal cells of benign prostatic glands do not express Dicer at detectable levels. In contrast, basal cells were most commonly strongly positive. The functional significance of this expression pattern may be linked to the antiapoptotic function of Dicer (most likely via miR-mediated action).41 Stem cells, their immediate progeny, and intermediate cells are known to express bcl-2, which prevents apoptosis, and are crucial for self-renewal of the normal prostatic luminal epithelium.32 Other proteins whose expression in benign prostate is limited to the basal cells and whose expression in luminal cells has also been associated with prostate cancer are ezrin,42 c-met,43 and prostate stem cell antigen PSCA.44 The expression pattern of these proteins supports the concept that prostate cancers arise from the transformation of basal/intermediate cells.45 The presence of Dicer in normal basal cells of prostate and its up-regulation in neoplastic luminal cells suggests that Dicer may play a role in the early steps of prostate cancer development, probably by potentiating an almost global miR up-regulation.
Dicer and miR/siRNA-Mediated Therapy
Hundreds of human miR are predicted to regulate 10 to 30% of protein-encoding genes by interactions with their 3' untranslated regions.46,47 Perfectly complementary interactions between small RNA (such as siRNA or miRNA) and target mRNA result in mRNA degradation. In contrast, mismatched bp interactions between either siRNA or miRNA and the target mRNA can result in translational repression.48,49
Because mammalian Dicer functions in both siRNA and miR pathways,50,51 prostate cancer may become a useful model to study the competition between small RNA pathways: an endogenous miR pathway and experimental, or exogenous, RNAi pathway.50 With the development of RNAi-based treatments, it might be crucial to learn what branch of small RNA pathway is potentiated by increased levels of the protein components of the miR machinery.
When confirmed by a further validation study in an independent and larger cohort, increased expression of Dicer may be clinically useful for the prognosis of prostate adenocarcinoma patients. The significant up-regulation of Dicer in prostate cancers, together with its relative lack of expression in benign luminal cells, suggests that Dicer may become an attractive therapeutic target. Finally, understanding Dicer expression patterns will be required to predict the susceptibility to potential miRNA- and siRNA-based therapy.52
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Acknowledgements |
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Footnotes |
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Supported by grants from the Postdoctoral Pathology Research Training Program (PPRTP, RT-00147, CC-99FUND), the Department of Pathology, the University of Pittsburgh Medical Center (S.C.), and a Research Scholar grant (RSG-05-246-01-GMC) from the American Cancer Society (R.W.S.).
Accepted for publication July 27, 2006.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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