We had previously examined genome-wide expression of microRNAs and mRNAs in 60 primary human prostate tumors and 16 non-tumor prostate tissues in collaboration with Dr. Carlo Croce at Ohio State University. The analysis revealed that both key components of microRNA processing and numerous microRNAs were significantly altered in prostate tumors when compared with surrounding non-cancerous tissue. Tumor microRNAs were up- and down-regulated when compared with non-cancerous tissue and the expression profile of the tumors yielded a diagnostic microRNA signature. Notably, prostate tumors tended to express all members of the miR-106b-25 cluster at significantly higher levels than non-tumor prostate, which is consistent with the miR-106b-25 cluster having oncogenic properties in prostate tumor biology. The expression of miR-1 and miR-133 was consistently lower in tumors than in non-tumor prostate, indicating that these microRNAs may act as tumor suppressors. In a follow-up study for miR-1-133 cluster expression in human prostate tumors, we further corroborated this finding in an independent dataset and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the candidate tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer cells and the analysis of tumors indicated promoter hypermethylation of the miR-1 locus in a subset of human tumors. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair, and actin dynamics. This observation was further corroborated with protein expression analysis and 3-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters gamma-H2A.X marker expression and affects the cellular organization of F-actin and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.We also continued to study the specific functions of the miR-106b-25 cluster. Analyzing a large microRNA expression dataset for prostate tumor from patients with recurrent disease status revealed increased expression of the miR-106b-25 cluster in these tumors. Moreover, the cluster was found to be up-regulated in distant metastases of the prostate and in prostate tumors of TRAMP mice. Moreover, increased tumor miR-106b expression was associated with early disease recurrence and the combination of high miR-106b and low CASP7 (caspase-7) expression in primary tumors was an independent predictor of early disease recurrence (adjusted hazard ratio = 4.1;95% confidence interval: 1.6 to 12.3). To identify yet unknown oncogenic functions of miR-106b, we overexpressed miR-106b in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that caspase-7 is a direct target of miR-106b, which was confirmed by Western blot analysis and a 3-UTR reporter assay. Moreover, selected phenotypes induced by miR-106b knockdown in DU145 human prostate cancer cells did not develop when both miR-106b and caspase-7 expression were inhibited. Further analyses showed that caspase-7 is down-regulated in primary prostate tumors and metastatic lesions across multiple datasets and is by itself associated with disease recurrence and disease-specific survival. Using bioinformatics, we also observed that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally examined using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and may do so by altering apoptosis- and focal adhesion-related pathways. Our findings indicate that the miR-106b-25 cluster acts like an oncogene in human prostate cancer, which is recapitulated in the TRAMP mouse model, and is a predictor of early recurrence.We have previously characterized the expression profile of ultraconserved region-derived non-coding RNAs (ucRNAs) in prostate tumors and surrounding normal tissue using microarrays. We also found that some key ucRNAs are silenced by epigenetic mechanisms in human prostate cancer cells. Our lab is the first to explore genome-wide expression profiles of ucRNAs in prostate cancer and one of the first to explore regulatory mechanisms of these RNAs. However, quantification of ucRNAs by other methods than microarrays has been challenging. Recently, the use of the NanoString nCounter Analysis System allowed us the direct quantification of ucRNA transcripts with this newly introduced technology that may rival quantitative real-time PCR and array based assays because of its ability to measure RNAs in a direct and absolute manner. Other experiments showed that ucRNA transcription patterns can be modified by (synthetic) androgen exposure and siRNA-based down-regulation of candidate ucRNAs can lead to global mRNA expression changes, indicating that ucRNAs may have a broad functional role in prostate cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010886-05
Application #
8552878
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$167,211
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Hudson, Robert S; Yi, Ming; Volfovsky, Natalia et al. (2013) Transcription signatures encoded by ultraconserved genomic regions in human prostate cancer. Mol Cancer 12:13
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