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. 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 gammaH2A.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 recurrence status revealed that increased expression of the miR-106b-25 cluster in these tumors is associated with early disease recurrence. Moreover, the cluster was found to be up-regulated in distant metastases of the prostate and in prostate tumors of TRAMP mice. Other experiments showed that miR-106b-25 expression alters expression of cell adhesion proteins and increases soft agar invasion of human prostate cancer cells. Together, these 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. For further evaluation of the miR-106b-25 cluster, we generated mice with prostate-specific over-expression of this cluster. It is our hypothesis that constitutively elevated expression of microRNAs encoded by this cluster will lead to neoplastic alteration in the prostate. We have screened male founders carrying germline expression cassettes for probasin promoter-driven expression of the miR-106b-25 cluster and of either a luciferase (pBSn-miR106 IRES Luc) or a luciferase eGFP fusion gene reporter (pBSn-miR106 ffLuc2 egfp). Screening identified potential founders with high, prostate-specific luciferase expression. These founders are currently used to establish colonies of male offspring with miR-106b-25 transgene expression in their prostate. We have not observed any prostate abnormalities in transgene-positive mice at 3-4 months of age and are extending the life span of these mice to see if they develop any visible abnormalities at 1 year of age, specifically pre-neoplastic or neoplastic alterations like PIN. 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. A number of problems make ucRNAs difficult to measure including their overall low expression in the tissues, small differential expression, and the tendency of ultraconserved regions to transcribe RNAs in sense and antisense direction, thereby interfering with the accuracy of PCR-based quantitative assays. Thus, we are exploring a new quantitative detection system called the NanoString nCounter Analysis System. The NanoString nCounter Analysis System for the direct quantification of UCR transcripts is a 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. No study to date has profiled ucRNAs in such detail, and these experiments, if successful, would allow for additional studies exploring ucRNAs in human biofluids and tissues for biomarker discovery.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010886-04
Application #
8349221
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2011
Total Cost
$255,457
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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