miR-105 Inhibits Prostate Tumour Growth by Suppressing CDK6 Levels

Addison CL (2013) miR-105 Inhibits Prostate Tumour Growth by Suppressing CDK6 Levels. PLoS ONE 8(8): e70515. doi:10.1371/journal.pone.0070515 miR-105 Inhibits Prostate Tumour Growth by Suppressing CDK6 Levels D. Rice Honeywell 0 1 Miguel A. Cabrita 0 1 Huijun Zhao 0 1 Jim Dimitroulakos 0 1 Christina L. Addison 0 1 Moray Campbell, Roswell Park Cancer Institute, United States of America 0 Current address: Department of Cellular and Molecular Medicine, University of Ottawa , Ottawa, Ontario , Canada 1 1 Cancer Therapeutics Program, Ottawa Hospital Research Institute , Ottawa, Ontario , Canada , 2 Departments of Biochemistry Microbiology and Immunology, University of Ottawa , Ontario , Canada , 3 Department of Medicine, University of Ottawa , Ontario , Canada A significant role for micro (mi)RNA in the regulation of gene expression in tumours has been recently established. In order to further understand how miRNA expression may contribute to prostate tumour growth and progression, we evaluated expression of miRNA in two invasive prostate tumour lines, PC3 and DU145, and compared it to that in normal prostate epithelial cells. Although a number of miRNAs were differentially expressed, we focused our analysis on miR-105, a novel miRNA not previously linked to prostate cancer. miR-105 levels were significantly decreased in both tumour cell lines in comparison to normal prostate epithelial cells. To determine its potential role in prostate cancer pathogenesis, we overexpressed miR-105 in both PC3 and DU145 cells and determined its effect on various tumourigenic properties. miR-105 overexpression inhibited tumour cell proliferation, tumour growth in anchorage-independent three-dimensional conditions and tumour invasion in vitro, properties of highly aggressive tumour cells. Of potential clinical significance, miR-105 overexpression inhibited tumour growth in vivo in xenograft models using these cell lines. We further identified CDK6 as a putative target of miR-105 which is likely a main contributor to the inhibition of tumour cell growth observed in our assays. Our results suggest that miR-105 inhibits tumour cell proliferation and hence may represent a novel therapeutically relevant cellular target to inhibit tumour growth or a marker of aggressive tumours in prostate cancer patients. - Funding: This work was supported by operating grants to CLA from the Canadian Institutes in Health Research (MOP-84369) and the Prostate Fight Foundation through funds raised by the Motorcycle Ride for Dad. DRH was also a recipient of an Ontario Graduate Scholarship from the Ontario Ministry of Training, Colleges and Universities. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Micro(mi) RNAs are small RNA inhibitors which have been shown to play an important role in regulating gene expression in a number of organisms. miRNAs were first discovered in Caenorhabditis elegans (C. elegans) [1,2], and were subsequently shown to block gene expression by post-transcriptional binding and silencing of specific mRNA sequences. The transcription and processing of miRNA into its mature form has been recently described in extensive detail elsewhere [35]. Briefly, miRNA molecules are ,1922 nucleotides long and can be transcribed from either intergenic regions or within gene introns or exons [6]. Mature miRNA is then bound and unwound by the Argonaute protein and incorporated into the RISC, a protein complex which helps guide the mature miRNA strand to its target mRNA binding site [7] via a 57 nucleotide seed sequence within the mature miR strand [8]. After binding its target, the complex causes either inhibition of translation or degradation of the target mRNA molecule [9]. In recent years, miRNAs have been shown to play a role in many diseases, such as cancer [10], heart disease [11], and diabetes [12,13]. Prostate cancer is no exception, and studies have shown that miRNA dysregulation occurs in the majority of prostate tumours [1315]. Some of these dysregulated miRNAs have been shown to modulate the prostate cancer phenotype by affecting crucial growth regulatory targets such as K-Ras [16], androgen receptor [17] and cyclin D1 [18]. Other miRNAs, for example, miR-21, have been dubbed oncomirs as a result of their overexpression leading to the promotion of tumour phenotypes in numerous cancer types [1921]. There are also miRNAs that function as tumour suppressors, and inhibit the tumourigenic phenotype in cancer cells. For example, miR-143 is downregulated in many cancers, as it has been shown to regulate, and allow expression of genes that promote proliferation and migration, such as K-Ras and cyclinD1 [16]. Moreover, modulating cellular miRNA expression using depletion strategies [22] or overexpression of miRNAs [23], can regulate tumour phenotype both in vitro and in vivo. Thus increasing our understanding of miRNA dysregulation in prostate cancer will facilitate our understanding of prostate cancer progression and could also identify important novel therapeutic targets and predictive miRNA signatures for advanced and/or aggressive disease. Given their important role in modulating the tumourigenic phenotype, we set out to identify miRNAs that play a significant role in mediating growth and invasion of aggressive prostate tumour cells. To this end, we performed a miRNA microarray experiment to compare the miRNA expression profiles of normal prostate epithelial cells (PrEC) with those of two well characterized metastatic prostate tumour cell lines (PC3 and DU145). Although a number of significant differences were uncovered, we focused our study on determining the role of differentially expressed miRNAs not previously reported to regulate prostate tumour progression. Hence, we examined the role of miR-105 in this study, which demonstrated significantly decreased expression in both metastatic prostate tumour cell lines when compared to PrEC. miR-105 belongs to a group of miRs whose levels are regulated in cancer cells by preventing export of their Drosha/DGCR8 processed pre-miR forms from the nucleus into the cytoplasm [24]. Overexpression of miR-105 was also shown to be associated with changes in proliferation markers in primary ovarian granulosa cells [25]. Given these observations together with our array data, we evaluated the role of miR-105 as a potential novel regulator of prostate cancer cell proliferation, anchorage-independent growth and invasion in vitro and tumour growth of xenografted prostate cancer cells in vivo. Overall our data suggests that miR-105 inhibits growth of prostate tumours both in vitro and in vivo. Materials and Methods Cells and media PC3 and DU145 human prostate carcinoma cells were obtained from ATCC (Manassas, VA). These cells were grown in Dulbeccos Modified Eagle Medium (DMEM, Mediatech Inc, Manassas, (...truncated)