miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis

RESEARCH ARTICLE miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis S. J. Seashols-Williams1¤a*, W. Budd2¤b, G. C. Clark1, Q. Wu3, R. Daniel1, E. Dragoescu4, Z. E. Zehner1* a11111 1 Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, United States of America, 2 Department of Bioinformatics, Virginia Commonwealth University, Richmond, Virginia, United States of America, 3 Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia, United States of America, 4 Department of Pathology, Virginia Commonwealth University, Richmond, Virginia, United States of America ¤a Current address: Department of Forensic Science, Virginia Commonwealth University, Richmond, Virginia, United States of America ¤b Current address: American International Biotechnology, LLC, Richmond, Virginia, United States of America * (SJSW); (ZEZ) OPEN ACCESS Citation: Seashols-Williams SJ, Budd W, Clark GC, Wu Q, Daniel R, Dragoescu E, et al. (2016) miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis. PLoS ONE 11(7): e0159601. doi:10.1371/journal.pone.0159601 Editor: Irina U Agoulnik, Florida International University, UNITED STATES Received: January 8, 2016 Accepted: July 5, 2016 Published: July 22, 2016 Copyright: © 2016 Seashols-Williams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Raw and processed high-throughput sequencing data files are publically available through the Gene Expression Omnibus (GEO) database (accession number GSE79365). Funding: The project described was supported by grant number R21CA152349 from the National Cancer Institute to ZEZ. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. Human Abstract Identification of dysregulated microRNAs (miRNAs) in prostate cancer is critical not only for diagnosis, but also differentiation between the aggressive and indolent forms of the disease. miR-9 was identified as an oncomiR through both miRNA panel RT-qPCR as well as high-throughput sequencing analysis of the human P69 prostate cell line as compared to its highly tumorigenic and metastatic subline M12, and found to be consistently upregulated in other prostate cell lines including DU-145 and PC3. While miR-9 has been characterized as dysregulated either as an oncomiR or tumour suppressor in a variety of other cancers including breast, ovarian, and nasopharyngeal carcinomas, it has not been previously evaluated and proven as an oncomiR in prostate cancer. miR-9 was confirmed an oncomiR when found to be overexpressed in tumour tissue as compared to adjacent benign glandular epithelium through laser-capture microdissection of radical prostatectomy biopsies. Inhibition of miR-9 resulted in reduced migratory and invasive potential of the M12 cell line, and reduced tumour growth and metastases in male athymic nude mice. Analysis showed that miR-9 targets e-cadherin and suppressor of cytokine signalling 5 (SOCS5), but not NF-ĸB mRNA. Expression of these proteins was shown to be affected by modulation in expression of miR-9. Introduction Prostate cancer (CaP) is the most common cancer for men in the United States other than skin cancer, and the second leading cause of cancer deaths in the US, with over 29,000 fatalities each PLOS ONE | DOI:10.1371/journal.pone.0159601 July 22, 2016 1 / 20 miR-9 Acts as an OncomiR in Prostate Cancer tissues and patient consent were provided by the VCU Tissue and Data Acquisition and Analysis Core (TDAAC) Facility, supported, in part, with the funding from NIH-NCI Cancer Center Core Support Grant P30 CA016059, as well as through the Dept. of Pathology, School of Medicine, and Massey Cancer Center of Virginia Commonwealth University. The authors would also like to thank the Virginia Commonwealth University Massey Cancer Center (MCC) and Initiative for Maximizing Student Diversity Scholars Program (IMSD) for support for QW. WEB performed this work as a postdoctoral associate at VCU with ZEZ. His current employer (American International Biotechnology, LLC) was not associated with and did not fund the research in any way. Competing Interests: The authors have declared that no competing interests exist. year [1]. The current standard for diagnosis of a potential prostate cancer is a rise in prostatespecific antigen (PSA) levels as a screening test, followed by manual examination, and ultrasound-guided transrectal biopsy [2]. Interventions tend to have drastic consequences for the diagnosed male; radical prostatectomies, cryosurgery, and androgen ablation therapy significantly affect patient quality of life through high levels of incontinence, psychological, and sexual side effects [3]. Thus, the identification of new screening practices is critical to diagnosing not only prostate cancer, but also differentiating between the aggressive and indolent forms of the disease. microRNAs are a class of small RNAs that were described in C. Elegans in 1993 [4], and have since emerged as major regulators of protein levels through attenuation of translation at the ribosome [5–8]. microRNAs, or miRs, are 19–22 nucleotide single-stranded RNA sequences that are guided by a protein complex to their mRNA targets, typically in the 3’-UTR of the mRNA. microRNA transcription is often driven by standard transcription factor activation, including c-Myc and NF-kB [9–11], and most miRs are transcribed by RNA Polymerase II [12]. microRNAs can bind either perfectly or imperfectly to an mRNA target, thus making it possible for one miR to attenuate the translation of tens to hundreds of different targets. Additionally, miRs have been shown to impact all aspects of the proteome, from cell proliferation and apoptosis to mitochondrial and metabolic processes, to cytoskeleton and secreted products [8,13]. Thus, microRNAs have recently been subject to intense scrutiny as modulators of protein levels in cancer, as they are increasingly being shown to influence carcinogenesis and tumour progression. The previously described progression cell lines P69 and M12 are a unique model for prostate cancer, in that P69, having been immortalized from a human non-neoplastic prostate epithelium section [14,15], is poorly tumorigenic and non-metastatic in nature. Having originated from a basal cell lineage, P69 and its sublines do not express the androgen receptor, and are thus androgen independent [15]. In contrast to its poorly tumorigenic P69 parent line, the M12 cell line, which was de (...truncated)