Abstract 3982: Deregulation of tumor-suppressive miR-16 through progestin-mediated oncogenic signaling contributes to breast cancer development
Authors: Rivas Martin A. , Huang Yi-Wen , Schillaci Roxana , Huang Tim H-M , Elizalde Patricia V. 
Published: June 2012

Abstract
Breast cancer is the commonest type of cancer among women in the US and the second leading cancer-death cause. Understanding its biology will help to design new clinical approaches to achieve a better outcome of the disease. microRNA (miRNA) are short ribonucleic acids with important regulatory functions and with an increasingly acknowledged role in cancer. The present study was designed to characterize progestin-induced miRNA regulation in breast cancer. Primary cultures of the murine breast cancer C4HD were treated or not with 10nM of the synthetic progestin medroxiprogesterone acetate (MPA) for 24 h and total RNA extracted for miRNA profiling using Applied Biosystems low density qPCR arrays. Analysis of data rendered a total of 16 miRNA significantly regulated (P&lt;0.05): miR-378*, miR-376a, miR-224, miR-190b, miR-16, miR-410 and miR-197 were downregulated and miR-191*, miR-17*, miR-470*, miR-451, miR-702, miR-434-3p, miR-493, miR-23a* and miR-485* were upregulated under treatment with MPA. Interestingly, miR-16 has already been shown to be a tumor suppressor in leukemia and so we focused our further study on it. We validated the downregulation of miR-16 by qPCR (0.4 fold against control) and observed that small interference RNA (siRNA) to progesterone receptor (PR) or to signal transducer and activator of signal 3 (STAT3) overcame miR-16 downregulation by MPA. The oncogene c-Myc was a reported negative regulator of miR-16, and we observed that siRNA to PR or STAT3 inhibited MPA-induced c-Myc upregulation. A query for miR-16 targets on miRecords search engine showed that the cell cycle promoter gene Cyclin E has conserved putative target sites on 3′UTR mRNA. Consistently, siRNA to PR and to STAT3 inhibited MPA-induced CCNE mRNA and Cyclin E protein increase as assessed by qPCR and Western blot. In addition, transfection with pre-miR-16 abrogated MPA capacity of inducing Cyclin E upregulation. Most importantly, pre-miR-16 inhibited MPA-induced proliferation, as observed in [3H]-Thymidine incorporation assays and Trypan-blue cell counts. C4HD is a progestin-dependent tumor since it is unable to grow in vivo under exogenous progestin's absence. Therefore, 1 × 106 C4HD cells transfected with a pre-miR-control (CTRL) or with pre-miR-16 were injected s.c. in 8-week female Balb/c mice which had been previously injected with a MPA slow-release depot. Tumors became palpable at day 15 but the ones from pre-miR-16 group grew at a lower rate than the ones from pre-miR-CTRL group, and the mean tumor volume at day 40 was significantly smaller (312.4 ± 42.3 mm3 for CTRL group against 22.2 ± 34.9 mm3 for pre-miR-16 group). To sum up, these results suggest that progestin induce breast cancer growth through a pathway involving PR/STAT3/c-Myc and miR-16 which acts on mRNA targets such as Cyclin E to control cell cycle progression.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3982. doi:10.1158/1538-7445.AM2011-3982