title: Downregulation of the tumor-suppressor miR-16 via progestin-mediated oncogenic signaling contributes to breast cancer development.
abstract: BACKGROUND
  Experimental and clinical evidence points to a critical role of progesterone and the nuclear progesterone receptor (PR) in controlling mammary gland tumorigenesis. However, the molecular mechanisms of progesterone action in breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are short ribonucleic acids which have also been found to play a pivotal role in cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is poorly explored. In this study we explored progestin modulation of miRNA expression in mammary tumorigenesis.
  
  
  METHODS
  We performed a genome-wide study to explore progestin-mediated regulation of miRNA expression in breast cancer. miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. Breast cancer cells were transfected with the precursor of miR-16 and proliferation assays, Western blots or in vivo experiments were performed. Target genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E. Reporter gene assays were performed to confirm that cyclin E 3'UTR is a direct target of miR-16.
  
  
  RESULTS
  We found that nine miRNAs were upregulated and seven were downregulated by progestin in mammary tumor cells. miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as one of the downregulated miRNAs in murine and human breast cancer cells. Progestin induced a decrease in miR-16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription factor c-Myc. A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR region. We found that, similar to the molecular mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of cyclin E expression by progestin. Moreover, overexpression of miR-16 abrogated the ability of progestin to induce cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role for miR-16 as a tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand heregulin (HRG) downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast tumor cells.
  
  
  CONCLUSIONS
  In this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel role for miR-16 as a tumor suppressor in progestin- and growth factor-induced growth in breast cancer.
authors.affiliation: Laboratory of Molecular Mechanisms of Carcinogenesis, Instituto de Biología y Medicina Experimental (IBYME), CONICET, Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Rivas
authors.forename: Leandro
authors.initials: L
authors.lastname: Venturutti
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Huang
authors.forename: Roxana
authors.initials: R
authors.lastname: Schillaci
authors.forename: Tim Hui-Ming
authors.initials: TH
authors.lastname: Huang
authors.forename: Patricia V
authors.initials: PV
authors.lastname: Elizalde
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D055106:Genome-Wide Association Study; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D011372:Progestins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation; D000099053:Histidine-Rich Glycoprotein
chemical_list: D020413:3' Untranslated Regions; C475820:CCNE1 protein, human; D019927:Cyclin E; C505362:MIRN16 microRNA, human; C489427:MYC protein, human; D035683:MicroRNAs; D015513:Oncogene Proteins; D011372:Progestins; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D011980:Receptors, Progesterone; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D000099053:Histidine-Rich Glycoprotein