title: MicroRNA profiling of the pubertal mouse mammary gland identifies miR-184 as a candidate breast tumour suppressor gene.
abstract: BACKGROUND
  The study of mammalian development has offered many insights into the molecular aetiology of cancer. We previously used analysis of mammary morphogenesis to discover a critical role for GATA-3 in mammary developmental and carcinogenesis. In recent years an important role for microRNAs (miRNAs) in a myriad of cellular processes in development and in oncogenesis has emerged.
  
  
  METHODS
  microRNA profiling was conducted on stromal and epithelial cellular subsets microdissected from the pubertal mouse mammary gland. miR-184 was reactivated by transient or stable overexpression in breast cancer cell lines and examined using a series of in vitro (proliferation, tumour-sphere and protein synthesis) assays. Orthotopic xenografts of breast cancer cells were used to assess the effect of miR-184 on tumourigenesis as well as distant metastasis. Interactions between miR-184 and its putative targets were assessed by quantitative PCR, microarray, bioinformatics and 3' untranslated region Luciferase reporter assay. The methylation status of primary patient samples was determined by MBD-Cap sequencing. Lastly, the clinical prognostic significance of miR-184 putative targets was assessed using publicly available datasets.
  
  
  RESULTS
  A large number of microRNA were restricted in their expression to specific tissue subsets. MicroRNA-184 (miR-184) was exclusively expressed in epithelial cells and markedly upregulated during differentiation of the proliferative, invasive cells of the pubertal terminal end bud (TEB) into ductal epithelial cells in vivo. miR-184 expression was silenced in mouse tumour models compared to non-transformed epithelium and in a majority of breast cancer cell line models. Ectopic reactivation of miR-184 inhibited the proliferation and self-renewal of triple negative breast cancer (TNBC) cell lines in vitro and delayed primary tumour formation and reduced metastatic burden in vivo. Gene expression studies uncovered multi-factorial regulation of genes in the AKT/mTORC1 pathway by miR-184. In clinical breast cancer tissues, expression of miR-184 is lost in primary TNBCs while the miR-184 promoter is methylated in a subset of lymph node metastases from TNBC patients.
  
  
  CONCLUSIONS
  These studies elucidate a new layer of regulation in the PI3K/AKT/mTOR pathway with relevance to mammary development and tumour progression and identify miR-184 as a putative breast tumour suppressor.
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. y.phua@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. y.phua@garvan.org.au.
authors.forename: Yu Wei
authors.initials: YW
authors.lastname: Phua
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. a.nguyen@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. a.nguyen@garvan.org.au.
authors.forename: Akira
authors.initials: A
authors.lastname: Nguyen
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. d.roden@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. d.roden@garvan.org.au.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Roden
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. b.elsworth@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. b.elsworth@garvan.org.au.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Elsworth
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. n.deng@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. n.deng@garvan.org.au.
authors.forename: Niantao
authors.initials: N
authors.lastname: Deng
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. i.nikolic@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. i.nikolic@garvan.org.au.
authors.forename: Iva
authors.initials: I
authors.lastname: Nikolic
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. j.yang@garvan.org.au.
authors.forename: Jessica
authors.initials: J
authors.lastname: Yang
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. a.mcfarland@garvan.org.au.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mcfarland
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge, UK. Roslin.Russell@cruk.cam.ac.uk.
authors.forename: Roslin
authors.initials: R
authors.lastname: Russell
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. w.kaplan@garvan.org.au.
authors.forename: Warren
authors.initials: W
authors.lastname: Kaplan
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. m.cowley@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. m.cowley@garvan.org.au.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Cowley
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. r.nair@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. r.nair@garvan.org.au.
authors.forename: Radhika
authors.initials: R
authors.lastname: Nair
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. e.zotenko@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. e.zotenko@garvan.org.au.
authors.forename: Elena
authors.initials: E
authors.lastname: Zotenko
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. sandra.o'toole@sswahs.nsw.gov.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. sandra.o'toole@sswahs.nsw.gov.au.|Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia. sandra.o'toole@sswahs.nsw.gov.au.|Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia. sandra.o'toole@sswahs.nsw.gov.au.
authors.forename: Sandra
authors.initials: S
authors.lastname: O'Toole
authors.affiliation: Metabolism in Human Diseases Unit, Institute of Molecular and Cell Biology, A*STAR, 61 Biopolis Drive, Proteos, Singapore. shtan@imcb.a-star.edu.sg.
authors.forename: Shi-Xiong
authors.initials: SX
authors.lastname: Tan
authors.affiliation: Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia. david.james@sydney.edu.au.|The Charles Perkins Centre, School of Molecular Bioscience, University of Sydney, Camperdown, NSW, Australia. david.james@sydney.edu.au.
authors.forename: David E
authors.initials: DE
authors.lastname: James
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. s.clark@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. s.clark@garvan.org.au.
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Clark
authors.affiliation: Agensys, affiliate of Astellas Pharmaceuticals, 1800 Stewart St, Santa Monica, CA, 90403, USA. kouros.mehr@gmail.com.
authors.forename: Hosein
authors.initials: H
authors.lastname: Kouros-Mehr
authors.affiliation: The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, NSW, Sydney, Australia. a.swarbrick@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, Sydney, UNSW, Australia. a.swarbrick@garvan.org.au.
authors.forename: Alexander
authors.initials: A
authors.lastname: Swarbrick
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D016000:Cluster Analysis; D044127:Epigenesis, Genetic; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D006801:Humans; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009362:Neoplasm Metastasis; D011379:Prognosis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012741:Sexual Maturation / Q000235:genetics*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: C552233:MIRN184 microRNA, mouse; D035683:MicroRNAs; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases