MicroRNA profiling of the pubertal mouse mammary gland identifies miR-184 as a candidate breast tumour suppressor gene

Yu Wei Phua, Akira Nguyen, Daniel L Roden, Benjamin Elsworth, Niantao Deng, Iva Nikolic, Jessica Yang, Andrea Mcfarland, Roslin Russell, Warren Kaplan, Mark J Cowley, Radhika Nair, Elena Zotenko, Sandra O'Toole, Shi-Xiong Tan, David E James, Susan J Clark, Hosein Kouros-Mehr, Alexander Swarbrick

Research output: Contribution to journalArticle (Academic Journal)peer-review

33 Citations (Scopus)

Abstract

INTRODUCTION: 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.

Original languageEnglish
Pages (from-to)83
JournalBreast Cancer Research
Volume17
DOIs
Publication statusPublished - 2015

Keywords

  • Animals
  • Breast Neoplasms
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Transformation, Neoplastic
  • Cluster Analysis
  • Epigenesis, Genetic
  • Female
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic
  • Gene Silencing
  • Genes, Tumor Suppressor
  • Humans
  • Mammary Glands, Animal
  • Mice
  • MicroRNAs
  • Neoplasm Metastasis
  • Prognosis
  • Proto-Oncogene Proteins c-akt
  • RNA Interference
  • Sexual Maturation
  • TOR Serine-Threonine Kinases

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