Abstract
IntroductionThe 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.MethodsmicroRNA 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.ResultsA 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0593-0) contains supplementary material, which is available to authorized users.
Highlights
The study of mammalian development has offered many insights into the molecular aetiology of cancer
Identification of microRNAs enriched in mammary cellular compartments We performed miRNA expression profiling on microdissected stroma, mature ducts and terminal end bud (TEB) of pubertal 5-week-old GFP+ mice to identify miRNAs involved in mammary gland development (Fig. 1a)
We identified a set of miRNAs that were specific to the stroma, ducts, TEBs or both epithelial fractions (Fig. 1b; Table 1). miR-31 was the most highly enriched miRNA expressed in the TEBs, approximately 10.7-fold upregulated versus ducts
Summary
The study of mammalian development has offered many insights into the molecular aetiology of cancer. A large body of evidence has identified augmented receptor tyrosine kinase (RTK)-PI3K-Akt-mTOR activity in the basal like and TBNCs either due to mutations in RTKs or PIK3CA or loss of phosphatase and tensin homologue (PTEN) expression [6,7,8,9]. This pathway has become a major focus of breast cancer drug development, patient responses to these novel drug compounds in clinical trials have been variable, perhaps due to an incomplete understanding of pathway interactions and feedback loops. A thorough understanding of the regulation of these signalling pathways is essential to effectively personalise breast cancer treatment
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