title: MicroRNA-18a inhibits hypoxia-inducible factor 1α activity and lung metastasis in basal breast cancers.
abstract: BACKGROUND
  In breast cancer, distinct expression profiles of microRNAs (miRNAs) have been associated with molecular subgroups and clinicopathological characteristics, implicating a diagnostic and prognostic role of miRNAs. However, the biological functions of deregulated miRNAs in tumor progression are not yet completely defined. In this study, we investigated the function of miR-18a in regulating breast cancer metastasis through the hypoxia-inducible factor 1α (HIF1A)-dependent hypoxic response.
  
  
  METHODS
  An orthotopic metastatic breast cancer xenograft model (MDA-MB-231 cells) was used to identify miRNAs associated with spontaneous lung metastasis. The function of miR-18a in regulating HIF1A expression, as well as cellular responses to hypoxia and metastasis, were then studied in vitro and in vivo by assessing ectopic miR-18a expression or miR-18a inhibition. miRNA-mRNA interactions (AGO2 immunoprecipitation and 3' untranslated region Luciferase reporter assays), gene expression (quantitative PCR and microarray), cell migration and invasion, and cell growth were assessed under normoxic or hypoxic conditions, complemented by orthotopic xenograft of tumor cells to the mammary fat pad to investigate the effect of modulating miR-18a expression on primary tumor growth and lung metastasis. Last, clinically relevant correlations between miR-18a, HIF1A, hypoxia-responsive gene expression and distant metastasis-free survival (DMFS) were assessed using published expression array breast tumors data sets.
  
  
  RESULTS
  miRNAs encoded by the MIR17HG gene were downregulated in lung metastases compared to primary tumors. Ectopic expression of miR-18a, a MIR17HG family member, in a metastatic variant of MDA-MB-231 cells reduced primary tumor growth and lung metastasis, whereas miR-18a inhibition in the parental cells promoted tumor growth and lung metastasis. We identified HIF1A as a direct target of miR-18a. Modulating miR-18a expression significantly affected hypoxic gene expression, cell invasiveness and sensitivity to anoikis and hypoxia in vitro in a HIF1A-dependent manner. Analysis of previously published data revealed that higher expression of HIF1A and a panel of hypoxic genes is associated with shorter DMFS interval in patients with basal-like breast tumors, and that, within this subtype, miR-18a expression is inversely correlated with hypoxic gene expression. Together, these data support a role of miR-18a in repressing distant metastasis through a HIF1A-dependent pathway.
  
  
  CONCLUSIONS
  The results of this study reveal a novel role for miR-18a in targeting HIF1A and repressing metastasis of basal-like breast tumors.
authors: Departments of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. rkrutili@uthsc.edu.|Center for Cancer Research, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. rkrutili@uthsc.edu.
authors: Raisa
authors: R
authors: Krutilina
authors: Department of Pharmacology, University of Tennessee Health Science Center, 424 Crowe research building, 874 Union Avenue, Memphis, TN, 38163, USA. wsun5@uthsc.edu.
authors: Wenlin
authors: W
authors: Sun
authors: Departments of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. asethur1@uthsc.edu.|Center for Cancer Research, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. asethur1@uthsc.edu.
authors: Aarti
authors: A
authors: Sethuraman
authors: Center for Cancer Research, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. martinmbrown@gmail.com.
authors: Martin
authors: M
authors: Brown
authors: Departments of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. tseagro1@uthsc.edu.|Center for Cancer Research, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. tseagro1@uthsc.edu.
authors: Tiffany N
authors: TN
authors: Seagroves
authors: Departments of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. lpfeffer@uthsc.edu.|Center for Cancer Research, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. lpfeffer@uthsc.edu.
authors: Lawrence M
authors: LM
authors: Pfeffer
authors: Department of Neurosurgery, University of Tennessee Health Science Center, 847 Monroe Avenue, Memphis, TN, 38163, USA. tnignatova@yahoo.com.
authors: Tatyana
authors: T
authors: Ignatova
authors: Departments of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. mfan2@uthsc.edu.|Center for Cancer Research, University of Tennessee Health Science Center, 19 South Manassas Street, Memphis, TN, 38163, USA. mfan2@uthsc.edu.
authors: Meiyun
authors: M
authors: Fan
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015687:Cell Hypoxia; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000556:secondary; D061986:MCF-7 Cells; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D035683:MicroRNAs / Q000502:physiology*; D018295:Neoplasms, Basal Cell / Q000235:genetics / Q000378:metabolism* / Q000556:secondary; D034622:RNA Interference
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C522576:MIRN18A microRNA, human; D035683:MicroRNAs