Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

Background MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. Methodology/Principal Findings We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR–92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells. Conclusions miR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer.

[1]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[2]  A. Sacchi,et al.  The microRNA miR‐92 increases proliferation of myeloid cells and by targeting p63 modulates the abundance of its isoforms , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[3]  Carmen Z. Cantemir-Stone,et al.  Epigenetic regulation of miR-17~92 contributes to the pathogenesis of pulmonary fibrosis. , 2013, American journal of respiratory and critical care medicine.

[4]  V. Speirs,et al.  Gene Expression of ERβ Isoforms in Laser Microdissected Human Breast Cancers: Implications for Gene Expression Analyses , 2009, Cellular oncology : the official journal of the International Society for Cellular Oncology.

[5]  A. Blais,et al.  Overexpression of a dominant negative type II bone morphogenetic protein receptor inhibits the growth of human breast cancer cells. , 2003, Cancer research.

[6]  Sofie Nilsson,et al.  Downregulation of miR-92a Is Associated with Aggressive Breast Cancer Features and Increased Tumour Macrophage Infiltration , 2012, PloS one.

[7]  George A. Calin,et al.  GAM/ZFp/ZNF512B is central to a gene sensor circuitry involving cell-cycle regulators, TGFβ effectors, Drosha and microRNAs with opposite oncogenic potentials , 2010, Nucleic acids research.

[8]  Yixuan Hou,et al.  MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer. , 2012, The international journal of biochemistry & cell biology.

[9]  T. Tlsty,et al.  Know thy neighbor: stromal cells can contribute oncogenic signals. , 2001, Current opinion in genetics & development.

[10]  Dennis C. Sgroi,et al.  Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion , 2005, Cell.

[11]  C. Croce,et al.  MicroRNA gene expression deregulation in human breast cancer. , 2005, Cancer research.

[12]  Victoria Kristina Perry,et al.  Gene expression abnormalities in histologically normal breast epithelium of breast cancer patients , 2007, International journal of cancer.

[13]  Y Magnusson,et al.  TGF-beta receptor type-2 expression in cancer-associated fibroblasts regulates breast cancer cell growth and survival and is a prognostic marker in pre-menopausal breast cancer , 2013, Oncogene.

[14]  Reuven Agami,et al.  miR-148 targets human DNMT3b protein coding region. , 2008, RNA.

[15]  Raghu Kalluri,et al.  Fibroblasts in cancer , 2006, Nature Reviews Cancer.

[16]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

[17]  V. Speirs,et al.  Expression of oestrogen receptor beta isoforms is regulated by transcriptional and post-transcriptional mechanisms. , 2010, The Biochemical journal.

[18]  J. Steitz,et al.  Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR , 2007, Proceedings of the National Academy of Sciences.

[19]  Hsien-Da Huang,et al.  miRTarBase update 2014: an information resource for experimentally validated miRNA-target interactions , 2013, Nucleic Acids Res..

[20]  Anwar Hossain,et al.  Mir-17-5p Regulates Breast Cancer Cell Proliferation by Inhibiting Translation of AIB1 mRNA , 2006, Molecular and Cellular Biology.

[21]  I. Ellis,et al.  Nuclear and Cytoplasmic Expression of ERβ1, ERβ2, and ERβ5 Identifies Distinct Prognostic Outcome for Breast Cancer Patients , 2008, Clinical Cancer Research.

[22]  V. Speirs,et al.  Differential regulation of oestrogen receptor β isoforms by 5′ untranslated regions in cancer , 2009, Journal of cellular and molecular medicine.

[23]  A. Harris,et al.  Gene Expression Analysis in Human Breast Cancer Associated Blood Vessels , 2012, PloS one.

[24]  S. Lawler,et al.  MicroRNAs in cancer: biomarkers, functions and therapy. , 2014, Trends in molecular medicine.

[25]  V. Speirs,et al.  Estrogen receptor {beta}1 expression is regulated by miR-92 in breast cancer. , 2010, Cancer research.

[26]  Y. Yatabe,et al.  A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. , 2005, Cancer research.

[27]  Nectarios Koziris,et al.  TarBase 6.0: capturing the exponential growth of miRNA targets with experimental support , 2011, Nucleic Acids Res..

[28]  S. Ropero,et al.  A microRNA DNA methylation signature for human cancer metastasis , 2008, Proceedings of the National Academy of Sciences.

[29]  P. Tan,et al.  Identification of Circulating MicroRNA Signatures for Breast Cancer Detection , 2013, Clinical Cancer Research.

[30]  Jiri Zavadil,et al.  Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation , 2011, The EMBO journal.

[31]  Robert A. Weinberg,et al.  Autocrine TGF-β and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts , 2010, Proceedings of the National Academy of Sciences.

[32]  Valerie Speirs,et al.  MiR-26b is down-regulated in carcinoma-associated fibroblasts from ER-positive breast cancers leading to enhanced cell migration and invasion , 2013, The Journal of pathology.

[33]  Metin N. Gurcan,et al.  Pten in Stromal Fibroblasts Suppresses Mammary Epithelial Tumors , 2009, Nature.

[34]  L. Shea,et al.  Transcription Factor Networks in Invasion-Promoting Breast Carcinoma-Associated Fibroblasts , 2013, Cancer Microenvironment.

[35]  M J Bissell,et al.  Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction. , 1996, Physiological reviews.

[36]  D. Allred,et al.  Prognostic and predictive factors in breast cancer by immunohistochemical analysis. , 1998, Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc.

[37]  H. Moses,et al.  Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators , 2011, Proceedings of the National Academy of Sciences.

[38]  Tara L. Naylor,et al.  microRNAs exhibit high frequency genomic alterations in human cancer. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Jun Yao,et al.  Distinct epigenetic changes in the stromal cells of breast cancers , 2005, Nature Genetics.

[40]  Peter A. Jones,et al.  Epigenetic Activation of Tumor Suppressor MicroRNAs in Human Cancer Cells , 2006, Cell cycle.

[41]  Yingjian Chen,et al.  Circulating microRNA-92a and microRNA-21 as novel minimally invasive biomarkers for primary breast cancer , 2012, Journal of Cancer Research and Clinical Oncology.

[42]  Xabier Agirre,et al.  Epigenetic regulation of microRNA expression in colorectal cancer , 2009, International journal of cancer.

[43]  G. Scambia,et al.  Prognostic significance of the estrogen receptor beta (ERβ) isoforms ERβ1, ERβ2, and ERβ5 in advanced serous ovarian cancer. , 2014, Gynecologic oncology.

[44]  Rudolf Jaenisch,et al.  Targeted Deletion Reveals Essential and Overlapping Functions of the miR-17∼92 Family of miRNA Clusters , 2008, Cell.

[45]  Kathryn A. O’Donnell,et al.  c-Myc-regulated microRNAs modulate E2F1 expression , 2005, Nature.

[46]  Terry Hyslop,et al.  A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation , 2008, The Journal of cell biology.

[47]  John N Weinstein,et al.  A stromal gene signature associated with inflammatory breast cancer , 2008, International journal of cancer.

[48]  Jarret Glasscock,et al.  Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank , 2014, Breast Cancer Research.

[49]  Martin Clynes,et al.  BreastMark: An Integrated Approach to Mining Publicly Available Transcriptomic Datasets Relating to Breast Cancer Outcome , 2013, Breast Cancer Research.

[50]  Bo Zhang,et al.  Distinct MicroRNA Subcellular Size and Expression Patterns in Human Cancer Cells , 2012, International journal of cell biology.

[51]  V. Speirs,et al.  Clinical and functional significance of loss of caveolin‐1 expression in breast cancer‐associated fibroblasts , 2012, The Journal of pathology.