Targeting of syndecan-1 by microRNA miR-10b promotes breast cancer cell motility and invasiveness via a Rho-GTPase- and E-cadherin-dependent mechanism Sherif A. Ibrahim 1,2 , George W. Yip 3 , Christian Stock 4 , Jun-Wei Pan 3 , Claudia Neubauer 1 , Michaela Poeter 5 , Danute Pupjalis 5 , Chuay Yeng Koo 3 , Reinhard Kelsch 6 , Roland Schu ¨le 7 , Ursula Rescher 5 , Ludwig Kiesel 1 and Martin G€ otte 1 1 Department of Gynecology and Obstetrics, Mu ¨nster University Hospital, Mu ¨nster, Germany 2 Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt 3 Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4 Institute of Physiology II, Mu ¨nster University Hospital, Mu ¨nster, Germany 5 Institute of Medical Biochemistry, ZMBE, University of Mu ¨nster, Mu ¨nster, Germany 6 Institute of Transfusion Medicine and Transplantation Immunology, Mu ¨nster University Hospital, Mu ¨nster, Germany 7 Women’s Hospital and Center for Clinical Research, University Medical Center, Freiburg, Germany microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3 0 UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin c 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/ RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase- dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer. The transmembrane proteoglycan syndecan-1 modulates a multitude of physiological processes via binding of its hepa- ran sulfate carbohydrate chains to multiple ligands relevant to tumor progression. 1 Syndecan-1 is a classical coreceptor for growth factors, angiogenic factors and chemokines, and acts as a cell and matrix adhesion receptor. 1,2 Syndecan-1 modulates integrin function, 3 proteolysis 4 and tumor angio- genesis. 2,3,5 The striking resistance of syndecan-1-deficient mice to mammary tumorigenesis has been linked to a poten- tial role in cancer stem cell function. 6 Stromal syndecan-1 is significantly downregulated upon preoperative systemic ther- apy of breast cancer, 7 consistent with a possible predictive value in neoadjuvant chemotherapy. 8 Although several stud- ies demonstrated that high syndecan-1 expression is associ- ated with negative prognostic parameters 9 and reduced breast cancer-specific overall survival, 10 additional work showed a shift of high epithelial to high stromal syndecan-1 expression during tumor progression, 11–13 suggesting that reduced epi- thelial expression of syndecan-1 in breast cancer may favor Key words: syndecans, heparan sulfate proteoglycan, CD138, breast cancer, cell motility, microRNA, oncomiR, ATF-2, RUNX1 Additional Supporting Information may be found in the online version of this article. Grant sponsors: German Academic Exchange Service DAAD A/06/ 90277, Maria M€ oller Stiftung, F€ orderkreis Universit€ at Mu ¨nster, DFG GRK 1549/1 IRTG ‘Molecular and Cellular Glycosciences’, ’Innovative Medizinische Forschung’ IMF GO ¨ 1 1 11 10, G.I.F. I- 1004-136.11/2008, National Medical Research Council, Singapore Grant NMRC/1023/2005 DOI: 10.1002/ijc.27629 History: Received 10 Feb 2012; Accepted 25 Apr 2012; Online 10 May 2012 M.G. and G.W.Y. contributed equally to this work. Correspondence to: Martin G€ otte, Department of Gynecology and Obstetrics, Mu ¨nster University Hospital, Albert-Schweitzer-Campus 1, Geb€ aude D11, D-48149 Mu ¨nster, Germany, E-mail: martingotte@ uni-muenster.de or George W. Yip, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Block MD 10, Singapore 117597, Singapore, E-mail: georgeyip@nus.edu.sg Cancer Cell Biology Int. J. Cancer: 000, 000–000 (2012) V C 2012 UICC International Journal of Cancer IJC