microRNA miR-142-3p Inhibits Breast Cancer Cell Invasiveness by Synchronous Targeting of WASL, Integrin Alpha V, and Additional Cytoskeletal Elements

MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3’-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer.

[1]  F. Sarkar,et al.  Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. , 1995, The Journal of clinical investigation.

[2]  L. Kiesel,et al.  HS3ST2 modulates breast cancer cell invasiveness via MAP kinase‐ and Tcf4 (Tcf7l2)‐dependent regulation of protease and cadherin expression , 2014, International journal of cancer.

[3]  D. M. Pereira,et al.  Delivering the promise of miRNA cancer therapeutics. , 2013, Drug discovery today.

[4]  H. Oberleithner,et al.  Bradykinin shifts endothelial fluid passage from para- to transcellular routes , 2006, Pflügers Archiv.

[5]  T. Tuschl,et al.  Combined Characterization of microRNA and mRNA Profiles Delineates Early Differentiation Pathways of CD133+ and CD34+ Hematopoietic Stem and Progenitor Cells , 2011, Stem cells.

[6]  Katsuo Suzuki,et al.  WAVE2, N‐WASP, and mena facilitate cell invasion via phosphatidylinositol 3‐kinase‐dependent local accumulation of actin filaments , 2011, Journal of cellular biochemistry.

[7]  C. Barbas,et al.  Alphav integrins mediate adhesion and migration of breast carcinoma cell lines. , 1998, Clinical & experimental metastasis.

[8]  J. Chen,et al.  Alpha(v) integrin, p38 mitogen-activated protein kinase, and urokinase plasminogen activator are functionally linked in invasive breast cancer cells. , 2001, The Journal of biological chemistry.

[9]  Steffi Ketelhut,et al.  Quantitative Stain-Free and Continuous Multimodal Monitoring of Wound Healing In Vitro with Digital Holographic Microscopy , 2014, PloS one.

[10]  Shuang Huang,et al.  αv Integrin, p38 Mitogen-activated Protein Kinase, and Urokinase Plasminogen Activator Are Functionally Linked in Invasive Breast Cancer Cells* , 2001, The Journal of Biological Chemistry.

[11]  R. Adams,et al.  Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor–specific angiogenic signaling , 2013, Molecular biology of the cell.

[12]  Toshimichi Yoshida,et al.  Binding of αvβ1 and αvβ6 integrins to tenascin-C induces epithelial–mesenchymal transition-like change of breast cancer cells , 2013, Oncogenesis.

[13]  L. Kiesel,et al.  Defective glycosylation of decorin and biglycan, altered collagen structure, and abnormal phenotype of the skin fibroblasts of an Ehlers–Danlos syndrome patient carrying the novel Arg270Cys substitution in galactosyltransferase I (β4GalT-7) , 2006, Journal of Molecular Medicine.

[14]  M. Götte,et al.  MicroRNA regulation of proteoglycan function in cancer , 2014, The FEBS journal.

[15]  G. Shen,et al.  miR-1423 p restricts cAMP production in CD 4 þ CD 25 T cells and CD 4 þ CD 25 þ TREG cells by targeting AC 9 mRNA , 2013 .

[16]  M. Felcht,et al.  Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation* , 2010, The Journal of Biological Chemistry.

[17]  Ying Shi,et al.  Deficiency of Kruppel‐like factor KLF4 in mammary tumor cells inhibits tumor growth and pulmonary metastasis and is accompanied by compromised recruitment of myeloid‐derived suppressor cells , 2013, International journal of cancer.

[18]  A. Hollestelle,et al.  BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants. , 2006, Cancer research.

[19]  D. Wirtz,et al.  The Arp2/3 complex mediates multigeneration dendritic protrusions for efficient 3‐dimensional cancer cell migration , 2013, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

[21]  L. Nusblat,et al.  The non-redundant role of N-WASP in podosome-mediated matrix degradation in macrophages. , 2011, European journal of cell biology.

[22]  Valerie Speirs,et al.  Choosing the right cell line for breast cancer research , 2011, Breast Cancer Research.

[23]  L. Silengo,et al.  Cross talk between beta(1) and alpha(V) integrins: beta(1) affects beta(3) mRNA stability. , 2001, Molecular biology of the cell.

[24]  F. McCabe,et al.  CNTO 95, a fully human anti αv integrin antibody, inhibits cell signaling, migration, invasion, and spontaneous metastasis of human breast cancer cells , 2007, Clinical & Experimental Metastasis.

[25]  C. Croce,et al.  Breast cancer signatures for invasiveness and prognosis defined by deep sequencing of microRNA , 2012, Proceedings of the National Academy of Sciences.

[26]  V. Quaranta,et al.  Migration of breast epithelial cells on Laminin-5: differential role of integrins in normal and transformed cell types , 1998, Breast Cancer Research and Treatment.

[27]  S. Strömblad,et al.  p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration , 2002, The Journal of cell biology.

[28]  R. Weinberg,et al.  Tumour invasion and metastasis initiated by microRNA-10b in breast cancer , 2007, Nature.

[29]  Doron Betel,et al.  The microRNA.org resource: targets and expression , 2007, Nucleic Acids Res..

[30]  Marie-France Carlier,et al.  IQGAP1 Stimulates Actin Assembly through the N-Wasp-Arp2/3 Pathway* , 2007, Journal of Biological Chemistry.

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

[32]  L. Kiesel,et al.  Targeting of syndecan‐1 by microRNA miR‐10b promotes breast cancer cell motility and invasiveness via a Rho‐GTPase‐ and E‐cadherin‐dependent mechanism , 2012, International journal of cancer.

[33]  Daniel Carl,et al.  Investigation of living pancreas tumor cells by digital holographic microscopy. , 2006, Journal of biomedical optics.

[34]  J. Baak,et al.  Biologic profiling of lymph node negative breast cancers by means of microRNA expression , 2010, Modern Pathology.

[35]  C. Albrecht,et al.  In vitro inhibition of SKOV-3 cell migration as a distinctive feature of progesterone receptor membrane component type 2 versus type 1 , 2012, Steroids.

[36]  Frank J. Sørensen,et al.  Diagnostic and prognostic microRNAs in stage II colon cancer. , 2008, Cancer research.

[37]  Min Liu,et al.  MicroRNA‐142‐3p, a new regulator of RAC1, suppresses the migration and invasion of hepatocellular carcinoma cells , 2011, FEBS letters.

[38]  Olli Kallioniemi,et al.  Identification of MicroRNAs Inhibiting TGF-β-Induced IL-11 Production in Bone Metastatic Breast Cancer Cells , 2012, PloS one.

[39]  D. Sanoudou,et al.  Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle , 2009, Molecular and Cellular Biology.

[40]  Kimito Yamada,et al.  Epidermal growth factor‐dependent enhancement of invasiveness of squamous cell carcinoma of the breast , 2010, Cancer science.

[41]  Andrea Riccardo Genazzani,et al.  Estrogen Receptor- (cid:1) Promotes Breast Cancer Cell Motility and Invasion via Focal Adhesion Kinase and N-WASP , 2010 .

[42]  C. Otey.  Faculty Opinions recommendation of P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration. , 2002 .

[43]  M. Lacroix,et al.  Relevance of Breast Cancer Cell Lines as Models for Breast Tumours: An Update , 2004, Breast Cancer Research and Treatment.

[44]  G. Mills,et al.  miR-145 participates with TP53 in a death-promoting regulatory loop and targets estrogen receptor-α in human breast cancer cells , 2010, Cell Death and Differentiation.

[45]  M. Brandi,et al.  MicroRNA Role in Thyroid Cancer Development , 2011, Journal of thyroid research.

[46]  N. Tritsch,et al.  Deleted in Colorectal Cancer Binding Netrin-1 Mediates Cell Substrate Adhesion and Recruits Cdc42, Rac1, Pak1, and N-WASP into an Intracellular Signaling Complex That Promotes Growth Cone Expansion , 2005, The Journal of Neuroscience.

[47]  Daya Luo,et al.  A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells , 2013, Journal of Translational Medicine.

[48]  L. Kiesel,et al.  miR-142-3p is a novel regulator of cell viability and proinflammatory signalling in endometrial stroma cells. , 2015, Reproductive biomedicine online.

[49]  G. Bhanot,et al.  Identification of a microRNA panel for clear-cell kidney cancer. , 2010, Urology.

[50]  J. Sturge,et al.  N-WASP activation by a beta1-integrin-dependent mechanism supports PI3K-independent chemotaxis stimulated by urokinase-type plasminogen activator. , 2002, Journal of cell science.

[51]  T. Meyer,et al.  Expression of alphav integrins and vitronectin receptor identity in breast cancer cells. , 1998, British Journal of Cancer.

[52]  Stefano Volinia,et al.  MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets , 2009, The Journal of pathology.

[53]  Patrik Langehanenberg,et al.  Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy. , 2007, Journal of biomedical optics.

[54]  C. Kittas,et al.  Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer , 2007, Cancer Cell International.

[55]  Z. Chang,et al.  Regulation of RhoA-dependent ROCKII activation by Shp2 , 2008, The Journal of cell biology.

[56]  Shunsuke Noguchi,et al.  Microvesicle-mediated RNA molecule delivery system using monocytes/macrophages. , 2011, Molecular therapy : the journal of the American Society of Gene Therapy.

[57]  I. Ng,et al.  Rho‐kinase 2 is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion , 2009, Hepatology.

[58]  G. Shen,et al.  miR‐142‐3p restricts cAMP production in CD4+CD25− T cells and CD4+CD25+ TREG cells by targeting AC9 mRNA , 2009, EMBO reports.

[59]  J. Condeelis,et al.  N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells. , 2009, Cell motility and the cytoskeleton.

[60]  William P Schiemann,et al.  β3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells , 2006, Breast Cancer Research.

[61]  W. Jiang,et al.  Claudin-5 is involved in breast cancer cell motility through the N-WASP and ROCK signalling pathways , 2012, Journal of experimental & clinical cancer research : CR.

[62]  Rudolf Jaenisch,et al.  DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal , 2007, Nature Genetics.

[63]  L. Kiesel,et al.  miR-145-dependent targeting of Junctional Adhesion Molecule A and modulation of fascin expression are associated with reduced breast cancer cell motility and invasiveness , 2010, Oncogene.

[64]  J. Condeelis,et al.  N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors , 2012, Journal of Cell Science.

[65]  Monilola A. Olayioye,et al.  miR149 functions as a tumor suppressor by controlling breast epithelial cell migration and invasion. , 2014, Cancer research.

[66]  William Ritchie,et al.  Micro-RNA response to imatinib mesylate in patients with chronic myeloid leukemia , 2010, Haematologica.

[67]  George Oster,et al.  Force generation by actin polymerization II: the elastic ratchet and tethered filaments. , 2003, Biophysical journal.

[68]  D. Kraemer,et al.  Targeting αV-integrins decreased metastasis and increased survival in a nude rat breast cancer brain metastasis model , 2012, Journal of Neuro-Oncology.

[69]  H. Oberleithner,et al.  Nanotopography follows force in TGF-β1 stimulated epithelium , 2010, Nanotechnology.

[70]  A. Pasquinelli,et al.  A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA , 2001, Science.

[71]  J. Benítez,et al.  MicroRNA expression signatures for the prediction of BRCA1/2 mutation‐associated hereditary breast cancer in paraffin‐embedded formalin‐fixed breast tumors , 2014, International journal of cancer.

[72]  B. Moerschbacher,et al.  Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells , 2015, Scientific Reports.

[73]  N. Hamilton,et al.  N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway , 2011, Nature Cell Biology.