title: ITIH5 mediates epigenetic reprogramming of breast cancer cells.
abstract: Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive.
  
  
  
  ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement.
  
  
  
  ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility.
  
  
  
  Our results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Rose
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Kloten
authors.affiliation: Institute of Complex Systems, ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Jülich, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Noetzel
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Gola
authors.affiliation: Department of Experimental Molecular Imaging (ExMI), Helmholtz Institute for Biomedical Engineering, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Ehling
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Timon
authors.initials: T
authors.lastname: Heide
authors.affiliation: Experimental Gene Therapy and Clinical Chemistry, Institute of Molecular Pathobiochemistry, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Steffen K
authors.initials: SK
authors.lastname: Meurer
authors.affiliation: Institute of Complex Systems, ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Jülich, Germany.
authors.forename: Aljona
authors.initials: A
authors.lastname: Gaiko-Shcherbak
authors.affiliation: Institute for Biomedical Engineering-Cell Biology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Antonio S
authors.initials: AS
authors.lastname: Sechi
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Huth
authors.affiliation: Experimental Gene Therapy and Clinical Chemistry, Institute of Molecular Pathobiochemistry, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Weiskirchen
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Klaas
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Antonopoulos
authors.affiliation: Institute for Biomedical Engineering-Cell Biology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.|Helmholtz-Institute for Biomedical Engineering-Stem Cell Biology and Cellular Engineering, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Lin
authors.affiliation: Institute for Biomedical Engineering-Cell Biology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.|Helmholtz-Institute for Biomedical Engineering-Stem Cell Biology and Cellular Engineering, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wagner
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.|Division of Medical Oncology, Department of Internal Medicine, Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Veeck
authors.affiliation: Department of Experimental Molecular Imaging (ExMI), Helmholtz Institute for Biomedical Engineering, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Gremse
authors.affiliation: Institute for Laboratory Animal Science, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Steitz
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Knüchel
authors.affiliation: Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany. edahl@ukaachen.de.
authors.forename: Edgar
authors.initials: E
authors.lastname: Dahl
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D064550:Death-Associated Protein Kinases / Q000235:genetics*; D044127:Epigenesis, Genetic; D005109:Extracellular Matrix; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000556:secondary*; D051379:Mice; D009368:Neoplasm Transplantation; D011379:Prognosis; D053491:Proteinase Inhibitory Proteins, Secretory / Q000235:genetics*; D016019:Survival Analysis
chemical_list: C482680:ITIH5 protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases
keywords: Breast cancer; Cancer stem cells; DAPK1; Epigenetic reprogramming; Extracellular matrix; ITIH5