title: TNF-α promotes breast cancer cell migration and enhances the concentration of membrane-associated proteases in lipid rafts.
abstract: OBJECTIVE
  Tumor progression is associated with cell migration, invasion and metastasis. These processes are accompanied by the activation of specific proteases that are either linked to cellular membranes or are secreted into extracellular spaces. TNF-α is known to play an important role in various aspects of tumor progression. The aim of this work was to assess the effect of TNF-α on the migration of breast cancer cells and, in addition, to assess its association with the location of membrane-associated proteases in lipid rafts.
  
  
  METHODS
  Wound scratch healing and Transwell migration assays were used to study the effect of TNF-α on the migration of both hormone-dependent and hormone-independent breast cancer-derived cells, i.e., MCF7 and MDA-MB-231, respectively. The expression and secretion of three matrix metalloproteases, MMP9, MMP2 and MT1-MMP, and two dipeptidyl peptidases, CD26 and FAP-α, was investigated using RT-PCR, Western blotting and gelatin zymography. In addition, activation of the MAPK/ERK signaling pathway was investigated by Western blotting.
  
  
  RESULTS
  We found that a TNF-α-induced enhancement of breast cancer cell migration was accompanied by an increased secretion of MMP9, but not MMP2, into the culture media. We also found that TNF-α upregulated the expression of the dipeptidyl peptidases CD26 and FAP-α in a dose-dependent manner and, in addition, enhanced the concentration of all five proteases in lipid rafts in the breast cancer-derived cells tested, regardless of cell type. Furthermore, we found that TNF-α activated the MAPK/ERK signaling pathway by increasing the ERK1/2 phosphorylation level. Application of the MEK/ERK1/2 inhibitor U-0126 resulted in down-regulation of TNF-α-induced MMP9 secretion and abrogation of the enhanced concentration of proteases in the lipid rafts.
  
  
  CONCLUSIONS
  From our results we conclude that TNF-α-induced activation of the MAPK/ERK signaling pathway may promote breast cancer cell migration via both upregulation of MMP9, CD26 and FAP-α and concentration of these proteases, as also MT1-MMP and MMP2, in the lipid rafts. TNF-α may serve as a potential therapeutic target in breast cancers susceptible to TNF-α stimulation.
authors.affiliation: Laboratory of Cytobiochemistry, Biotechnology Faculty, University of Wroclaw, Wroclaw, Poland.
authors.forename: Dominika
authors.initials: D
authors.lastname: Wolczyk
authors.affiliation: Department of Gastrointestinal and General Surgery, Wroclaw Medical University, Wroclaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zaremba-Czogalla
authors.affiliation: Laboratory of Cytobiochemistry, Biotechnology Faculty, University of Wroclaw, Wroclaw, Poland.
authors.forename: Anita
authors.initials: A
authors.lastname: Hryniewicz-Jankowska
authors.affiliation: Department of Gastrointestinal and General Surgery, Wroclaw Medical University, Wroclaw, Poland.
authors.forename: Renata
authors.initials: R
authors.lastname: Tabola
authors.affiliation: Department of Gastrointestinal and General Surgery, Wroclaw Medical University, Wroclaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Grabowski
authors.affiliation: Laboratory of Cytobiochemistry, Biotechnology Faculty, University of Wroclaw, Wroclaw, Poland.
authors.forename: Aleksander F
authors.initials: AF
authors.lastname: Sikorski
authors.affiliation: Department of Gastrointestinal and General Surgery, Wroclaw Medical University, Wroclaw, Poland. katarzyna.augoff@umed.wroc.pl.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Augoff
mesh_terms: D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D005260:Female; D006801:Humans; D021962:Membrane Microdomains / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D014409:Tumor Necrosis Factor-alpha; D010447:Peptide Hydrolases
keywords: DPPIV/CD26; DRM; FAP-α/F19/seprase; MMP2; MMP9; MT1-MMP/MMP14; TNF-α