title: Interleukin-like epithelial-to-mesenchymal transition inducer activity is controlled by proteolytic processing and plasminogen-urokinase plasminogen activator receptor system-regulated secretion during breast cancer progression.
abstract: BACKGROUND
  Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) is an essential cytokine in tumor progression that is upregulated in several cancers, and its altered subcellular localization is a predictor of poor survival in human breast cancer. However, the regulation of ILEI activity and the molecular meaning of its altered localization remain elusive.
  
  
  METHODS
  The influence of serum withdrawal, broad-specificity protease inhibitors, different serine proteases and plasminogen depletion on the size and amount of the secreted ILEI protein was investigated by Western blot analysis of EpRas cells. Proteases with ILEI-processing capacity were identified by carrying out an in vitro cleavage assay. Murine mammary tumor and metastasis models of EpC40 and 4T1 cells overexpressing different mutant forms of ILEI were used-extended with in vivo aprotinin treatment for the inhibition of ILEI-processing proteases-to test the in vivo relevance of proteolytic cleavage. Stable knockdown of urokinase plasminogen activator receptor (uPAR) in EpRas cells was performed to investigate the involvement of uPAR in ILEI secretion. The subcellular localization of the ILEI protein in tumor cell lines was analyzed by immunofluorescence. Immunohistochemistry for ILEI localization and uPAR expression was performed on two human breast cancer arrays, and ILEI and uPAR scores were correlated with the metastasis-free survival of patients.
  
  
  RESULTS
  We demonstrate that secreted ILEI requires site-specific proteolytic maturation into its short form for its tumor-promoting function, which is executed by serine proteases, most efficiently by plasmin. Noncleaved ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through a propeptide-dependent interaction. In addition to ILEI processing, plasmin rapidly increases ILEI secretion by mobilizing its intracellular protein pool in a uPAR-dependent manner. Elevated ILEI secretion correlates with an altered subcellular localization of the protein, most likely representing a shift into secretory vesicles. Moreover, altered subcellular ILEI localization strongly correlates with high tumor cell-associated uPAR protein expression, as well as with poor survival, in human breast cancer.
  
  
  CONCLUSIONS
  Our findings point out extracellular serine proteases, in particular plasmin, and uPAR as valuable therapeutic targets against ILEI-driven tumor progression and emphasize the prognostic relevance of ILEI localization and a combined ILEI-uPAR marker analysis in human breast cancer.
authors.forename: Agnes
authors.initials: A
authors.lastname: Csiszar
authors.forename: Betül
authors.initials: B
authors.lastname: Kutay
authors.forename: Silvia
authors.initials: S
authors.lastname: Wirth
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schmidt
authors.forename: Sabine
authors.initials: S
authors.lastname: Macho-Maschler
authors.forename: Martin
authors.initials: M
authors.lastname: Schreiber
authors.forename: Memetcan
authors.initials: M
authors.lastname: Alacakaptan
authors.forename: Georg F
authors.initials: GF
authors.lastname: Vogel
authors.forename: Karin
authors.initials: K
authors.lastname: Aumayr
authors.forename: Lukas A
authors.initials: LA
authors.lastname: Huber
authors.forename: Hartmut
authors.initials: H
authors.lastname: Beug
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D045744:Cell Line, Tumor; D016207:Cytokines / Q000502:physiology*; D018450:Disease Progression; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D053208:Kaplan-Meier Estimate; D019272:Leukocyte Elastase / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism* / Q000401:mortality / Q000556:secondary; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000502:physiology*; D009368:Neoplasm Transplantation; D020842:Plasma Kallikrein / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D059748:Proteolysis; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism*
chemical_list: D016207:Cytokines; C514209:FAM3C protein, human; D009363:Neoplasm Proteins; D055293:Receptors, Urokinase Plasminogen Activator; D020842:Plasma Kallikrein; D019272:Leukocyte Elastase; D005341:Fibrinolysin