title: Phosphatidylinositol 3-Kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin-1 depletion in breast cancer cells.
abstract: BACKGROUND
  Flotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of flotillins in breast cancer.
  
  
  METHODS
  Human adenocarcinoma MCF7 breast cancer cells were stably depleted of flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant flotillin proteins.
  
  
  RESULTS
  We here show that stable knockdown of flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression.
  
  
  CONCLUSIONS
  This study demonstrates that flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon flotillin knockdown which may even facilitate cancer cell growth and proliferation.
authors.forename: Nina
authors.initials: N
authors.lastname: Kurrle
authors.forename: Wymke
authors.initials: W
authors.lastname: Ockenga
authors.forename: Melanie
authors.initials: M
authors.lastname: Meister
authors.forename: Frauke
authors.initials: F
authors.lastname: Völlner
authors.forename: Sina
authors.initials: S
authors.lastname: Kühne
authors.forename: Bincy A
authors.initials: BA
authors.lastname: John
authors.forename: Antje
authors.initials: A
authors.lastname: Banning
authors.affiliation: Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany. Ritva.Tikkanen@biochemie.med.uni-giessen.de.
authors.forename: Ritva
authors.initials: R
authors.lastname: Tikkanen
mesh_terms: D001943:Breast Neoplasms; D049109:Cell Proliferation; D002867:Chromones / Q000494:pharmacology; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000502:physiology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D020935:MAP Kinase Signaling System; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport; D015854:Up-Regulation
chemical_list: D002867:Chromones; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C106354:flotillins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C512478:EGFR protein, human