title: Remodeling of purinergic receptor-mediated Ca2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells.
abstract: BACKGROUND
  The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment.
  
  
  RESULTS
  We assessed changes in intracellular Ca(2+) in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR(TETRA)) and observed significant changes in the potency of ATP (EC(50) 0.175 µM (-EGF) versus 1.731 µM (+EGF), P<0.05), and the nature of the ATP-induced Ca(2+) transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca(2+) signaling, indicating that these alterations are not simply a consequence of changes in global Ca(2+) homeostasis. To determine whether changes in ATP-mediated Ca(2+) signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X(5) ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X(5) leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression.
  
  
  CONCLUSIONS
  The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.
authors.affiliation: School of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Felicity M
authors.initials: FM
authors.lastname: Davis
authors.forename: Paraic A
authors.initials: PA
authors.lastname: Kenny
authors.forename: Eliza T-L
authors.initials: ET
authors.lastname: Soo
authors.forename: Bryce J W
authors.initials: BJ
authors.lastname: van Denderen
authors.forename: Erik W
authors.initials: EW
authors.lastname: Thompson
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cabot
authors.forename: Marie-Odile
authors.initials: MO
authors.lastname: Parat
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Roberts-Thomson
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Monteith
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005260:Female; D006801:Humans; D011983:Receptors, Purinergic / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D011983:Receptors, Purinergic; D004815:Epidermal Growth Factor; D002118:Calcium