title: Stimulus-dependent differences in signalling regulate epithelial-mesenchymal plasticity and change the effects of drugs in breast cancer cell lines.
abstract: BACKGROUND
  The normal process of epithelial mesenchymal transition (EMT) is subverted by carcinoma cells to facilitate metastatic spread. Cancer cells rarely undergo a full conversion to the mesenchymal phenotype, and instead adopt positions along the epithelial-mesenchymal axis, a propensity we refer to as epithelial mesenchymal plasticity (EMP). EMP is associated with increased risk of metastasis in breast cancer and consequent poor prognosis. Drivers towards the mesenchymal state in malignant cells include growth factor stimulation or exposure to hypoxic conditions.
  
  
  METHODS
  We have examined EMP in two cell line models of breast cancer: the PMC42 system (PMC42-ET and PMC42-LA sublines) and MDA-MB-468 cells. Transition to a mesenchymal phenotype was induced across all three cell lines using epidermal growth factor (EGF) stimulation, and in MDA-MB-468 cells by hypoxia. We used RNA sequencing to identify gene expression changes that occur as cells transition to a more-mesenchymal phenotype, and identified the cell signalling pathways regulated across these experimental systems. We then used inhibitors to modulate signalling through these pathways, verifying the conclusions of our transcriptomic analysis.
  
  
  RESULTS
  We found that EGF and hypoxia both drive MDA-MB-468 cells to phenotypically similar mesenchymal states. Comparing the transcriptional response to EGF and hypoxia, we have identified differences in the cellular signalling pathways that mediate, and are influenced by, EMT. Significant differences were observed for a number of important cellular signalling components previously implicated in EMT, such as HBEGF and VEGFA. We have shown that EGF- and hypoxia-induced transitions respond differently to treatment with chemical inhibitors (presented individually and in combinations) in these breast cancer cells. Unexpectedly, MDA-MB-468 cells grown under hypoxic growth conditions became even more mesenchymal following exposure to certain kinase inhibitors that prevent growth-factor induced EMT, including the mTOR inhibitor everolimus and the AKT1/2/3 inhibitor AZD5363.
  
  
  CONCLUSIONS
  While resulting in a common phenotype, EGF and hypoxia induced subtly different signalling systems in breast cancer cells. Our findings have important implications for the use of kinase inhibitor-based therapeutic interventions in breast cancers, where these heterogeneous signalling landscapes will influence the therapeutic response.
authors.affiliation: Systems Biology Laboratory, Melbourne School of Engineering, University of Melbourne, Building 193, Parkville, VIC, 3010, Australia. joseph.cursons@unimelb.edu.au.|ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Melbourne School of Engineering, University of Melbourne, Parkville, VIC, 3010, Australia. joseph.cursons@unimelb.edu.au.
authors.forename: Joseph
authors.initials: J
authors.lastname: Cursons
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. leuchowius@wehi.edu.au.|Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3010, Australia. leuchowius@wehi.edu.au.
authors.forename: Karl-Johan
authors.initials: KJ
authors.lastname: Leuchowius
authors.affiliation: St. Vincent's Institute, Melbourne, VIC, Australia. mwaltham@unimelb.edu.au.
authors.forename: Mark
authors.initials: M
authors.lastname: Waltham
authors.affiliation: St. Vincent's Institute, Melbourne, VIC, Australia. evatc@uow.edu.au.
authors.forename: Eva
authors.initials: E
authors.lastname: Tomaskovic-Crook
authors.affiliation: Systems Biology Laboratory, Melbourne School of Engineering, University of Melbourne, Building 193, Parkville, VIC, 3010, Australia. mforoutan@student.unimelb.edu.au.
authors.forename: Momeneh
authors.initials: M
authors.lastname: Foroutan
authors.affiliation: Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, SA, 5000, Australia. Cameron.Bracken@health.sa.gov.au.|Discipline of Medicine, University of Adelaide, Adelaide, SA, 5005, Australia. Cameron.Bracken@health.sa.gov.au.
authors.forename: Cameron P
authors.initials: CP
authors.lastname: Bracken
authors.affiliation: Royal Perth Hospital, Perth, Australia. Andrew.Redfern@health.wa.gov.au.
authors.forename: Andrew
authors.initials: A
authors.lastname: Redfern
authors.affiliation: Systems Biology Laboratory, Melbourne School of Engineering, University of Melbourne, Building 193, Parkville, VIC, 3010, Australia. edmund.crampin@unimelb.edu.au.|ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Melbourne School of Engineering, University of Melbourne, Parkville, VIC, 3010, Australia. edmund.crampin@unimelb.edu.au.|School of Mathematics and Statistics, Faculty of Science, University of Melbourne, Parkville, VIC, 3010, Australia. edmund.crampin@unimelb.edu.au.|School of Medicine, Faculty of Medicine Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, 3010, Australia. edmund.crampin@unimelb.edu.au.
authors.forename: Edmund J
authors.initials: EJ
authors.lastname: Crampin
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. istreet@wehi.EDU.AU.|Department of Medical Biology, The University of Melbourne, Parkville, VIC, 3010, Australia. istreet@wehi.EDU.AU.
authors.forename: Ian
authors.initials: I
authors.lastname: Street
authors.affiliation: Systems Biology Laboratory, Melbourne School of Engineering, University of Melbourne, Building 193, Parkville, VIC, 3010, Australia. melissa.davis@unimelb.edu.au.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Davis
authors.affiliation: St. Vincent's Institute, Melbourne, VIC, Australia. e2.thompson@qut.edu.au.|Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland Institute of Technology, Brisbane, Australia. e2.thompson@qut.edu.au.|University of Melbourne Department of Surgery, St. Vincent's Hospital, Melbourne, Australia. e2.thompson@qut.edu.au.
authors.forename: Erik W
authors.initials: EW
authors.lastname: Thompson
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D000068338:Everolimus; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D009362:Neoplasm Metastasis; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D007166:Immunosuppressive Agents; D011743:Pyrimidines; D011758:Pyrroles; D004815:Epidermal Growth Factor; D000068338:Everolimus; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus; C575618:capivasertib