title: The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro.
abstract: Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.
authors.affiliation: Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland. Electronic address: olgapiskareva@rcsi.ie.
authors.forename: Olga
authors.initials: O
authors.lastname: Piskareva
authors.affiliation: Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Harry
authors.initials: H
authors.lastname: Harvey
authors.affiliation: Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: John
authors.initials: J
authors.lastname: Nolan
authors.affiliation: Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Ross
authors.initials: R
authors.lastname: Conlon
authors.affiliation: Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
authors.forename: Leah
authors.initials: L
authors.lastname: Alcock
authors.affiliation: Molecular Pathology Laboratory, Beaumont Hospital, Dublin 9, Ireland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Buckley
authors.affiliation: Department of Biology, The National University of Ireland Maynooth, Maynooth, County Kildare, Ireland.
authors.forename: Paul
authors.initials: P
authors.lastname: Dowling
authors.forename: Michael
authors.initials: M
authors.lastname: Henry
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland.
authors.forename: Finbarr
authors.initials: F
authors.lastname: O'Sullivan
authors.affiliation: Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Isabella
authors.initials: I
authors.lastname: Bray
authors.affiliation: Cancer Genetics Group, Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Raymond L
authors.initials: RL
authors.lastname: Stallings
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002648:Child; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006801:Humans; D007223:Infant; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D040901:Proteomics
chemical_list: D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D002945:Cisplatin
keywords: Cisplatin; Drug resistance; Epithelial-to-mesenchymal transition (EMT); Invasion; Neuroblastoma; Proteomics