title: Anoikis-resistant subpopulations of human osteosarcoma display significant chemoresistance and are sensitive to targeted epigenetic therapies predicted by expression profiling.
abstract: BACKGROUND
  Osteosarcoma (OS) is the most common type of solid bone cancer, with latent metastasis being a typical mode of disease progression and a major contributor to poor prognosis. For this to occur, cells must resist anoikis and be able to recapitulate tumorigenesis in a foreign microenvironment. Finding novel approaches to treat osteosarcoma and target those cell subpopulations that possess the ability to resist anoikis and contribute to metastatic disease is imperative. Here we investigate anchorage-independent (AI) cell growth as a model to better characterize anoikis resistance in human osteosarcoma while using an expression profiling approach to identify and test targetable signaling pathways.
  
  
  METHODS
  Established human OS cell lines and patient-derived human OS cell isolates were subjected to growth in either adherent or AI conditions using Ultra-Low Attachment plates in identical media conditions. Growth rate was assessed using cell doubling times and chemoresistance was assessed by determining cell viability in response to a serial dilution of either doxorubicin or cisplatin. Gene expression differences were examined using quantitative reverse-transcription PCR and microarray with principal component and pathway analysis. In-vivo OS xenografts were generated by either subcutaneous or intratibial injection of adherent or AI human OS cells into athymic nude mice. Statistical significance was determined using student's t-tests with significance set at α=0.05.
  
  
  RESULTS
  We show that AI growth results in a global gene expression profile change accompanied by significant chemoresistance (up to 75 fold, p<0.05). AI cells demonstrate alteration of key mediators of mesenchymal differentiation (β-catenin, Runx2), stemness (Sox2), proliferation (c-myc, Akt), and epigenetic regulation (HDAC class 1). AI cells were equally tumorigenic as their adherent counterparts, but showed a significantly decreased rate of growth in-vitro and in-vivo (p<0.05). Treatment with the pan-histone deacetylase inhibitor vorinostat and the DNA methyltransferase inhibitor 5-azacytidine mitigated AI growth, while 5-azacytidine sensitized anoikis-resistant cells to doxorubicin (p<0.05).
  
  
  CONCLUSIONS
  These data demonstrate remarkable plasticity in anoikis-resistant human osteosarcoma subpopulations accompanied by a rapid development of chemoresistance and altered growth rates mirroring the early stages of latent metastasis. Targeting epigenetic regulation of this process may be a viable therapeutic strategy.
authors.affiliation: Helen DeVos Children's Hospital, Spectrum Health System, Grand Rapids, MI, USA. jessica.foley@vai.org.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Foley
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. donald.scholten@vai.org.|Michigan State University College of Human Medicine, Grand Rapids, MI, USA. donald.scholten@vai.org.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Scholten
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. monksn@medimmune.com.
authors.forename: Noel R
authors.initials: NR
authors.lastname: Monks
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. david.cherba@vai.org.
authors.forename: David
authors.initials: D
authors.lastname: Cherba
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. david.monsma@vai.org.
authors.forename: David J
authors.initials: DJ
authors.lastname: Monsma
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. paula.davidson@vai.org.
authors.forename: Paula
authors.initials: P
authors.lastname: Davidson
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. dawna.dylewski@vai.org.
authors.forename: Dawna
authors.initials: D
authors.lastname: Dylewski
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. karl.dykema@vai.org.
authors.forename: Karl
authors.initials: K
authors.lastname: Dykema
authors.affiliation: Van Andel Research Institute, Grand Rapids, MI, USA. mary.winn@vai.org.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Winn
authors.affiliation: Helen DeVos Children's Hospital, Spectrum Health System, Grand Rapids, MI, USA. matt.steensma@vai.org.|Van Andel Research Institute, Grand Rapids, MI, USA. matt.steensma@vai.org.|Michigan State University College of Human Medicine, Grand Rapids, MI, USA. matt.steensma@vai.org.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Steensma
mesh_terms: D023102:Anoikis; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001859:Bone Neoplasms / Q000188:drug therapy / Q000235:genetics*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D006801:Humans; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000970:Antineoplastic Agents