title: miR Profiling Identifies Cyclin-Dependent Kinase 6 Downregulation as a Potential Mechanism of Acquired Cisplatin Resistance in Non-Small-Cell Lung Carcinoma.
abstract: To identify the mechanisms of cisplatin resistance, global microRNA (miR) expression was tested. The expression of miR-145 was consistently higher in resistant cells. The expression of cyclin-dependent kinase 6 (CDK6), a potential target of miR-145, was lower in resistant cells, and inhibition of CDK4/6 protected cells from cisplatin. Cell cycle inhibition, currently being tested in clinical trials, might be antagonistic to cisplatin and other cytotoxic drugs.
  
  
  BACKGROUND
  Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Platinum-based chemotherapeutic drugs are the most active agents in treating advanced disease. Resistance to these drugs is common and multifactorial; insight into the molecular mechanisms involved will likely enhance efficacy.
  
  
  METHODS
  A set of NSCLC platinum-resistant sublines was created from the Calu6 cell line. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentially expressed microRNAs (miRs) in these lines were identified using Affymetrix miR arrays. The potential genes targeted by these miRs were searched using the TargetScan algorithm. The expression levels of miRs and mRNA were tested using real-time polymerase chain reaction.
  
  
  RESULTS
  miR-145 was reproducibly elevated in all the resistant sublines tested; however, modulation of miR-145 levels alone in these cells did not affect their response to cisplatin. A potential target of miR-145 is cyclin-dependent kinase 6 (CDK6), an important regulator of cell proliferation. The mRNA and protein levels of CDK6 were both downregulated in the resistant sublines. An inhibitor of CDK4/6 (PD0332991) protected parental NSCLC cells from cisplatin cytotoxicity.
  
  
  CONCLUSIONS
  In the present study, we identified miRs differentially expressed in cisplatin-resistant cell lines, including miR-145. A predicted target of miR-145 is CDK6, and its expression was found to be downregulated in the resistant sublines, although not directly by miR-145. Inhibition of CDK6 antagonizes cisplatin-induced NSCLC cell cytotoxicity, suggesting that agents that inhibit CDK6 should be avoided during cisplatin therapy.
authors.affiliation: Division of Medical Oncology, University of Ottawa Faculty of Medicine and The Ottawa Hospital Cancer Centre, Ottawa, Ontario, Canada.
authors.forename: Jair
authors.initials: J
authors.lastname: Bar
authors.affiliation: Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
authors.forename: Ivan
authors.initials: I
authors.lastname: Gorn-Hondermann
authors.affiliation: Division of Medical Oncology, University of Ottawa Faculty of Medicine and The Ottawa Hospital Cancer Centre, Ottawa, Ontario, Canada.
authors.forename: Patricia
authors.initials: P
authors.lastname: Moretto
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Department of Biochemistry, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada.
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Perkins
authors.affiliation: Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
authors.forename: Nima
authors.initials: N
authors.lastname: Niknejad
authors.affiliation: Division of Medical Oncology, University of Ottawa Faculty of Medicine and The Ottawa Hospital Cancer Centre, Ottawa, Ontario, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: Stewart
authors.affiliation: Division of Medical Oncology, University of Ottawa Faculty of Medicine and The Ottawa Hospital Cancer Centre, Ottawa, Ontario, Canada.
authors.forename: Glenwood D
authors.initials: GD
authors.lastname: Goss
authors.affiliation: Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Department of Biochemistry, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada. Electronic address: jdimitroulakos@ohri.ca.
authors.forename: Jim
authors.initials: J
authors.lastname: Dimitroulakos
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000188:drug therapy; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002945:Cisplatin / Q000627:therapeutic use*; D051361:Cyclin-Dependent Kinase 6 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000188:drug therapy; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012189:Retrospective Studies
chemical_list: C541010:MIRN145 microRNA, human; D035683:MicroRNAs; D010879:Piperazines; D011725:Pyridines; D051361:Cyclin-Dependent Kinase 6; C500026:palbociclib; D002945:Cisplatin
keywords: CDK6; Cell cycle inhibition; NSCLC; Predictive biomarkers; microRNA