title: Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition.
abstract: BACKGROUND
  Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied.
  
  
  METHODS
  RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity.
  
  
  RESULTS
  In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential.
  
  
  CONCLUSIONS
  These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment.
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. francesca.megiorni@uniroma1.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Megiorni
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. hdominici@tin.it.|Department of Oncology, Alder Hey Children's NHS Foundation Trust, Eaton Road, L12 2AP, Liverpool, UK. hdominici@tin.it.
authors.forename: Heather P
authors.initials: HP
authors.lastname: McDowell
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. simona.camero@uniroma1.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Camero
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. o.mannarino@libero.it.
authors.forename: Olga
authors.initials: O
authors.lastname: Mannarino
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161, Rome, Italy. simona.ceccarelli@uniroma1.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Ceccarelli
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. milena.paiano@uniroma1.it.
authors.forename: Milena
authors.initials: M
authors.lastname: Paiano
authors.affiliation: Department of Paediatric Surgery, Alder Hey Children's NHS Foundation Trust; Academic Paediatric Surgery Unit, University of Liverpool, Eaton Road, L12 2AP, Liverpool, UK. paul.losty@liverpool.ac.uk.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Losty
authors.affiliation: Department of Oncology, Alder Hey Children's NHS Foundation Trust, Eaton Road, L12 2AP, Liverpool, UK. barry.pizer@alderhey.nhs.uk.
authors.forename: Barry
authors.initials: B
authors.lastname: Pizer
authors.affiliation: Department of Perinatal and Paediatric Pathology, Alder Hey Children's NHS Foundation Trust, Eaton Road, L12 2AP, Liverpool, UK. rajeev.shukla@alderhey.nhs.uk.
authors.forename: Rajeev
authors.initials: R
authors.lastname: Shukla
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161, Rome, Italy. antonio.pizzuti@uniroma1.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pizzuti
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. anna.clerico@uniroma1.it.
authors.forename: Anna
authors.initials: A
authors.lastname: Clerico
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. carlo.dominici@uniroma1.it.
authors.forename: Carlo
authors.initials: C
authors.lastname: Dominici
mesh_terms: D000293:Adolescent; D000077548:Anaplastic Lymphoma Kinase; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D016203:CDC2 Protein Kinase; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002648:Child; D000077547:Crizotinib; D056744:Cyclin B1 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors* / Q000235:genetics; D011720:Pyrazoles / Q000627:therapeutic use*; D011725:Pyridines / Q000627:therapeutic use*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D017526:Receptor, IGF Type 1; D017451:Receptors, Somatomedin / Q000037:antagonists & inhibitors*; D018232:Rhabdomyosarcoma, Alveolar / Q000188:drug therapy*; D014410:Tumor Stem Cell Assay
chemical_list: D000970:Antineoplastic Agents; C534699:CCNB1 protein, human; D056744:Cyclin B1; C000597236:IGF1R protein, human; C118585:P62 protein, human; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; D017451:Receptors, Somatomedin; D000077547:Crizotinib; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D051057:Proto-Oncogene Proteins c-akt; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D053148:Caspase 3