title: Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population.
abstract: BACKGROUND
  The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD).
  
  
  METHODS
  Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium.
  
  
  RESULTS
  MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells.
  
  
  CONCLUSIONS
  Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.
authors.affiliation: Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Via Vetoio, Coppito 2, 67100, L'Aquila, Italy. carmela.ciccarelli@univaq.it.
authors.forename: Carmela
authors.initials: C
authors.lastname: Ciccarelli
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. francesca.vulcano@iss.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Vulcano
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. luisa.milazzo@iss.it.
authors.forename: Luisa
authors.initials: L
authors.lastname: Milazzo
authors.affiliation: Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Via Vetoio, Coppito 2, 67100, L'Aquila, Italy. giovanniluca.gravina@libero.it.
authors.forename: Giovanni Luca
authors.initials: GL
authors.lastname: Gravina
authors.affiliation: Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Via Vetoio, Coppito 2, 67100, L'Aquila, Italy. f.marampon@gmail.com.
authors.forename: Francesco
authors.initials: F
authors.lastname: Marampon
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. giampiero.macioce@iss.it.
authors.forename: Giampiero
authors.initials: G
authors.lastname: Macioce
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. adele.giampaolo@iss.it.
authors.forename: Adele
authors.initials: A
authors.lastname: Giampaolo
authors.affiliation: Department of Radiotherapy, University of Rome "Sapienza", Rome, Italy. vincenzo.tombolini@uniroma1.it.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Tombolini
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopedic, Section of Histology, Sapienza University of Rome, Rome, Italy. virginia.dipaolo@uniroma1.it.
authors.forename: Virginia
authors.initials: V
authors.lastname: Di Paolo
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. jane.hassan@iss.it.
authors.forename: Hamisa Jane
authors.initials: HJ
authors.lastname: Hassan
authors.affiliation: Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Via Vetoio, Coppito 2, 67100, L'Aquila, Italy. biancamaria.zani@univaq.it.
authors.forename: Bianca Maria
authors.initials: BM
authors.lastname: Zani
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002070:Butadienes / Q000494:pharmacology; D063646:Carcinogenesis / Q000473:pathology*; D045744:Cell Line, Tumor; D020935:MAP Kinase Signaling System / Q000187:drug effects; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D009570:Nitriles / Q000494:pharmacology; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011836:Radiation Tolerance / Q000187:drug effects; D018233:Rhabdomyosarcoma, Embryonal / Q000201:enzymology* / Q000473:pathology*; D018874:Spheroids, Cellular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D023041:Xenograft Model Antitumor Assays; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D002070:Butadienes; D009570:Nitriles; D047428:Protein Kinase Inhibitors; C113580:U 0126; D048051:p38 Mitogen-Activated Protein Kinases