title: Inhibition of the Notch-Hey1 axis blocks embryonal rhabdomyosarcoma tumorigenesis.
abstract: OBJECTIVE
  Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and remains refractory to combined-modality therapy in patients with high risk disease. In skeletal myogenesis, Notch signaling prevents muscle differentiation and promotes proliferation of satellite cell progeny. Given its physiologic role in myogenesis and oncogenic role in other human cancers, we hypothesized that aberrant Notch signaling may contribute to RMS tumorigenesis and present novel therapeutic opportunities.
  
  
  METHODS
  Human RMS cell lines and tumors were evaluated by immunoblot, IHC, and RT-PCR to measure Notch ligand, receptor, and target gene expression. Manipulation of Notch signaling was accomplished using genetic and pharmacologic approaches. In vitro cell growth, proliferation, and differentiation were assessed using colorimetric MTT and BrdU assays, and biochemical/morphologic changes after incubation in differentiation-promoting media, respectively. In vivo tumorigenesis was assessed using xenograft formation in SCID/beige mice.
  
  
  RESULTS
  Notch signaling is upregulated in human RMS cell lines and tumors compared with primary skeletal muscle, especially in the embryonal (eRMS) subtype. Inhibition of Notch signaling using Notch1 RNAi or γ-secretase inhibitors reduced eRMS cell proliferation in vitro. Hey1 RNAi phenocopied Notch1 loss and permitted modest myogenic differentiation, while overexpression of an activated Notch moiety, ICN1, promoted eRMS cell proliferation and rescued pharmacologic inhibition. Finally, Notch inhibition using RNAi or γ-secretase inhibitors blocked tumorigenesis in vivo.
  
  
  CONCLUSIONS
  Aberrant Notch-Hey1 signaling contributes to eRMS by impeding differentiation and promoting proliferation. The efficacy of Notch pathway inhibition in vivo supports the development of Notch-Hey1 axis inhibitors in the treatment of eRMS.
authors.affiliation: Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Belyea
authors.forename: Sarasija
authors.initials: S
authors.lastname: Naini
authors.forename: Rex C
authors.initials: RC
authors.lastname: Bentley
authors.forename: Corinne M
authors.initials: CM
authors.lastname: Linardic
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D002219:Carbamates / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D024510:Muscle Development / Q000187:drug effects / Q000502:physiology; D009132:Muscles / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D018233:Rhabdomyosarcoma, Embryonal / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D023041:Xenograft Model Antitumor Assays
chemical_list: D053829:Amyloid Precursor Protein Secretases; D051792:Basic Helix-Loop-Helix Proteins; D002219:Carbamates; D018797:Cell Cycle Proteins; D004151:Dipeptides; D034741:RNA, Small Interfering; D051880:Receptors, Notch; C485631:HEY1 protein, human; C410009:L 685458; C523463:benzyloxycarbonyl-isoleucyl-leucinal