title: LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma.
abstract: LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Gabriella Cunha
authors.initials: GC
authors.lastname: Vieira
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: S
authors.initials: S
authors.lastname: Chockalingam
authors.affiliation: Department of Cellular Pathology, Southmead Hospital, Bristol, UK.
authors.forename: Zsombor
authors.initials: Z
authors.lastname: Melegh
authors.affiliation: Colorectal Cancer Laboratory, School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Greenhough
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Sally
authors.initials: S
authors.lastname: Malik
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Marianna
authors.initials: M
authors.lastname: Szemes
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Ji Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Abderrahmane
authors.initials: A
authors.lastname: Kaidi
authors.affiliation: The Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: The Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Catchpoole
authors.affiliation: Colorectal Cancer Laboratory, School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Rhys
authors.initials: R
authors.lastname: Morgan
authors.affiliation: Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, UK.
authors.forename: David O
authors.initials: DO
authors.lastname: Bates
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Peter David
authors.initials: PD
authors.lastname: Gabb
authors.affiliation: Cancer Epigenetics Laboratory and School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK.
authors.forename: Karim
authors.initials: K
authors.lastname: Malik
mesh_terms: D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D007231:Infant, Newborn; D018613:Microscopy, Confocal; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D060449:Wnt Signaling Pathway / Q000235:genetics*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: C116884:LGR5 protein, human; D043562:Receptors, G-Protein-Coupled; D051176:beta Catenin; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases
keywords: LGR5; MEK/ERK; Wnt/β-catenin; cell survival; neuroblastoma