LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma

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...

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Published in:Oncotarget 2015-11, Vol.6 (37), p.40053-40067
Main Authors: Vieira, Gabriella Cunha, Chockalingam, S, Melegh, Zsombor, Greenhough, Alexander, Malik, Sally, Szemes, Marianna, Park, Ji Hyun, Kaidi, Abderrahmane, Zhou, Li, Catchpoole, Daniel, Morgan, Rhys, Bates, David O, Gabb, Peter David, Malik, Karim
Format: Article
Language:English
Subjects:
Apoptosis - genetics
beta Catenin - genetics
beta Catenin - metabolism
Cell Line, Tumor
Cell Proliferation - genetics
Cell Survival - genetics
Child
Child, Preschool
Extracellular Signal-Regulated MAP Kinases - metabolism
Gene Expression Regulation, Neoplastic
Humans
Immunoblotting
Infant
Infant, Newborn
Microscopy, Confocal
Mitogen-Activated Protein Kinase Kinases - metabolism
Neuroblastoma - genetics
Neuroblastoma - metabolism
Neuroblastoma - pathology
Receptors, G-Protein-Coupled - genetics
Receptors, G-Protein-Coupled - metabolism
Research Paper
Reverse Transcriptase Polymerase Chain Reaction
RNA Interference
Wnt Signaling Pathway - genetics
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Summary: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.
ISSN:1949-2553
1949-2553
DOI:10.18632/oncotarget.5548