LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma
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Gabriella Cunha Vieira1,*, S. Chockalingam1,*, Zsombor Melegh2, Alexander Greenhough3, Sally Malik1, Marianna Szemes1, Ji Hyun Park1, Abderrahmane Kaidi1, Li Zhou4, Daniel Catchpoole4, Rhys Morgan3, David O. Bates5, Peter David Gabb1, Karim Malik1,*
1Cancer Epigenetics Laboratory and, School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK
2Department of Cellular Pathology, Southmead Hospital, Bristol, UK
3Colorectal Cancer Laboratory, School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK
4The Kids Research Institute, The Children’s Hospital at Westmead, Westmead, New South Wales, Australia
5Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, UK
*These authors have contributed equally to this work
Karim Malik, e-mail: firstname.lastname@example.org
Keywords: neuroblastoma, LGR5, Wnt/β-catenin, MEK/ERK, cell survival
Received: July 17, 2015 Accepted: October 19, 2015 Published: October 23, 2015
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.
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