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

// Gabriella Cunha Vieira 1, * , S. Chockalingam 1, * , Zsombor Melegh 2 , Alexander Greenhough 3 , Sally Malik 1 , Marianna Szemes 1 , Ji Hyun Park 1 , Abderrahmane Kaidi 1 , Li Zhou 4 , Daniel Catchpoole 4 , Rhys Morgan 3 , David O. Bates 5 , Peter David Gabb 1 , Karim Malik 1, * 1 Cancer Epigenetics Laboratory and, School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK 2 Department of Cellular Pathology, Southmead Hospital, Bristol, UK 3 Colorectal Cancer Laboratory, School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK 4 The Kids Research Institute, The Children’s Hospital at Westmead, Westmead, New South Wales, Australia 5 Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, UK * These authors have contributed equally to this work Correspondence to: Karim Malik, e-mail: k.t.a.malik@bristol.ac.uk Keywords: neuroblastoma, LGR5, Wnt/β-catenin, MEK/ERK, cell survival Received: July 17, 2015 Accepted: October 19, 2015 Published: October 23, 2015 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.