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.
Highlights
Neuroblastoma (NB) is one of the commonest extracranial paediatric solid tumours, arising from neural crest progenitor cells of the sympathetic nervous system which have failed to undergo regulated differentiation and development as a result of aberrant gene expression programmes instigated by critical oncoproteins [1, 2]
The increased expression of LGR5 in poorly differentiated and high stage primary NBs and NB cell lines suggests that certain subsets of NBs are competent for Wnt signalling dependent on Wnt and R-Spondin ligands
LGR5 has been shown to be highly expressed in a number of cancers, including colorectal cancer [34], breast cancer [20], cervical cancer [35] and glioblastoma [36], where it is associated with positive modulation of Wnt signalling in cancer stem cells
Summary
Neuroblastoma (NB) is one of the commonest extracranial paediatric solid tumours, arising from neural crest progenitor cells of the sympathetic nervous system which have failed to undergo regulated differentiation and development as a result of aberrant gene expression programmes instigated by critical oncoproteins [1, 2]. The oncogenic p.F1174L ALK mutant has been shown to potentiate the tumorigenic effect of MYCN in a mouse model, leading to higher penetrance, earlier onset and increased lethality [7]. This pronounced effect on tumorigenicity was accompanied by dramatic activation of the mitogenactivated protein kinase (MAPK) and phosphoinositide 3-kinase/Akt (PI3K/Akt) pathways. Both PI3K/Akt and MAPK signalling pathways are frequently deregulated in cancer, and represent targets for therapeutic intervention [8, 9]. Two very recent studies have demonstrated that mutations in this pathway are more frequent in relapsing NB [13, 14]
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