LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma - PubMed (original) (raw)

. 2015 Nov 24;6(37):40053-67.

doi: 10.18632/oncotarget.5548.

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

Affiliations

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

Gabriella Cunha Vieira et al. Oncotarget. 2015.

Erratum in

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.

Keywords: LGR5; MEK/ERK; Wnt/β-catenin; cell survival; neuroblastoma.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1

Figure 1. Expression of LGR5 in neuroblastoma

A. Kaplan-Meier survival curves derived from 2 independent microarray analyses of neuroblastomas showing the association of high LGR5 mRNA expression with poor prognosis. Datasets used are from reference [27] (left) and [26] (right), obtained using the R2 Genomics Analysis and Visualization Platform (

http://r2.amc.nl

). B. Immunoblotting showing elevated expression of LGR5 in primary NBs. Fetal Adrenal protein is shown as a normal control(FA). C. Immunohistochemistry demonstrating prominent cytoplasmic staining in poorly differentiated NBs (top) and little or no staining in differentiating NBs (bottom). A section of normal colon where interspersed LGR5 positive cells are located at the bottom of crypts is shown as a control. D. Immunoblotting showing elevated expression of LGR5 in cell-lines. The colorectal cancer cell line LOVO is shown for comparison.

Figure 2

Figure 2. Wnt3a and R-spondin responsiveness of neuroblastoma cell lines

A. TOPFLASH reporter assay for SK-N-BE(2)-C and SH-SY5Y showing increased luciferase activity upon co-stimulation with Wnt3a and Rspos 1–3 ( p < 5.0e-04 and p < 5.0e-03 respectively). B. TOPFLASH reporter assay combined with control siRNA, LGR5 or β-catenin targeting siRNAs, demonstrating requirement for LGR5 for reporter activity ( p < 0.05). Reporter assays were conducted at least twice in triplicate. PBS: Phosphate-buffered saline, WR2: Wnt3a/Rspo2 treatment. C. Confocal microscopy of β-catenin nuclear translocation in SK-N-BE(2)-C, SH-SY5Y and SK-N-AS cells on addition of Wnt3a/Rspo2. Green fluorescent staining shows β-catenin and cells were counterstained with DAPI (blue) for nuclei control D. Flow cytometry analysis of NB lines treated with Wnt3a/Rspo2 and labelled with Ki67 to measure proliferation. Note the shift and increased median fluorescence intensity (MFI) accompanying treatment. Note that x-axes are in log scale.

Figure 3

Figure 3. LGR5 knockdown-induced cell death is independent of Wnt/β-catenin signalling pathway

A. SK-N-BE(2)-C, SK-N-AS and SH-SY5Y cells were transfected with siRNAs targeting LGR5 or β-catenin. LGR5 knockdown led to dramatic cell death in all three cell lines tested ( p < 0.05), but β-catenin depletion did not. B. Immunoblots confirming knockdowns and the absence of dephosphorylated (active) β-catenin. C: cytoplasmic, N: nuclear.

Figure 4

Figure 4. Depletion of LGR5 induces apoptosis in neuroblastoma cells

Knockdown of LGR5 with independent siRNAs induces apoptosis shown by cell-counts, increased cleaved PARP (cPARP), and rescue by the caspase 3 inhibitor QVD A. SK-N-AS cells and B. SH-SY5Y cells. Asterisks denote p < 0.05, and assays are representative of at least 3 biological replicates. Membrane asymmetry assessment of apoptosis in C. SK-N-AS cells and D. SH-SY5Y cells. EA, early apoptosis; LA, late apoptosis.

Figure 5

Figure 5. LGR5 regulates MEK/ERK and Akt signalling

Immunoblotting demonstrating A. decreases in p-ERK1/2 (T202/Y204) and p-MEK1/2 (S217/221), B. Elevated Bim-EL and C. altered Akt phosphorylation and Rictor levels accompanying LGR5 knockdown.

Figure 6

Figure 6. LGR5 is required for G1 to S phase transition

A. Increased sub-G0/G1 cells following LGR5 knockdown of SK-N-AS and SH-SY5Y cells demonstrated by flow cytometry. Asterisks denote p < 0.05. B. Cell cycle analysis of LGR5 knockdowns. After 48 hrs of transfection, G1 arrest of cells is apparent through comparison of G1-S phase ratios. Asterisks denote p < 0.05. C. Immunoblotting analysis of cell cycle regulatory proteins confirming G1 arrest accompanying LGR5 knockdown.D. Immunoblot demonstrating cell cycle dependent changes in LGR5 expression in SH-SY5Y cells. E. Quantification of LGR5 protein levels (relative to β-actin) plotted against percentage of cells in G1- or S-phase.

Figure 7

Figure 7. A model showing regulatory modalities for LGR5 in neuroblastoma

Pro-survival signalling of MEK/ERK activities which are R-Spondin independent are shown on the left, and may be inhibited by MEK/ERK inhibitors such as Trametinib. Proliferative Wnt signalling in the presence of Wnt/Rspos is shown on the right. FZD: Frizzled receptors; LRP: Low Density Lipoprotein Receptor-Related Proteins, β-cat: β-catenin, TCF: T-cell factor/lymphoid enhancer factor.

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