LGR5 is Expressed by Ewing Sarcoma and Potentiates Wnt/β-Catenin Signaling

Abstract

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of putative stem cell origin that predominantly occurs in children and young adults. Although most patients with localized ES can be cured with intensive therapy, the clinical course is variable and up to one third of patients relapse following initial remission. Unfortunately, little is yet known about the biologic features that distinguish low-risk from high-risk disease or the mechanisms of ES disease progression. Recent reports have suggested that putative cancer stem cells exist in ES and may contribute to an aggressive phenotype. The cell surface receptor leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a somatic stem cell marker that functions as an oncogene in several human cancers, most notably colorectal carcinoma. LGR5 is a receptor for the R-spondin (RSPO) family of ligands and RSPO-mediated activation of LGR5 potentiates Wnt/β-catenin signaling, contributing to stem cell proliferation and self-renewal. Given its presumed stem cell origin, we investigated whether LGR5 contributes to ES pathogenesis. We found that LGR5 is expressed by ES and that its expression is relatively increased in cells and tumors that display a more aggressive phenotype. In particular, LGR5 expression was increased in putative cancer stem cells. We also found that neural crest-derived stem cells express LGR5, raising the possibility that expression of LGR5 may be a feature of ES cells of origin. LGR5-high ES cells showed nuclear localization of β-catenin and robust activation of TCF reporter activity when exposed to Wnt ligand and this was potentiated by RSPO. However, modulation of LGR5 or exposure to RSPO had no impact on proliferation confirming that Wnt/β-catenin signaling in ES cells does not recapitulate signaling in epithelial cells. Together these studies show that the RSPO-LGR5-Wnt-β-catenin axis is present and active in ES and may contribute to tumor pathogenesis.

Keywords: Ewing sarcoma; LGR5; R-spondin; Wnt; stem cell; β-catenin.

Figure 1

Leucine-rich repeat-containing G-protein coupled receptor 5 is expressed by Ewing sarcomas. qRT-PCR analysis revealed low to very high-level expression of LGR5 in (A) 49 primary Ewing sarcoma (ES) and (B) 15 ES cell lines. For primary tumors levels of expression relative to GAPDH were <0.001% in 1 tumor, 0.001–0.01% in 7 tumors, 0.01–0.1% in 17 tumors, 0.1–1% in 12 tumors, 1–10% in 9 tumors, and 10–100% in 3 tumors.

Figure 2

Leucine-rich repeat-containing G-protein coupled receptor 5 is expressed by neural crest stem cells (NCSC). (A) qRT-PCR analysis of human embryonic stem cells (hESC), hESC-derived NCSC (hNCSC), hNCSC-derived mesenchymal stem cells (hNC-MSC), bone marrow-derived mesenchymal stem cells (hBM-MSC), and human lung embryo fibroblasts (MRC5) showed that undifferentiated hNCSC express the highest levels of LGR5. (B) Murine bone marrow stromal cells of neural crest origin express higher levels of Lgr5 than cells of mesodermal origin (from publically available microarray data GEO accession GSE30419) (Wislet-Gendebien et al., 2012). N = 3 ± SEM. Lines represent arithmetic means of replicate samples.

Figure 3

Leucine-rich repeat-containing G-protein coupled receptor 5 is increased in aggressive disease and putative cancer stem cells. (A) Metastatic tumor-derived CHLA10 cells express higher levels of LGR5 than CHLA9 cells, which were derived from the primary tumor at diagnosis. (B) LGR5 expression was found by microarray analysis to be increased in tumors from 4 patients with rapidly progressive and fatal primary ES (DOD-dead of disease) compared to 10 patients with at least 48 months disease free survival (LTS-long term survivors). (C) LGR5 levels are higher in CD133⁺ compared to CD133⁻ STA-ET-8.2 cells. Data from two independent sorting experiments are shown and expression on the y-axis is of CD133⁺ cells relative to the corresponding CD133⁻ cells. (D) LGR5 expression is increased in ALDH^high compared to ALDH^low MHH-ES and TC71 cells. Data from three independent sorts are shown and expression on the y-axis is that of ALDH^high relative to the corresponding ALDH^low cells. The horizontal line in (B–D) all represent arithmetic mean values.

Figure 4

No correlation exists between LGR5 expression and Wnt/β-catenin activity in ES cells in standard culture. (A) Canonical Wnt/β-catenin target gene expression was measured by qRT-PCR in ES cell lines. There was no apparent correlation between LGR5 expression and Wnt/β-catenin target genes AXIN2 and CD44. (B) No consistent or significant change occurs in the expression of a panel of Wnt/β-catenin target genes following LGR5 knockdown. The dotted line represents a ratio of 1 between shLGR5#1 and shNS cells. (C) LGR5 knockdown does not measurably decrease Wnt/β-catenin transcriptional activity. Luciferase activity was determined using TCF reporter-transduced cell lines as described in Section “Material and Methods.” All data shown is from three independent experiments and error bars are SEM.

Figure 5

Exogenous Wnt3a induces nuclear localization of β-catenin and is potentiated by RSPO2. (A) CHLA25 cells grown in the presence of Wnt3a conditioned medium (CM) induced nuclear localization of β-catenin and this was robustly potentiated by the addition of RSPO2. RSPO2 alone had no effect on β-catenin nuclear localization. (B) The percentage of CHLA25, A673, and TC71 cells with nuclear β-catenin all increased with Wnt3a CM, but CHLA25 showed the greatest increase in nuclear localization with the addition of RSPO2. Data from three independent experiments and error bars are SEM.

Figure 6

RSPO2 potentiates Wnt/β-catenin signaling in LGR5-high ES cells. (A) Exposure of ES cell lines to Wnt3a conditioned medium (CM) increased AXIN2 expression, but expression was only potentiated by RSPO2 in the LGR5-high cell line, CHLA25. (B) TCF reporter activity was induced in ES cells exposed to Wnt3a CM, but was only potentiated in CHLA25 cells by the addition of RSPO2. (C) TCF reporter activity was measured in control and LGR5 knockdown CHLA25 cells following exposure to Wnt3a with/without RSPO2. Reporter activity in the presence of RSPO2 was significantly diminished in LGR5 knockdown cells. All data shown is from three independent experiments and error bars are SEM.

Figure 7

Leucine-rich repeat-containing G-protein coupled receptor 5 does not promote ES cell proliferation. (A) qRT-PCR confirmation of LGR5 knockdown in ES cell lines transduced with LGR5-targeted shRNAs (shNS = non-silencing control). (B) Confirmation of LGR5 over-expression in A673 cells transduced with LGR5 over-expression construct (Empty, empty vector control). (C) ES cell expansion was measured by MTS assay and was not affected by LGR5 knockdown. (D) LGR5 over-expression did affect cell growth in A673 cells. (E) Parental cell growth was not affected by the addition of WNT3A and/or RSPO2 to standard culture conditions. All data shown is from two independent experiments and error bars are SEM.