Stem Cell Markers in Neuroblastoma-An Emerging Role for LGR5

Abstract

The prognostic value of cancer stem cell markers in various cancer subtypes is a well documented research area. Our findings show that the stem cell marker Lgr5 is associated with an aggressive phenotype in neuroblastoma. Here, we discuss these findings within the context of recent studies in several cancers such as lung, colorectal and intestinal cancer, glioblastoma and ewing's sarcoma. Neuroblastoma continues to be an elusive disease, due to its heterogeneous presentation ranging from spontaneous regression to aggressive metastatic disease and intertwined genetic variability. Currently, the most significant prognostic marker of high risk disease and poor prognosis is amplification of the MYCN oncogene, which is found in approximately 25% of cases (Huang and Weiss, 2013). With this in mind, there is still much to learn about the driving mechanisms of this aggressive pediatric tumor. Neuroblastoma development is thought to be the result of aberrant differentiation of the cell of origin, embryonic neural crest cells which then migrate and invade during the developmental stage (Joshi et al., 2007). Aberrant cells are those which would, under normal conditions form the mature tissues of the sympathetic ganglia and adrenal medulla. Tumors are known to develop indiscriminately along the radius of the sympathetic ganglia, although it is well established that the adrenal glands are fundamentally the most common primary site (Jessen and Mirsky, 2005).

Keywords: LGR5; chemoresistance; neuroblastoma; relapse.

Figure 1

Quantification of Lgr5 mRNA expression using quantitative RT-PCR. (A) LGR5 mRNA expression in a panel of 11 neuroblastoma cell lines established at diagnosis or relapse (N = 3). Elevated expression of LGR5 was shown in BE2c, SKNAS and ^****SKNSH (p < 0.0001), ^**SKNAS (p < 0.01) and ^*BE2C (p < 0.05) (One-way ANOVA). Human foreskin fibroblasts were used as a control cell line. (B) LGR5 mRNA expression in p53 mutant and wild-type neuroblastoma cell lines (p = 0.487, Paired t-test). (C) LGR5 mRNA expression in MYCN amplified and non-amplified sub groups. Cell lines were divided into MYCN amplified (IMR32, BE2c, and LAN-1) and MYCN non-amplified (p = 0.339, Paired t-test).

Figure 2

Immunofluorescence staining of LGR5 in neuroblastoma cell lines. (A) Immunofluorescence staining of Lgr5 in BE2c cells (B) Overlay shows that it is primarily located in the cytoplasm of BE2c neuroblastoma cells (C) NB69 cells show an absence of Lgr5 staining when stained under the same conditions as the BE2c cell line, indicating low expression (D) Overlay top right shows the morphology of the cell, with the absence of stain by phase contrast microscopy.