Differential Expression of Insulin-like Growth Factor 2 (IGF2) but not Insulin-like Growth Factor Receptors (IGF1R/IGF2R) in Neuroblastoma Cell Lines

Introduction

Neuroblastoma is the most common solid tumor in young children that arises from neural crest cells. Cellular heterogeneity is found in both primary neuroblastoma tumor and isolated neuroblastoma cell lines. NB tumor cells can be classified according to the phenotype into N-type (neuroblastic), S-type (substrate-adherent), and I-type (intermediate) (Ross et al. 1995, Ross et al. 2007). I-type NB cells were characterized as cancer stem cells, which keep the self-renewal ability and also the potential to differentiate along both neuron and glial pathways. Previous study by J. Walton showed that the I-type is the most malignant phenotype, which has much higher tumorigenicity than N- type NB cells, and S-type NB cells are not tumorigenic (Walton et al. 2004). Comparing among these three phenotypes can help our understanding the molecular mechanism of the malignancy of I-type cells as cancer stem cell, and the nature of the cellular heterogeneity of neuroblastoma.
Insulin-like growth factors (IGFs) play important roles in many tumors (Baserga, 1995; Satyamoorthy et al., 2001; Wu et al., 2002), including neuroblastoma (Sullivan et al., 1995; Zumkeller and Schwab, 1999; Misawa et al., 2000; van Golen and Feldman, 2000; Kim et al., 2004). IGF2 is an autocrine signal known to be involved in tumorigenesis directly (Werner and LeRoith, 1993). The effect of IGFs is mediated by insulin-like growth factors receptor 1 (IGF1R) that possesses tyrosine kinase activity. Ligand binding causes autophosphorylation of IGF1R which initiates a cascade of cellular signaling including Ras-MAP signaling pathway (Kim et al. 1997) and PI3K pathway (Czech, 1998; Pollak et al., 2004). It is shown that inhibition of IGF1R in neuroblastoma cells induces the regression of established tumor in mice (Liu et al., 1998). Furthermore, IGF1R overexpression causes less apoptosis and enhances neuroblastoma tumorigenesis (Singleton et al. 1996).
Previous studies suggest that IGF signaling pathway is differentially activated in neuroblastoma cell lines (Kim et al., 2004). In this study, I compared the expression of IGF2 ligand, IGF1R, and IGF2R in seven neuroblastoma cell lines with three different phenotypes. The result showed that the most malignant I-type cell lines express the highest level of IGF2 mRNA compared to N- and S-type cell lines, and IGF1R and IGF2R expression is phenotype-independent, suggesting a possible role of IGF signal pathway in the malignant property of I-type cancer stem cells.

Figures

Figure 1-IGF1R mRNA level in NB cell lines. (A). RT-PCR showing that IGF1R mRNA level expressed in all cell lines, and the mRNA levels are similar (B). Gel pictures are scanned. Density of each band is measured by Kodak gel imaging system. IGF1R mRNA level is normalized to GAPDH. Three independent experiments are done. Average and standard error are shown.

Figure 2-IGF2 mRNA level in NB cell lines. (A). RT-PCR showing that IGF2 mRNA level is high in both I-type cell line, intermediate in both N-type cell lines, and low in S-type cell lines, except for LA1-5s. (B). Gel pictures are scanned. Density of each band is measured by Kodak gel imaging system. IGF2 mRNA level is normalized to GAPDH. Three independent experiments are done. Average and standard error are shown.

Figure 3-IGF2R mRNA level in NB cell lines. (A). RT-PCR showing that IGF2R mRNA levels are similar in all cell lines (B). Gel pictures are scanned. Density of each band is measured by Kodak gel imaging system. IGF1R mRNA level is normalized to GAPDH. Three independent experiments are done. Average and standard error are shown.

Figure 4-IGF2 mRNA level in BE(2)-C cell line treated with BU. (A). BU treatment induces S-type like differentiation of I-type BE(2)-C cell and decreases IGF2 mRNA level. IGF2R mRNA level is not changed with BU treatment. (B). Differentiation induced by BU causes IGF2 mRNA level change is further supported by quantitative real-time PCR.

IGF signaling pathway plays key roles in cell cycle progression, cell proliferation and tumor progression. In the study three important genes: IGF2, IGF1R, and IGF2R in IGF signaling were examined in seven neuroblastoma cell lines with three different phenotypes. IGF1R and IGF2R were found expressed in all seven cell lines at the same level regardless the phenotype. Interestingly, IGF2 was found expressed at the highest level in the most malignant phenotype, I-type neuroblastoma cell lines, indicating a possible important role of this signaling pathway in the malignancy of these neuroblastoma cancer stem cells.

Full Paper

Acknowledgments

I would like to thank Dr. Robert Ross for providing neuroblastoma cell lines, and thank Leleesha Samaraweera and Bo Liu for the help in the lab work of this project. I also would like to thank Barbara Spengler for her valuable advices. I appreciate Dr. Berish Rubin for his guidance. Finally, I would like to thank my husband, Dan Han for he gives me so much help and encouragement during this year.

This document was last modified 05/12/2009.
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