N-Cadherin in Neuroblastoma Disease: Expression and Clinical Significance

One of the first and most important steps in the metastatic cascade is the loss of cell-cell and cell-matrix interactions. N-cadherin, a crucial mediator of homotypic and heterotypic cell-cell interactions, might play a central role in the metastasis of neuroblastoma (NB), a solid tumor of neuroectodermal origin. Using Reverse Transcription Quantitative PCR (RT-qPCR), Western blot, immunocytochemistry and Tissue MicroArrays (TMA) we demonstrate the expression of N-cadherin in neuroblastoma tumors and cell lines. All neuroblastic tumors (n = 356) and cell lines (n = 10) expressed various levels of the adhesion protein. The N-cadherin mRNA expression was significantly lower in tumor samples from patients suffering metastatic disease. Treatment of NB cell lines with the N-cadherin blocking peptide ADH-1 (Exherin, Adherex Technologies Inc.), strongly inhibited tumor cell proliferation in vitro by inducing apoptosis. Our results suggest that N-cadherin signaling may play a role in neuroblastoma disease, marking involvement of metastasis and determining neuroblastoma cell viability

N-cadherin protein levels correlate with metastatic disease.

<p>Visualized is the distribution of samples (percentage) staining weak, moderate and strong in each class (metastasized N = 34, or non-metastasized tumors N = 39). The number of samples in each class was set to 100 percent.</p

N-cadherin mRNA levels negatively correlate to metastasis and risk definition.

<p>A. N-cadherin mRNA expression and the presence/absence of metastasis at diagnosis. B. N-cadherin mRNA expression according to risk group (high risk vs. others). The median (horizontal line), the interquartile range (box), and the upper and lower range of the data (whiskers) are shown.</p

Immunohistochemical expression of N-cadherin on TMA.

<p>Immunohistochemical staining was performed and results scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031206#s2" target="_blank">Materials and Methods</a>. Images are representative for strong (a), moderate (b) to weak level (c) or, in one case, negative staining (d). Note: All four tumors are stained simultaneously on the same TMA slide facilitating a comparison of staining intensities. Images were taken with an ×60 dry lens and are presented at identical magnification.</p

Summary of patient characteristics.

<p>*Remark that not for all patients included in the TMA cohort complete information was available.</p

ADH-1 induces cell death in vitro.

<p>ADH-1 (1 mg/ml) was added to exponentially growing cultures of epithelial cells, fibroblasts and the indicated NB cell lines. Cell survival was measured at 12, 24 and 48 h after addition of ADH-1, using the CellTiter-Glo luminescent Cell Viability assay (Promega). Data were normalized to no drug treatment. Data are mean ± standard deviation (n = 3).</p