Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma.

Shunpei Satoh,Miki Ohira,Tomoaki Taguchi,Yoshiaki Kinoshita,Md Shamim Hossain,Akira Nakagawara,Atsushi Takatori,Kenichi Kohashi,Ryota Souzaki,Yohko Nakamura,Atsushi Ogura

doi:10.1038/srep32682

Abstract

In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1−/− mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB.

Highlights

To reveal the expressional correlation at the mRNA level, we performed qPCR analysis using 87 human NB cDNA samples including all International Neuroblastoma Staging System (INSS) stages with MYCN-amplified and non-amplified tumours
The homogenous properties of cultured cell lines might result in the opposite expression patterns of Neuronal leucine-rich repeat 1 (NLRR1) and ALK at the mRNA level, which were not confirmed in clinical samples, they exhibited no statistical significance
We provide the first evidence that NLRR1 and ALK exhibit mutually exclusive expression patterns in NB tissues and murine DRGs, and that NLRR1 binds to ALK through its extracellular region to impair ALK functions in cell proliferation

Summary

Introduction

Our recent studies have revealed that NLRR1 is a direct transcriptional target of MYCN, and NLRR1 potentiates epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor (IGFR) signals, which vice versa initiates MYCN transcription[35,36]. It is still unclear how NLRR1 influences other RTKs in NB. We examined the functional relationship between NLRR1 and ALK, and found that NLRR1 suppressed ALK signalling through a direct physical interaction Immunohistochemistry of both human primary NBs and mouse dorsal root ganglia (DRGs) revealed a mutually exclusive expression pattern of NLRR1 and ALK in vivo, which suggested an inhibitory effect of NLRR1 on ALK

Methods

Results

Conclusion