Abstract 5000: The heterogeneous expression and functional relationship of ALK and NLRR1 in neuroblastoma
Authors: Satoh Shunpei , Takatori Atsushi , Ogura Atsushi , Ohira Miki , Hossain Shamim Md. , Koh Katsuyoshi , Kishimoto Hiroshi , Hanada Ryoji , Nakagawara Akira 
Published: August 2015

Abstract
Introduction: Neuroblastoma (NB) is one of the most common extracranial solid tumors in childhood, which arises from neural crest cells in sympathoadrenal lineage. Although recent studies have revealed that anaplastic lymphoma kinase (ALK) signal is aberrantly activated in high-risk group of NB due to nonsynonymous mutations, gene amplification and mutation-independent overexpression, the regulatory mechanism of the signal remains elusive. Recently, we have identified neuronal leucine rich repeat 1 (NLRR1), a type I transmembrane protein, as a poor prognostic marker in NB and, notably, it accelerates epidermal growth factor receptor (EGFR) signal to enhance cell proliferation. Here we have revealed that NLRR1 functionally affects ALK signal and vice versa in NB.
Material & Methods: Cell growth of NB cell lines was evaluated by WST-8 assay or living cell imaging system. Immunoprecipitation was performed to check protein-protein interactions and concentrate secreted proteins. Further binding studies were performed by utilizing alkaline phosphatase-tagged and Fc-tagged proteins. Quantification of secreted proteins was assessed by sandwich ELISA assay. For ALK and EGFR signal inhibition, NB cells were treated with crizotinib, alectinib and erlotinib. The expression levels of ALK and NLRR1 were evaluated in embryonic Nlrr1 knockout mice as well as in the NB clinical samples by immunohistochemistry.
Results: In the binding assays including immunoprecipitation, we found that NLRR1 extracellular domain physically interacted with the full-length ALK. Unexpectedly, NLRR1 expression suppressed ALK phosphorylation and cell proliferation in ALK-positive NB cells. Similar inhibitory effect was also observed by the treatment of the conditioned medium from the culture of NLRR1-expressing cells, suggesting a soluble form of NLRR1 protein. Certainly, the additional investigation confirmed that NLRR1 was cleaved to generate a soluble fragment of the extracellular domain. Conversely ALK inhibitors (ALKi) treatment resulted in up-regulation of NLRR1 and EGFR. Furthermore, EGF treatment restored ALKi-induced cell growth suppression. Additional erlotinib treatment certainly competed with the EGF rescue effect against ALKi. Finally, immunohistochemical analysis revealed that NLRR1 and ALK showed mutually exclusive expression pattern in NB tissues and murine embryonic dorsal root ganglia.
Conclusion: We have identified the bilateral regulatory system between ALK and NLRR1, in which NLRR1 suppresses ALK phosphorylation whereas the inhibition of ALK results in the elevated expression of NLRR1 and EGFR. These findings in part support their mutually exclusive expression pattern in developing neural tissues and human NB. Further study should clarify the pathological meaning of the heterogeneity between ALK and NLRR1/EGFR pathways to develop a novel dual inhibition therapy in NB.
Citation Format: Shunpei Satoh, Atsushi Takatori, Atsushi Ogura, Miki Ohira, Shamim Md. Hossain, Katsuyoshi Koh, Hiroshi Kishimoto, Ryoji Hanada, Akira Nakagawara. The heterogeneous expression and functional relationship of ALK and NLRR1 in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5000. doi:10.1158/1538-7445.AM2015-5000