Flotillin-1 Regulates Oncogenic Signaling in Neuroblastoma Cells by Regulating ALK Membrane Association

Arata Tomiyama,Kentaro Mori,Junko Takita,Miki Ohira,Akira Nakagawara,Kazuki Sasaki,Ryuichi Sakai,Hideki Yamaguchi,Takamasa Uekita,Chifumi Kitanaka,Reiko Kamata

doi:10.1158/0008-5472.can-14-0241

Abstract

Neuroblastomas harbor mutations in the nonreceptor anaplastic lymphoma kinase (ALK) in 8% to 9% of cases where they serve as oncogenic drivers. Strategies to reduce ALK activity offer clinical interest based on initial findings with ALK kinase inhibitors. In this study, we characterized phosphotyrosine-containing proteins associated with ALK to gain mechanistic insights in this setting. Flotillin-1 (FLOT1), a plasma membrane protein involved in endocytosis, was identified as a binding partner of ALK. RNAi-mediated attenuation of FLOT1 expression in neuroblastoma cells caused ALK dissociation from endosomes along with membrane accumulation of ALK, thereby triggering activation of ALK and downstream effector signals. These features enhanced the malignant properties of neuroblastoma cells in vitro and in vivo. Conversely, oncogenic ALK mutants showed less binding affinity to FLOT1 than wild-type ALK. Clinically, lower expression levels of FLOT1 were documented in highly malignant subgroups of human neuroblastoma specimens. Taken together, our findings suggest that attenuation of FLOT1-ALK binding drives malignant phenotypes of neuroblastoma by activating ALK signaling.

Highlights

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is rather expressed in the nervous system during development in mice [1]
To identify the phosphotyrosine-containing proteins associated with ALK, we performed two-step affinity purification using TNB-1 neuroblastoma cells, which stably expresses the ALK protein tagged with FLAG at the C-terminus as described in Supplementary Fig. S1
We analyzed the expression of FLOT1 and ALK proteins in specimens from 45 clinical neuroblastoma cases, which belong to three clinical malignancy grades as classified by Brodeur's classification [35, 36], and demonstrated that the levels of FLOT1 expression inversely correlate with clinical malignancy grade (Fig. 1C)

Summary

Introduction

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is rather expressed in the nervous system during development in mice [1]. ALK was first identified in anaplastic large cell lymphoma as the fusion protein NPM-ALK caused by chromosomal translocation [2]. Genetic alterations of ALK have been identified in cell lines and clinical samples of neuroblastoma, which consist of gene amplifications, activating mutations, or N-terminus truncations [7,8,9,10,11,12]. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Authors' Affiliations: 1Division of Metastasis and Invasion Signaling, National Cancer Center Research Institute; 2Department of Cell Therapy and Transplantation Medicine, Graduate School of medicine, The University of Tokyo, Tokyo; 3Department of Neurosurgery, National Defense Medical College, Saitama; 4Department of Applied Chemistry, National Defense Academy, Kanagawa; 5Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center Research Institute, Osaka; 6Divisions of Cancer Genomics and 7Biochemistry and Innovative Cancer, Chiba Cancer Center Research Institute, Chiba; and 8Department of Molecular Cancer Science, Yamagata University School of Medicine, Yamagata, Japan

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Results

Conclusion