Abstract
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK(high) 12.8%, ALK(low) 73%, P = 0.0035; cell death: ALK(high) 56.4%, ALK(low) 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs.
Highlights
Neuroblastoma (NBL) is a common and highly aggressive childhood tumor resulting from the malignant transformation of immature neural crest-derived cells [1, 2]
We surveyed a panel of human NBL cell lines to identify cell lines with different levels of endogenous Anaplastic lymphoma kinase (ALK) protein (Fig. 1A and Supplementary S3B)
Http://www.ncbi.nlm.nih.gov/projects/SNP/ http://www.mrc-holland.com In UKF-NB3 and IMR-32 cell lines, the total amount of ALK receptor (200 kDa) was comparable with the level of NPM-ALK (80 kDa) present in the anaplastic large cell lymphomas (ALCL)-derived cell line SU-DHL1 characterized by high level of expression (Fig. 1A)
Summary
Neuroblastoma (NBL) is a common and highly aggressive childhood tumor resulting from the malignant transformation of immature neural crest-derived cells [1, 2]. In a study aimed at comparing gene expression in human fetal neuroblast and NBL, ALK has been detected among the genes up-regulated in unfavorable NBLs compared with fetal neuroblasts, suggesting a possible oncogenic role for ALK [12]. In this study gene gain, amplification and point mutations were identified in germline and somatic mutations in familial and high-risk patients, respectively [13]. Increasing evidence point to ALK as a crucial oncogene associated with NBL [14,15,16,17]. These results open new avenues to future investigations aimed at developing possible therapeutic strategies based on the inhibition of ALK-mediated signaling
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