Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines

BMC Cancer

Research Article

Roberto Luksch¹  Lorena Passoni²  Gian Paolo Tonini³  Federica Del Grosso³  Marilena De Mariano⁴  Luca Longo⁵ 

[1] Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133, Milano, Italy;Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133, Milano, Italy;Department of Medical Pharmacology, University of Milano, Via Vanvitelli 32, 20129, Milano, Italy;Translational Oncopathology, IRCSS A.O.U. San Martino-IST, National Cancer Research Institute, L.go R. Benzi 10, 16132, Genoa, Italy;Translational Oncopathology, IRCSS A.O.U. San Martino-IST, National Cancer Research Institute, L.go R. Benzi 10, 16132, Genoa, Italy;Department of Experimental Medicine, University of Genoa, Via L. Battista Alberti 2, 16132, Genoa, Italy;Translational Oncopathology, IRCSS A.O.U. San Martino-IST, National Cancer Research Institute, L.go R. Benzi 10, 16132, Genoa, Italy;Italian Neuroblastoma Foundation, L.go Gaslini 5, 16147, Genoa, Italy;

关键词: Anaplastic Lymphoma Kinase;    Tunicamycin;    Anaplastic Large Cell Lymphoma;    Anaplastic Lymphoma Kinase Inhibitor;    Tunicamycin Treatment;   

DOI  :  10.1186/1471-2407-11-525

 received in 2011-06-21, accepted in 2011-12-22,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundThe Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression.MethodsWe employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK).ResultsTunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3) were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death.ConclusionsIn this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.

【 授权许可】

Unknown   
© Grosso et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.