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

Federica Del Grosso1, Marilena De Mariano12, Lorena Passoni35, Roberto Luksch3, Gian Paolo Tonini1* and Luca Longo14

Author Affiliations

1 Translational Oncopathology, IRCSS A.O.U. San Martino-IST, National Cancer Research Institute, L.go R. Benzi 10, 16132 Genoa, Italy

2 Department of Experimental Medicine, University of Genoa, Via L. Battista Alberti 2, 16132 Genoa, Italy

3 Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133 Milano, Italy

4 Italian Neuroblastoma Foundation, L.go Gaslini 5, 16147 Genoa, Italy

5 Department of Medical Pharmacology, University of Milano, Via Vanvitelli 32, 20129 Milano, Italy

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BMC Cancer 2011, 11:525  doi:10.1186/1471-2407-11-525

Published: 22 December 2011



The 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.


We 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).


Tunicamycin 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.


In 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.