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

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BMC Cancer

, 11:525

Cell and molecular biology


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 t2;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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2407-11-525 contains supplementary material, which is available to authorized users.

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Autor: Federica Del Grosso - Marilena De Mariano - Lorena Passoni - Roberto Luksch - Gian Paolo Tonini - Luca Longo


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