title: Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Translational Oncopathology, IRCSS A.O.U. San Martino-IST, National Cancer Research Institute, L.go R. Benzi 10, 16132 Genoa, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Del Grosso
authors.forename: Marilena
authors.initials: M
authors.lastname: De Mariano
authors.forename: Lorena
authors.initials: L
authors.lastname: Passoni
authors.forename: Roberto
authors.initials: R
authors.lastname: Luksch
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Tonini
authors.forename: Luca
authors.initials: L
authors.lastname: Longo
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D014415:Tunicamycin; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases