Quantitative Phosphoproteomic Analysis Identifies Activation of the RET and IGF-1R/IR Signaling Pathways in Neuroblastoma

Abstract

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.

Genomic Copy Number Profiling Using Circulating Free Tumor DNA Highlights Heterogeneity in Neuroblastoma

“…Indeed, IGF1R signaling, which plays an important role in cancer cell proliferation, has been shown to play a role in the neuroblastoma malignant phenotype (41)(42)(43)(44). Furthermore, in 2 cases, an alteration targeting TERT, shown recently to characterize aggressive high-risk neuroblastoma (45,46), was identified in the cfDNA, with one case harboring this alteration also in the metastatic tumor cells of the bone marrow but not in the primary neuroblastoma.…”

Posttranslational modifications of neuroligins regulate neuronal and glial signaling

“…Consistent with this model, NLGN2/gephyrin interactions at GABAergic post-synaptic sites are increased and a concomitant enhancement of synaptic recruitment of γ2 subunit-containing GABA A Rs are observed in Pin1 knockout animals. Furthermore, a serine analogous to NLGN2 S714 in NLGN3 can be phosphorylated in human, mouse, and rat [25,27,30]. Serine phosphorylation at the consensus S-P motif in both NLGN2 and 3 is consistent with proline directed kinases playing an important regulatory role for multiple NLGN isoforms.…”

Single-cell RNA-sequencing of peripheral neuroblastic tumors reveals an aggressive transitional cell state at the junction of an adrenergic-mesenchymal transdifferentiation trajectory

“…From our cellcell interaction modelling we had also predicted that IGF1 ligands released specifically by noninflammatory macrophages could modulate the transitional gene target MYCN by binding to IGF1R receptors on neuroblasts. MYCN is a core pro-tumorigenic transcription factor in malignant neuroblasts and is strongly associated with poor patient prognosis, the upstream IGF1R receptor is highly expressed in neuroblastoma and is synonymous with a metastatic phenotype, suggesting an increased metastatic potential in neuroblasts after IGF1R mediated interactions with non-inflammatory macrophages [54][55][56][57][58] . This metastasis promoting interaction is further supported as tumor-associated macrophages are known to be more prevalent in neuroblastoma patients with metastatic disease compared with localized lesions 13 .…”