Transmembrane adaptor protein PAG1 is a novel tumor suppressor in neuroblastoma

Abstract

(NB) is the most common extracranial pediatric solid tumor with high mortality rates. The tyrosine kinase c-Src has been known to play an important role in differentiation of NB cells, but the mechanism of c-Src regulation has not been defined. Here, we characterize PAG1 (Cbp, Csk binding protein), a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in NB. Clinical cohort analysis demonstrate that low expression of PAG1 is a significant prognostic factor for high stage disease, increased relapse, and worse overall survival for children with NB. PAG1 knockdown in NB cells promotes proliferation and anchorage-independent colony formation with increased activation of AKT and ERK downstream of c-Src, while PAG1 overexpression significantly rescues these effects. In vivo, PAG1 overexpression significantly inhibits NB tumorigenicity in an orthotopic xenograft model. Our results establish PAG1 as a potent tumor suppressor in NB by inhibiting c-Src and downstream effector pathways. Thus, reactivation of PAG1 and inhibition of c-Src kinase activity represents an important novel therapeutic approach for high-risk NB.

PAG1 directs SRC-family kinase intracellular localization to mediate receptor tyrosine kinase-induced differentiation

“…A defect in differentiation, together with increased activity of total SFKs we observed in cells expressing PAG1 TM- (Figure 1) is consistent with previous observations that PAG1 acts as a tumor suppressor in neuroblastoma cells (Agarwal et al, 2016;Oneyama et al, 2008). Oneyama et al (2008) first demonstrated the interaction between PAG1 and SRC.…”

“…Consistent with our soft agar assay data (Figure 3), cells lacking PAG1 formed almost 20 times as many colonies as those re-expressing functional PAG1 in colony transformation assays (Oneyama et al, 2008). Using siRNA knockdown of PAG1, Agarwal et al also demonstrated increased SFK activity, increased anchorage independent growth, and increased ERK activity in neuroblastoma cell lines (Agarwal et al, 2016). A PAG1 mutant that lacked residues required for lipid raft localization failed to suppress SRC-mediated transformation (Oneyama et al, 2008), which emphasizes the importance of membrane localization of PAG1 for regulation of SFK activity.…”

“…PP2 treatment did not significantly affect colony formation for PAG1 TMexpressing cells, but did decrease the number and size of colonies formed by WT cells ( Figure 3A). These findings are consistent with previously reported 9 experiments using siRNA knockdown of PAG1 (Agarwal et al, 2016;Oneyama et al, 2008).…”

Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples

“…Orthotopic neuroblastoma xenografts were generated in four to six week old female athymic immunodeficient NCr nude mice as previously described31. Briefly, 1 × 10 6 human SH-SY5Y neuroblastoma cells were surgically implanted in beneath the renal capsule.…”

Domain interactions of C-terminal Src Kinase determined through NMR spectroscopy with segmental isotope labeling

“…Cbp could function as both a positive and negative regulator of mast cell signaling, depending upon the signaling pathway involved (Draberova et al, 2014). Cbp also serves as a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in neuroblastoma (Agarwal et al, 2016). Previously, we have studied the interaction of Csk L-SH2 with Cbp peptide using L-SH2 sample with and without binding with Cbp peptide (Liu and Cowburn, 2016).…”