Activation of the pituitary adenylate cyclase-activating polypeptide type I receptor promotes neuroblastoma proliferation and migration through distinct G protein pathways

The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) is a class B G protein-coupled receptor implicated in a variety of physiological and pathological processes, including cancer. While PAC1R has been reported to act as either an oncogene or a tumor suppressor in a context-dependent manner, its role in neuroblastoma remains largely unexplored. In this study, we identify PAC1R as being selectively overexpressed in neuroblastoma and strongly associated with poor patient prognosis, supporting its potential as a prognostic biomarker. Functionally, PAC1R activation by PACAP38 promoted neuroblastoma cell proliferation and migration through distinct G protein signaling pathways. Pharmacological inhibition and CRISPR/Cas9-mediated gene knockout revealed that proliferation is mediated by the Gαs/EPAC/AKT-ERK axis, whereas migration requires cooperative signaling through Gα12/13- and Gαq-dependent activation of the RhoA-ROCK pathway. Comprehensive signaling analyses using TRUPATH and BRET-based biosensors demonstrated broad activation of Gαs, Gαq/11, and Gα13 by PACAP38 and PACAP27, while maxadilan and VIP displayed biased agonism toward Gαs and Gα15. β-Arrestin recruitment assays further showed that PACAP38 exhibits greater potency and efficacy than PACAP27. Among three PAC1R inhibitors tested, AMG-301, a clinical-stage monoclonal antibody, most effectively inhibited PAC1R-mediated G protein activation, calcium mobilization, RhoA signaling, proliferation, and migration. Together, these findings establish PAC1R as a functionally significant and druggable target in neuroblastoma, highlight its context-dependent signaling plasticity, and support the rationale for PAC1R-targeted therapeutic strategies in high-risk neuroblastoma.