PROJECT SUMMARY/ABSTRACT GPCRs and RTKs are transmembrane receptors that control a host of physiological and pathological processes including cancer pain and other forms of chronic pain. This proposal focuses on signaling mechanisms of GPCRs and RTKs located in endosomes (eGPCRs and eRTKs) that might be responsible for cancer pain. The underlying hypothesis is that eGPCRs and eRTKs generate long-lasting signals in endosomes of human nociceptors and Schwann cells that induce sustained activation and sensitization to mediate pain. Cells within the oral cancer microenvironment (cancer, immune, and neuronal cells), release proteases, neuropeptides and growth factors that activate GPCRs (e.g., PAR2, CLR) and RTKs (e.g., TrkA, EGFR) on nociceptors and Schwann cells. GPCRs and RTKs undergo clathrin-dependent endocytosis and assemble signaling complexes that control activity and expression of ion channels and long-lasting sensitization of nociceptors and Schwann cells to drive pain. A reverse translational approach is used to study endosomal signaling of pain in 3 Specific Aims. Aim 1 determines the contribution of eGPCR and eRTK signaling to pain. Oral cancer patient pain will be evaluated (questionnaires, sensitivity testing of patient oral cancer tumors with mechanical and chemical stimuli, and assessment of comorbidities). Surgically resected tumors, trigeminal nerve, and cancer supernatant will be collected. Patient-specific Schwann cells will be isolated and cultured from the nerve. Nociceptors and Schwann cells will be obtained from organ donors. Nociceptive effects of cancer supernatant, proteases, neuropeptides, and growth factors that mediate activity of nociceptors and Schwann cells will be studied in mice. Endocytosis inhibitors will be used to probe the link between endocytosis and nociception. Sensitization and nociception will be correlated with individual patient pain phenotypes. Aim 2 Defines eGPCR and eRTK pain signaling mechanisms. Effects of cancer supernatant, and eGPCR and eRTK ligands will be studied. Endocytosis of GPCRs and RTKs that mediate oral cancer pain will be studied in human and mouse nociceptors and Schwann cells by confocal imaging and biophysical assays. Biosensors will be used to study the assembly of multiprotein signaling complexes in subcellular compartments of nociceptors and Schwann cells. Endosomal signaling will be correlated with pain phenotypes in individual patients. Aim 3 develops nanoparticles (NP) to antagonize eGPCR and eRTK signaling in preclinical pain models that recapitulate patient oral cancer pain. Endosomal acidity and light will be exploited to trigger NP disassembly and antagonist release, and test contributions of eGPCR and eRTK signaling. Their use in preclinical pain models will link endosomal signaling to pain. A deeper understanding of eGPCR and eRTK signaling mechanisms will provide insight into the neurobiology of oral cancer pain.

PROJECT NARRATIVE Oral cancer patients suffer from severe pain, especially while eating and talking. The proposal investigates whether G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) generate sustained signals from endosomes that mediate sensitization of human nociceptors and Schwann cells and thereby cause oral cancer pain. A deeper understanding of GPCR- and RTK- mediated pain signaling in humans will provide insights into the mechanism of oral cancer pain.