Project Summary/Abstract Oral cancer patients suffer from severe pain that is treated with opioids. However, opioid tolerance develops quickly. Currently, there are no other approaches to alleviate oral cancer pain or to forestall the development of opioid tolerance. In exploring a potential role of the epidermal growth factor receptor (EGFR) in oral cancer pain and opioid tolerance, we made an exciting discovery that one of the EGFR ligands, HB-EGF, is upregulated in patients with severe pain, and that EGFR interacts with both the mu opioid receptor (MOR) and the glutamate N-Methyl-D-aspartic acid receptor (NMDAR), two proteins important for chronic pain and opioid tolerance. This led us to hypothesize that HB-EGF mediated signaling in trigeminal (TG) neurons would: a) desensitize and down-regulate MOR and b) potentiate NMDAR-dependent synaptic transmission, contributing to oral cancer pain and opioid tolerance. We will test our hypothesis in three specific aims. Aim 1 will discover whether HB-EGF and trigeminal EGFR regulate pain, opioid tolerance, and NMDAR-mediated synaptic transmission in oral cancer. We will use pharmacological and genetic approaches to determine if HB-EGF and trigeminal EGFR are essential for oral cancer pain and opioid tolerance. Behaviors related to pain and opioid tolerance will be measured using a battery of assays. NMDAR mediated synaptic transmission will be studied using electrophysiological recordings in a newly developed brainstem preparation. We will quantify NMDAR expression in the mouse TG and brainstems using western blot. Aim 2 will discover whether HB-EGF and EGFR modulate MOR signaling and endocytosis. We will use BRET-based biosensors and super resolution imaging in model cell lines and TG neurons to study the impact of HB-EGF and EGFR on MOR cAMP activity, G-protein coupling, βARR recruitment and trafficking to endosomes. We will determine the effect of EGFR gene deletion and inhibition on MOR expression by qPCR, western blot, and fluorescence imaging in mouse TG neurons. Aim 3 will validate HB-EGF and EGFR as potential targets for pain and opioid tolerance in three oral cancer patient cohorts. First, we will model EGFR ligand expression in the tumor with self-reported pain, quantitative sensory testing scores, and opioid intake and explore the cellular origin of EGFR ligands by single cell RNA sequencing in tumor tissues. In the second cohort, we will model HB-EGF and EGFR expression in tumor tissues using immunofluorescence staining as a function of self-reported pain and opioid intake using data generated in a phase II anti-cancer trial examining the effect of an EGFR inhibitor erlotinib as an adjuvant to standard chemotherapy for cancer regression. In the third cohort, we will model peripheral EGFR and ligand(s) expression from self-reported pain. EGFR inhibitors are FDA-approved therapies for cancer management. HB-EGF is an emerging cancer target and a pain mediator. Targeting HB-EGF and EGFR therefo...