Targeting HB-EGF and trigeminal EGFR for oral cancer pain and opioid tolerance

项目总结/摘要 口腔癌患者患有严重的疼痛，用阿片类药物治疗。然而，阿片类药物耐受性的发展 快目前，还没有其他方法来减轻口腔癌疼痛或预防口腔癌的发展。 阿片耐受性在探索表皮生长因子受体（EGFR）在口腔癌中的潜在作用时， 疼痛和阿片类药物耐受，我们取得了一个令人兴奋的发现，EGFR配体之一，HB-EGF， EGFR在重度疼痛患者中上调，EGFR与μ阿片受体（莫尔）和 谷氨酸N-甲基-D-天冬氨酸受体（NMDAR），两种对慢性疼痛和阿片样物质重要蛋白 宽容这使我们假设HB-EGF介导的三叉神经（TG）神经元信号传导将： 脱敏和下调莫尔和B）增强NMDAR依赖性突触传递， 口腔癌疼痛和阿片类药物耐受性。我们将在三个具体目标中检验我们的假设。目标1将发现 HB-EGF和三叉神经EGFR是否调节疼痛、阿片耐受和NMDAR介导的突触 口腔癌的传播。我们将使用药理学和遗传学方法来确定HB-EGF和 三叉神经EGFR对于口腔癌疼痛和阿片耐受性是必需的。与疼痛和阿片类药物相关的行为 将使用一系列测定来测量耐受性。将研究NMDAR介导的突触传递 在新开发的脑干标本中使用电生理记录。我们将量化NMDAR 使用western blot检测小鼠TG和脑干中的表达。目的2将发现HB-EGF和 EGFR调节莫尔信号传导和内吞作用。我们将使用基于BRET的生物传感器和超分辨率 在模型细胞系和TG神经元中成像以研究HB-EGF和EGFR对莫尔cAMP活性的影响， G蛋白偶联、βARR募集和转运至内体。我们将确定EGFR的作用 通过qPCR、蛋白质印迹和荧光成像在小鼠中进行基因缺失和对莫尔表达的抑制 TG神经元。目标3将验证HB-EGF和EGFR作为疼痛和阿片类药物耐受的潜在靶点， 口腔癌患者队列。首先，我们将模拟EGFR配体在自我报告疼痛的肿瘤中的表达， 定量感觉测试评分和阿片类药物摄入量，并通过单次给药探索EGFR配体的细胞起源。 肿瘤组织中的细胞RNA测序。在第二个队列中，我们将模拟HB-EGF和EGFR的表达， 使用免疫荧光染色的肿瘤组织作为自我报告疼痛和阿片类药物摄入的函数， 在一项II期抗癌试验中生成的数据，该试验检查了EGFR抑制剂厄洛替尼作为佐剂的效果 癌症消退的标准化疗在第三个队列中，我们将模拟外周EGFR， 来自自我报告疼痛的配体表达。EGFR抑制剂是FDA批准的癌症治疗药物 管理HB-EGF是一种新兴的癌症靶点和疼痛介质。针对HB-EGF和EGFR 因此具有快速临床转化的潜力，以减少口腔癌疼痛和阿片类药物耐受性。

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 therefore has the potential for rapid clinical translation to reduce oral cancer pain and opioid tolerance.