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

项目摘要/摘要 口腔癌患者患有严重的疼痛，需要用阿片类药物治疗。然而，阿片类药物耐受性的形成 快点。目前，还没有其他方法来缓解口腔癌疼痛或预防口腔癌的发展 阿片耐受性。探讨表皮生长因子受体(EGFR)在口腔癌中的潜在作用 对于疼痛和阿片类药物的耐受，我们有了一个令人兴奋的发现，即EGFR的一个配体HB-EGF是 在剧烈疼痛的患者中，EGFR上调，并且EGFR与Mu阿片受体(MOR)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类药物至关重要的蛋白质 宽容。这导致我们假设HB-EGF介导的三叉神经(TG)神经元信号将：a) 脱敏和下调MOR和b)增强NMDAR依赖的突触传递，有助于 口腔癌疼痛和阿片类药物耐受性。我们将在三个具体目标上检验我们的假设。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在口腔癌疼痛和阿片类药物耐受中起重要作用。与疼痛和阿片类药物有关的行为 容忍度将使用一组分析方法进行测量。将研究NMDAR介导的突触传递 在一种新开发的脑干制剂中使用电生理记录。我们将量化NMDAR 免疫印迹法检测小鼠三叉神经节和脑脊髓区的表达。目标2将发现HB-EGF和 EGFR调节MOR信号和内吞作用。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神经元中进行成像，研究HB-EGF和EGFR对MOR cAMP活性的影响 G蛋白偶联、β受体募集和转运到内吞体内。我们将确定EGFR的效果 定量聚合酶链式反应、蛋白质印迹和荧光成像检测小鼠MOR基因缺失及表达抑制 三叉神经节神经元。AIM 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，我们将模拟有自我报告疼痛的肿瘤中EGFR配体的表达， 定量感觉测试评分和阿片类药物摄入量，并探索EGFR配体的细胞来源 肿瘤组织中的细胞RNA测序。在第二组中，我们将模拟HB-EGF和EGFR在 肿瘤组织使用免疫荧光染色作为自我报告疼痛和阿片类药物摄入量的函数使用 检测EGFR抑制剂厄洛替尼作为佐剂的II期抗癌试验中产生的数据 癌症消退的标准化疗。在第三组中，我们将模拟外周EGFR和 配体(S)表达自报疼痛。EGFR抑制剂是FDA批准的癌症治疗药物 管理层。HB-EGF是一个新兴的癌症靶点和疼痛调节剂。靶向HB-EGF和EGFR 因此有潜力用于临床快速翻译，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.