Endosomal mechanisms signaling oral cancer pain

项目总结/摘要 GPCR和RTK是跨膜受体，其控制宿主的生理和病理变化。 包括癌症疼痛和其他形式的慢性疼痛。该建议侧重于信号 位于核内体中的GPCR和RTK（eGPCR和eRTK）可能负责 治疗癌症疼痛潜在的假设是eGPCR和eRTK产生持久的信号， 人伤害感受器和雪旺细胞的内体，诱导持续的激活和致敏， 减轻疼痛。口腔癌微环境中的细胞（癌细胞、免疫细胞和神经细胞）， 激活GPCR的蛋白酶、神经肽和生长因子（例如，PAR 2，RTK）和RTK（例如，TrkA， EGFR）对伤害感受器和雪旺细胞的作用。GPCR和RTK经历网格蛋白依赖性内吞作用， 组装信号复合物，控制离子通道的活性和表达， 伤害感受器和雪旺氏细胞的敏感性来驱动疼痛。反向翻译方法用于 研究内体信号传导的疼痛在3个特定的目的。目标1确定了eGPCR的贡献， 疼痛的eRTK信号。将评估口腔癌患者的疼痛（问卷调查、 患者口腔癌肿瘤与机械和化学刺激，以及共病的评估）。 将收集手术切除的肿瘤、三叉神经和癌症上清液。患者特异 将从神经中分离和培养施万细胞。将获得伤害感受器和许旺细胞 器官捐赠者。肿瘤上清液、蛋白酶、神经肽和生长的伤害感受作用 将在小鼠中研究介导伤害感受器和许旺细胞活性的因子。内吞抑制剂 将被用来探测内吞作用和伤害感受之间的联系。致敏和伤害感受将 与个体患者疼痛表型相关。目的2定义eGPCR和eRTK疼痛信号传导 机制等将研究癌症上清液以及eGPCR和eRTK配体的作用。的内吞作用 将在人和小鼠伤害感受器中研究介导口腔癌疼痛的GPCR和RTK， 共聚焦成像和生物物理学分析的雪旺细胞。生物传感器将用于研究组装 多蛋白信号复合物在伤害感受器和许旺细胞的亚细胞区室。 内体信号传导将与个体患者的疼痛表型相关。Aim 3开发 纳米颗粒（NP）拮抗临床前疼痛模型中的eGPCR和eRTK信号传导， 患者口腔癌疼痛。内体酸性和光将被利用来触发NP分解， 拮抗剂释放，并测试eGPCR和eRTK信号传导的贡献。它们在临床前疼痛中的应用 模型将把内体信号传导与疼痛联系起来。更深入地了解eGPCR和eRTK信号 机制将提供深入了解口腔癌疼痛的神经生物学。

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.