Endosomal Signaling of PAR2 in Oral Cancer Pain

口腔癌疼痛是一种普遍的、使人衰弱的慢性疾病，它扰乱了患者的 边吃边说。患者通过持续使用阿片类药物来治疗癌症疼痛而产生耐受性，并要求 不断增加剂量以达到缓解的目的。阿片类药物还会产生严重的副作用，包括呼吸抑制， 上瘾和镇静。替代非阿片类药物战略是必要的。然而，口腔的病理生物学 癌症疼痛目前还没有得到很好的理解。我的研究计划探索了G蛋白 偶联受体(GPCRs)和瞬时受体电位(Trp)离子通道调节口腔癌痛。口头的 癌症和免疫细胞，如巨噬细胞，会分泌蛋白水解酶，裂解并激活细胞上的GPCRs。 浮出水面。GPCR激活和G蛋白偶联触发下游信号级联，从而使 Trp通道。具体地说，GPCR蛋白酶激活受体-2(PAR2)介导口腔癌的机制 痛觉异常并导致瞬时受体电位香草酸4(TRPV4)激活和兴奋性亢进。 痛觉感受器。然而，来自质膜的GPCR信号是暂时的。β-受体阻滞剂脱敏 GPCRs和偶联细胞骨架蛋白介导的内吞作用的受体，共同终止质膜 发信号。虽然内体传统上被认为是受体循环和降解的通道， 我的实验室最近的研究表明，内体中的GPCRs继续产生持续的信号， 调节疼痛的传递。然而，内体发出的PAR2信号是否能敏化TRPV4并引起 过度兴奋性还没有建立起来。本研究的目的是找出其作用机制。 口腔癌内体PAR2信号转导及其对TRPV4增敏作用的研究PH-刺激响应性 将开发将PAR2拮抗剂运送到三叉神经痛感觉神经元内体的纳米颗粒 来研究这条路径。目标1将开发pH刺激响应纳米颗粒，并表征 负载PAR2拮抗剂纳米粒的生物物理性质。目标2将探讨 内体PAR2信号及其通过基因编码的生物传感器和钙在TRPV4增敏中的作用 指标。目的研究PAR2信号转导通路在口腔癌临床前期小鼠模型中的作用机制。 通过pH刺激纳米颗粒研究内体PAR2信号将阐明内体GPCRs 是口腔癌疼痛的有效治疗靶点。在完成本提案中概述的目标和培训计划时， 这位名为Shavonne Teng的研究生将深入了解与慢性阻塞性肺疾病相关的GPCR信号。 疼痛。她将学习生物工程方法来开发纳米医学，学习生物物理学和细胞生物学 研究GPCR和Trp调节的方法，并学习如何在小鼠模型中研究口腔面部疼痛。这 研究培训将扩大她过去的经验，并为她的研究生涯做好准备 癌症疼痛领域的独立调查者。

Oral cancer pain is a prevalent, debilitating, and chronic condition that disrupts patients’ ability to eat and speak. Patients develop tolerance from continuous use of opioids to treat cancer pain and require escalating doses to achieve relief. Opioids also produce severe side effects, including respiratory depression, addiction, and sedation. Alternative non-opioid strategies are necessary. However, the pathobiology of oral cancer pain is currently not well understood. My research proposal explores the mechanisms by which G-protein coupled receptors (GPCRs) and transient receptor potential (TRP) ion channels regulate oral cancer pain. Oral cancers and immune cells such as macrophages secrete proteases that cleave and activate GPCRs at the cell surface. GPCR activation and G protein coupling triggers downstream signaling cascades, which can sensitize TRP channels. Specifically, the GPCR protease-activated receptor-2 (PAR2) mediates oral cancer mechanical allodynia and leads to transient receptor potential vanilloid 4 (TRPV4) activation and hyperexcitability of nociceptors. However, GPCR signaling from the plasma membrane is transient. Beta arrestins desensitize GPCRs and couple the receptors for clathrin-mediated endocytosis, which together terminate plasma membrane signaling. While endosomes were traditionally thought of as a conduit for receptor recycling and degradation, recent work from my lab demonstrated that GPCRs in endosomes continue to generate sustained signals that mediate pain transmission. However, whether PAR2 signals from endosomes to sensitize TRPV4 and evoke hyperexcitability has not been established. The purpose of this research is to identify the mechanisms of endosomal PAR2 signaling and its effect on TRPV4 sensitization in oral cancer. pH-stimulus responsive nanoparticles that deliver PAR2 antagonists to endosomes of pain-sensing trigeminal neurons will be developed to examine this pathway. Aim 1 will develop pH-stimulus responsive nanoparticles and characterize the biophysical properties of the nanoparticles loaded with PAR2 antagonist. Aim 2 will explore the mechanisms of endosomal PAR2 signaling and its role in TRPV4 sensitization via genetically-encoded biosensors and calcium indicators. Aim 3 seeks to assess mechanisms of PAR2 signaling in pre-clinical mice models of oral cancer. Investigating endosomal PAR2 signaling via pH-stimulus nanoparticle will elucidate whether endosomal GPCRs are valid therapeutic targets for oral cancer pain. In completing the aims and training plan outlined in this proposal, the graduate student, Shavonne Teng, will gain a deep understanding of GPCR signaling relevant to chronic pain. She will learn bioengineering approaches to develop nanomedicines, learn biophysical and cell biology approaches to study GPCR and TRP regulation, and learn how to study orofacial pain in mouse models. This research training will expand on her past experience and also prepare her for a research career as an independent investigator in the cancer pain field.