Protease/PAR2/TRPV4 Axis and Oral Cancer Pain

口腔癌会引起严重的疼痛，影响说话、进食和饮水。患者对 用于治疗这种疼痛（机械性异常性疼痛）的阿片类药物;需要逐渐增加剂量。不幸的是， 阿片类药物产生使人衰弱的副作用，包括深度镇静。一种非阿片类药物药理学策略， 减轻口腔癌疼痛势在必行;患者在手术切除前等待数天或数周， 复发或不能治愈。在前期工作中，我们发现蛋白酶激活受体2（PAR 2） 导致口腔癌机械性异常性疼痛。在最近的研究中，我们证明了口腔癌和 相关的巨噬细胞将蛋白酶分泌到口腔癌微环境中。我们发现两个很差的 表征的蛋白酶豆荚蛋白（Lgmn）和组织蛋白酶-S（Cat-S）表现出高度上调的表达， 在口腔癌微环境中的活性，并可能介导癌症疼痛。这些蛋白酶裂解 PAR 2对感觉神经元的影响。PAR 2上特定位点的切割稳定受体构象， 促进受体信号传导和运输途径的激活。这些信号通路导致TRPV 4 伤害感受器的激活和过度兴奋。我们现在提出的工作将定义Lgmn的机制， 口腔癌疼痛中的Cat-S。我们建议使用我们的方法来鉴定和定位口腔癌中的活化蛋白酶。 荧光猝灭的基于活性的探针（qABP），其与活化的蛋白酶共价相互作用。我们将 明确鉴定和确定患者肿瘤中存在的活化蛋白酶的细胞来源 患有口腔癌。然后，我们将分析蛋白酶活性和疼痛评分之间的相关性， 确定肿瘤蛋白酶是否诱导伤害感受器的PAR 2依赖性激活。使用缺乏 PAR 2或TRPV 4在Nav1.8伤害感受器中的作用，以及我们验证的口腔癌疼痛模型，我们将确定 肿瘤细胞和巨噬细胞蛋白酶是否通过激活伤害感受器上的PAR 2和TRPV 4引起疼痛。 此外，我们将使用我们的新的Par 2-muGFP小鼠进行高度特异性的PAR 2定位和运输 和时空保真度。最后，我们将定义肿瘤细胞和巨噬细胞 蛋白酶激活PAR 2和TRPV 4，诱导伤害感受器的过度兴奋性，并引起口腔伤害感受。我们 断言蛋白酶抑制剂以及PAR 2和TRPV 4拮抗剂对口服给药具有治疗潜力， 癌症疼痛;我们最终寻求利用这些机制来开发癌症疼痛疗法。

Oral cancer induces severe pain that disrupts talking, eating and drinking. Patients develop tolerance to the opioids used to treat this pain (mechanical allodynia); progressively larger doses are required. Unfortunately, opioids produce debilitating side effects including profound sedation. A non-opioid pharmacologic strategy to alleviate oral cancer pain is imperative; patients wait days or weeks before surgical resection, suffer from recurrence or cannot be cured. In previous work we found that protease-activated receptor-2 (PAR2) contributes to oral cancer mechanical allodynia. In more recent work we demonstrated that oral cancer and associated macrophages secrete proteases into the oral cancer microenvironment. We found that two poorly characterized proteases, legumain (Lgmn) and cathepsin-S (Cat-S), exhibit highly upregulated expression and activity in the oral cancer microenvironment and potentially mediate cancer pain. These proteases cleave PAR2 on sensory neurons. Cleavage at specific sites on PAR2 stabilizes receptor conformations which in turn promote activation of receptor signaling and trafficking pathways. These signaling pathways lead to TRPV4 activation and hyperexcitability of nociceptors. The work we now propose will define mechanisms of Lgmn and Cat-S in oral cancer pain. We propose to identify and localize activated proteases in oral cancers using our fluorescently-quenched activity-based probes (qABPs) that covalently interact with activated proteases. We will unequivocally identify and determine the cellular origin of activated proteases present in tumors from patients with oral cancer. We will then analyze the correlation between protease activity and pain scores, and determine whether tumor proteases induce PAR2-dependent activation of nociceptors. Using mice that lack PAR2 or TRPV4 in Nav1.8 nociceptors, and our validated models of oral cancer pain, we will determine whether tumor cell and macrophage proteases cause pain by activating PAR2 and TRPV4 on nociceptors. Furthermore, we will use our new Par2-muGFP mice for PAR2 localization and trafficking with high specificity and spatiotemporal fidelity. Lastly we will define the mechanisms by which tumor cell and macrophage proteases activate PAR2 and TRPV4, induce hyperexcitability of nociceptors, and cause oral nociception. We assert that protease inhibitors as well as PAR2 and TRPV4 antagonists hold therapeutic potential for oral cancer pain; we ultimately seek to exploit these mechanisms to develop cancer pain therapies.