Contribution of c-Jun N-terminal kinase activity to vestibular schwannoma growth

描述（由申请人提供）：前庭神经鞘瘤（VS）由前庭神经的许旺细胞（SC）发展而来。它们引起显著的发病率，包括耳聋、耳鸣、面瘫、失衡和脑干压迫。显微手术切除和立体定向放射外科/立体定向放射治疗（SRS/SRT）构成了目前唯一的VS治疗方法，通常会导致耳聋、耳鸣、面瘫和失衡。此外，一些需要治疗的患者不是显微手术或SRS/SRT的良好候选人。开发有效的替代疗法将对那些不适合当前治疗选择的患者大有益处。了解导致VS肿瘤发生和放射敏感性的因素将有助于确定潜在的治疗靶点。Merlin，VS中的缺陷基因产物，调节与肿瘤发生有关的激酶的活性，包括细胞外调节激酶（ERK）、磷脂酰肌醇-3-激酶（PI 3-K）/Akt和c-Jun N-末端激酶（JNK）。初步数据表明，VS细胞中ERK和PI-3 K/Akt的活化导致增殖增加，JNK的活化促进细胞增殖和存活。JNK活性的抑制增加了活性氧（ROS）的积累，包括线粒体超氧化物。进一步的ROS清除剂防止具有抑制的JNK的VS细胞中的凋亡，这意味着JNK的促存活作用可能是由于其降低氧化应激的能力。相反，JNK活性似乎促进失神经支配的SC的凋亡。最后，VS细胞表现出对辐射（IR）的高度抗性。这些观察结果导致以下假设：VS细胞中merlin功能的丧失导致持续的JNK激活，这反过来又抑制ROS的积累，促进细胞存活，并赋予辐射抗性。这些研究的目的是在原代人VS细胞培养物和植入裸鼠的人VS异种移植物中检验这一假设。第一个目的是确定merlin是否抑制人VS细胞和SC中的JNK活性，并确定导致JNK激活的上游信号。这将通过使用病毒介导的基因转移将功能性merlin替换到VS细胞中并确定JNK活性是否因此受到抑制来解决。平行研究将询问来自缺乏功能性merlin的转基因小鼠的SC是否表现出JNK活性增加。第二个目标将使用原代人VS培养物和裸鼠原位异种移植物来确定持续的JNK活性通过增加细胞增殖和存活来促进VS生长的程度。第三个目的是确定JNK抑制特定的活性氧（ROS）是否有助于VS细胞的存活，最终目的是确定JNK抑制剂是否增强3-照射（IR）诱导VS细胞凋亡和减少增殖的能力。这些研究的结果将为VS细胞生长和放射敏感性的基本机制提供见解，并可能有助于VS新疗法的开发。公共卫生相关性：前庭神经鞘瘤（VS）占所有颅内肿瘤的8-10%，经常导致听力损失，耳鸣，面瘫，失衡和脑干压迫。这些研究旨在确定导致肿瘤形成的潜在细胞异常，并确定专门限制神经鞘瘤生长的新疗法。

DESCRIPTION (provided by applicant): Vestibular schwannomas (VSs) develop from the Schwann cells (SCs) of the vestibular nerve. They cause significant morbidity including deafness, tinnitus, facial paralysis, imbalance, and brainstem compression. Microsurgical removal and stereotactic radiosurgery/stereotactic radiotherapy (SRS/SRT) constitute the only current treatments for VSs and often result in deafness, tinnitus, facial paralysis and imbalance themselves. Further, some patients that require treatment are not good candidates for either microsurgery or SRS/SRT. Development of effective alternative therapies will be of great benefit to those patients unsuitable for current therapeutic options. Understanding the factors that contribute to VS tumorigenesis and radiosensitivity will help identify potential therapeutic targets. Merlin, the defective gene product in VSs, regulates the activity of kinases implicated in tumorigenesis including extracellular regulated kinases (ERKs), phosphatidylinositol-3-kinase (PI3-K)/Akt, and c-Jun N-terminal kinases (JNKs). Preliminary data demonstrate activation of ERKs and PI-3K/Akt in VS cells leading to increased proliferation and activation of JNK promoting cell proliferation and survival. Inhibition of JNK activity increases accumulation of reactive oxygen species (ROS), including mitochondrial superoxides. Further ROS scavengers prevent apoptosis in VS cells with suppressed JNK, implying that the prosurvival effects of JNK may be due to its ability to reduce oxidative stress. Conversely, JNK activity appears to promote apoptosis in denervated SCs. Finally, VS cells appear highly resistant to irradiation (IR). These observations lead to the hypothesis that loss of merlin function in VS cells results in persistent JNK activation which, in turn, suppresses accumulation of ROS, promotes cell survival, and confers radioresistance. The goal of these studies is to test this hypothesis in primary human VS cell cultures and human VS xenografts implanted in nude mice. The first aim is to determine whether merlin inhibits JNK activity in human VS cells and in SCs and identify the upstream signaling leading to JNK activation. This will be addressed by replacing functional merlin into VS cells using viral-mediated gene transfer and determining if JNK activity is consequently suppressed. Parallel studies will ask if SCs derived from transgenic mice lacking functional merlin demonstrate increased JNK activity. The second aim will use primary human VS cultures and orthotopic xenografts in nude mice to determine the extent to which persistent JNK activity promotes VS growth by increasing cell proliferation and survival. The third aim seeks to determine if suppression of specific reactive oxygen species (ROS) by JNK contributes to VS cell survival and the final aim seeks to determine whether JNK inhibitors potentiate the ability of 3-irradiation (IR) to induce VS cell apoptosis and reduce proliferation. The results of these studies will provide insights into the fundamental mechanisms contributing to VS cell growth and radiosensitivity and will likely contribute to the development of novel therapies for VSs. PUBLIC HEALTH RELEVANCE: Vestibular schwannomas (VSs) comprise 8-10% of all intracranial tumors and frequently cause hearing loss, tinnitus, facial paralysis, imbalance, and brainstem compression. These studies seek to identify the underlying cellular abnormalities that lead to tumor formation and to identify novel therapies that specifically limit schwannoma growth.