Investigating drug targets and improving drug delivery for anti-cancer treatment of osteosarcoma

骨肉瘤是骨癌，最常见于儿童和年轻人。转移要么存在于 大多数患者在诊断时或在治疗过程中后期出现。存活率与 骨肉瘤的转移率非常低，因此需要新的治疗干预措施。我们实验室有 证明利鲁唑（Riluzole）是一种谷氨酸释放抑制剂，可有效抑制增殖并诱导 人和小鼠骨肉瘤中的细胞凋亡。此外，我们还证明利鲁唑可以阻断 mGluR5 受体信号传导抑制骨肉瘤细胞生长的活性。是 相关蛋白 (YAP) 是一种参与细胞增殖的转录辅激活因子。文献证据表明，YAP 丝氨酸 127 处的磷酸化促进 YAP 的细胞质隔离和降解。此外，亚普 在 DNA 损伤诱导的应激下，Y357 被 C-Abl 激酶磷酸化。有趣的是，磷酸化 Y357 处的 YAP 促进与转录辅激活因子 p73 的强烈相互作用，以诱导原转录 凋亡基因。我们的数据表明，利鲁唑降低了 YAP 丝氨酸 127 处的磷酸化，并且 增加 YAP 的核定位。此外，利鲁唑还改变了 YAP 突变体的定位， YAPS5A（61、109、127、164 和 397 处的五个丝氨酸残基更改为丙氨酸）从细胞质到细胞核 这表明定位的变化与这些位点的磷酸化无关。我们假设 YAP 核定位的增加促进促凋亡基因的转录。我们想要 确定YAP是否直接参与利鲁唑诱导的细胞凋亡以及YAP是否受C-Abl调节激活 骨肉瘤细胞中的促凋亡基因。我们将使用人类转移性骨肉瘤细胞 LM7 和 小鼠细胞系 OS482，用于研究 a) 利鲁唑对 YAP Y357 磷酸化的影响 b) C-Abl YAP Y357 磷酸化抑制剂和利鲁唑诱导的细胞凋亡 c) 利鲁唑治疗骨肉瘤 YAP 敲低或 C-Abl 敲低的细胞，以确认 YAP 和 C-Abl 在细胞凋亡中的作用 d) 利鲁唑 荧光素酶报告基因检测中 YAP 和 P73 结合以及 Bax 启动子转录的影响。 我们最近的体外数据表明，从氧化铁纳米笼中释放的利鲁唑更有效。 与游离利鲁唑或从细胞中释放的利鲁唑相比，可有效诱导 LM7 细胞凋亡 纳米球。我们假设从负载利鲁唑的纳米笼中释放利鲁唑会更有效， 与游离利鲁唑相比，在裸鼠模型中减少转移。我们将植入骨肉瘤细胞， LM7.eGFP.ffLuc，在裸鼠尾静脉中。我们将动物随机分为 4 组并进行 以下治疗。 1) 无处理，2) 游离利鲁唑，3) 单独的纳米笼，4) 负载利鲁唑的纳米笼。 我们将在所有组中使用生物发光成像来治疗动物并监测转移。我们将表演 整个动物磁共振成像（MRI）用于氧化铁纳米笼载体的生物分布，然后 使用定量磁化率图 (QSM) 来量化氧化铁纳米笼的浓度 转移部位。 我们的数据表明，通过 mGluR5 的谷氨酸信号传导对于 LM7 的集落形成能力非常重要 细胞。在目标 3 中，我们将评估 mGluR5 siRNA 递送在异种移植物中减小肿瘤大小的效率 鼠标模型。我们将使用载有 mGluR5 siRNA 的氧化铁纳米笼并使用以下方法诱导 siRNA 释放 小鼠磁热疗法。我们将进行全动物 MRI/QSM 和离体 MRI/QSM 以确定 每个器官和肿瘤部位的纳米笼浓度。我们将在 Aim 中执行和分析实验 2 和目标 3 是与我的同事 Hiroshi Matsui 博士（生物纳米技术专家）合作完成的。 简而言之，我们期望：a) 证明 YAP 在利鲁唑诱导的骨肉瘤细胞凋亡中的直接作用 b) 评估利鲁唑递送方法预防异种移植裸鼠转移的功效 模型 c) 通过纳米颗粒递送 mGluR5 siRNA，以防止异种移植小鼠模型中肿瘤的形成。 利鲁唑 (Riluzole) 被用作 FDA 批准的肌萎缩侧索硬化症 (ALS) 治疗药物。这项研究是一项 药物再利用研究，其中利鲁唑用作骨肉瘤的抗癌剂。 ！

Osteosarcoma is cancer of bone, most common in children and young adults. Metastasis is either present at the time of diagnosis or develops later during the course of treatment in most patients. The survival rate with metastatic osteosarcoma is very low, therefore new therapeutic interventions are needed. Our lab has demonstrated that Riluzole, a glutamate release inhibitor, is effective in inhibiting proliferation and inducing apoptosis both in human and mouse osteosarcoma. Furthermore, we have demonstrated that Riluzole blocks the activity of the mGluR5 receptor signaling to inhibit growth in osteosarcoma cells. Yes associated protein (YAP) is a transcription co-activator involved in cell proliferation. The evidence in literature shows that YAP phosphorylated at serine 127 facilitates cytoplasmic sequestration and degradation of YAP. Furthermore, YAP is phosphorylated at Y357 by C-Abl kinase under DNA damage-induced stress. Interestingly, phosphorylation of YAP at Y357 promotes strong interaction with p73, a transcription co-activator, to induce transcription of pro- apoptotic genes. Our data has shown that Riluzole decreased phosphorylation of YAP at serine 127 and increased nuclear localization of YAP. Furthermore, Riluzole also changed the localization of a YAP mutant, YAPS5A (five serine residues at 61, 109, 127, 164, and 397 changed to alanine) from cytoplasm to nucleus suggesting that change is localization is independent of phosphorylation at these sites. We hypothesize that the increase in nuclear localization of YAP facilitates transcription of pro-apoptotic genes. We want to determine if YAP is directly involved in Riluzole-induced apoptosis and if YAP is regulated by C-Abl to activate pro-apoptotic genes in osteosarcoma cells. We will use human metastatic osteosarcoma cells, LM7, and mouse cells lines, OS482, to study the effect of: a) Riluzole on phosphorylation of YAP at Y357 b) C-Abl inhibitors on phosphorylation of YAP at Y357 and Riluzole-induced apoptosis c) Riluzole on osteosarcoma cells with YAP knockdown or C-Abl knockdown to confirm the role of YAP and C-Abl in apoptosis d) Riluzole on YAP and P73 binding and on transcription of Bax promoter in a luciferase reporter assay. Our recent in vitro data has demonstrated that Riluzole released from the iron oxide nanocage is more effective in inducing apoptosis in LM7 cells compared to free Riluzole or Riluzole released from the nanosphere. We hypothesize that Riluzole released from Riluzole-loaded nanocage will be more effective, compared to free Riluzole, in reducing metastasis in a nude mouse model. We will implant osteosarcoma cells, LM7.eGFP.ffLuc, in the tail vein of nude mouse. We will randomly sort the animals in 4 groups and carry out the following treatments. 1) no treatment, 2) free Riluzole, 3) nanocage alone, 4) Riluzole-loaded nanocage. We will treat the animals and monitor metastasis using bioluminescence imaging in all groups. We will perform whole animal magnetic resonance imaging (MRI) for biodistribution of iron oxide nanocage carriers and then use quantitative susceptibility mapping (QSM) to quantify the concentration of iron oxide nanocages at metastasis sites. Our data has demonstrated that glutamate signaling via mGluR5 is important in colony forming ability of LM7 cells. In Aim 3, we will assess the efficiency of mGluR5 siRNA delivery to decrease tumor size in a xenograft mouse model. We will use mGluR5 siRNA-loaded iron oxide nanocage and induce siRNA release using magnetic hyperthermia in mice. We will perform whole animal MRI/QSM and ex vivo MRI/QSM to determine the nanocage concentration in each organ and tumor site. We will perform and analyze the experiments in Aim 2 and Aim 3 in collaboration with my colleague, Dr. Hiroshi Matsui, who is an expert in bionanotechnology. In brief, we expect to: a) demonstrate a direct role of YAP in Riluzole-induced apoptosis of osteosarcoma cells b) assess the efficacy of the delivery method of Riluzole in preventing metastasis in xenograft nude mouse model c) deliver mGluR5 siRNA via nanoparticle to prevent tumor establishment in a xenograft mouse model. Riluzole is used as a FDA approved therapeutic drug for Amyotrophic Lateral Sclerosis (ALS). This study is a drug- repurposing study in which Riluzole is used as an anti-cancer agent for osteosarcoma. !