Therapeutic disruption of Connexin43-mediated microtubule regulation to target glioblastoma cancer stem cells

项目摘要 胶质母细胞瘤（GBM）是人类最致命的不治之症之一。即使是积极的治疗 包括手术切除，然后使用替莫唑胺（TMZ）进行放疗和化疗， GBM患者的生存期仅为14个月。事实上，GBM癌细胞是高度浸润性的，并且包含亚细胞。 具有由肿瘤微环境调节的致瘤特性的神经胶质瘤干细胞（GSC）群体， 并且通常对化疗和放射治疗有抗性。剩余的GSC然后可以进入活动 自我更新的状态，和不对称分裂，概括了异质性肿瘤。因此，所有治疗 GBM患者将经历肿瘤复发和后续手术以及毒性放疗， 化疗方案对患者有害，并且常常是不充分的。因此迫切 需要新的治疗药物来靶向GSC并治疗这种毁灭性疾病。胶质母细胞瘤耐药 TMZ与差距连接蛋白连接蛋白43（Cx43）的表达相关，该蛋白使得能够 在GSC中观察到Cx43的水平增加。Cx43的功能不是 仅限于形成细胞间离子和小分子通过的通道，但也参与 细胞增殖、迁移和凋亡。因此，靶向Cx43活性有望治疗GBM， 防止肿瘤复发。在这项研究中，我们使用了一种名为JM 2的新型Cx43模拟肽 （微管膜2），其包含Cx43的微管结合序列。我们的初步数据显示 JM 2改变了Cx43与微管的结合，减少了Cx43缝隙连接的形成，并抑制了细胞的增殖。 GSC中的细胞通讯。最重要的是，我们揭示了JM 2在降低GSC中的治疗潜力。 体外和体内存活率。为了开发基于JM 2的靶向GBM中GSC的新疗法， 我们的总体目标是产生负载JM 2的聚酸酐生物可降解纳米颗粒（JM 2-NPs）， 将JM 2持续递送至GSC。我们将使用高分辨率显微镜技术，包括随机 光学重建显微镜（STORM）和生物化学测定以分析JM 2-NPs在GSC中的作用 来源于从解剖的患者肿瘤新鲜分离的人原代GBM细胞。最后，我们将评估 使用三维患者GBM衍生的类器官模型，JM 2-NP对离体GSC的治疗效果， 和体内使用原位GBM小鼠模型。这些结果将验证JM 2肽的有效作用 用于治疗高Cx43/化学抗性GSC，并提供预防GBM肿瘤的治疗机会 复发拟议的研究意义重大，因为这种创新方法不仅使我们能够 为致命的GBM开发新的疗法，但也将为新诊断的GBM的潜在临床试验奠定基础。 GBM患者在不久的将来。最后，我们的新的JM 2负载纳米颗粒可以扩展到其他CNS 这些疾病可能受益于靶向Cx43-微管相互作用。

Project Summary Glioblastoma (GBM) is one of the most lethal incurable human diseases. Even with aggressive therapies including surgical resection followed by radiotherapy and chemotherapy using temozolomide (TMZ), the median survival for GBM patients is only 14 months. In fact, GBM cancer cells are highly infiltrative and comprise a sub- population of glioma stem cells (GSCs) with tumorigenic properties regulated by the tumor microenvironment, and often resistant to chemotherapy and irradiation treatments. The remaining GSCs can then enter an active state of self-renewal, and asymmetric division that recapitulates the heterogeneous tumor. As a result, all treated GBM patients will experience tumor recurrence and subsequent surgeries and toxic radiotherapy and chemotherapy regimens are harmful for the patient and often remain insufficient. There is therefore an urgent need for new therapeutic drug to target GSCs and treat this devastating disease. Glioblastoma resistance to TMZ correlates with the expression of the gap junction protein Connexin43 (Cx43), a protein which enables communication between cells, and increased levels of Cx43 are observed in GSCs. The function of Cx43, is not limited to forming channels for the passage of ions and small molecules between cells, but also participates in cell proliferation, migration and apoptosis. Therefore, targeting Cx43 activity holds promise to treat GBM and prevent tumor recurrence. In this proposed research we use a novel Cx43 mimetic peptide named JM2 (juxtamembrane 2) that encompasses the microtubule binding sequence of Cx43. Our preliminary data show that JM2 alters Cx43 binding to microtubule, decreases the formation of Cx43 gap junctions, and inhibits cell- cell communication in GSCs. Most importantly, we reveal the therapeutic potential of JM2 in decreasing GSC survival in vitro and in vivo. With the goal of developing a new therapy based upon JM2 to target GSCs in GBM, our overall objective is to generate JM2-loaded polyanhydride biodegradable nanoparticles (JM2-NPs) for sustained delivery of JM2 to GSCs. We will use high-resolution microscopy techniques including stochastic optical reconstruction microscopy (STORM) and biochemistry assays to analyze the effect of JM2-NPs in GSCs derived from human primary GBM cells freshly isolated from dissected patient tumor. Finally, we will assess the therapeutic effect of JM2-NPs on GSCs ex vivo using a three dimensional patient GBM-derived organoid model, and in vivo using an orthotopic GBM mouse model. These results will validate the potent effect of JM2 peptide for treatment of high Cx43/chemoresistant GSCs and present a therapeutic opportunity to prevent GBM tumor recurrence. The proposed research is significant because this innovative approach will not only allow us to develop novel therapies for lethal GBM but also will lay foundation on potential clinical trials in newly diagnosed GBM patients in the near future. Finally, our new JM2-loaded nanoparticles may be scalable to other CNS diseases that could benefit from targeting Cx43-microtubule interaction.