Molecular Targeting of L1CAM to Inhibit Glioblastoma Stem Cell Invasion

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是最具侵袭性和致命性的脑癌。尽管积极的多模式治疗，GBM的治疗仍然在很大程度上无效。这些致命癌症的根本问题是其高度浸润性和耐药性，导致肿瘤快速复发。针对浸润性GBM细胞的改进疗法的开发需要对GBM侵袭和扩散的细胞和分子机制的新见解。我们已经证明，与神经干细胞具有相似特性的GBM癌细胞亚群显示出增强的侵袭能力、血管生成潜力和治疗抗性，这表明靶向这些GBM干细胞可以显著改善治疗。我们的长期目标是开发靶向GBM干细胞的新策略，以抑制GBM侵袭，减少治疗抗性并预防肿瘤复发。在寻找GBM干细胞特异性的潜在靶点时，我们将L1 CAM鉴定为GBM干细胞中相对于非干肿瘤细胞和神经祖细胞差异升高的表面蛋白。通过shRNA靶向L1 CAM有效地破坏神经球形成，抑制肿瘤生长并增加携带颅内GBM异种移植物的小鼠的存活率。在前期研究中，我们发现L1 CAM在体外敲低后显著降低了GBM干细胞的侵袭潜能，并降低了三种侵袭相关蛋白（RhoC，Cathepsin L2和S100 A4/Metastasin）的表达，提示L1 CAM在介导GBM干细胞侵袭中起着重要作用，L1 CAM可能成为开发新的抗侵袭策略的关键靶点。基于我们的初步研究，我们假设L1 CAM促进GBM干细胞的侵袭，并且在体内GBM干细胞中的L1 CAM的分子靶向抑制肿瘤侵袭。我们将通过以下三个具体目标来验证我们的假设并实现目标：1.通过异种移植模型中的可诱导敲低，确定L1 CAM在体内GBM干细胞侵袭中的作用; 2.使用L1 CAM阻断肽（L1 HM 06和L1 HT 08）开发GBM干细胞的治疗靶向以抑制GBM侵袭和生长; 3.使用下游过表达拯救实验来定义L1 CAM促进GBM干细胞侵袭的机制。我们将使用各种分子和细胞方法以及体内技术（如生物发光成像技术）来实现这些特定目标。这些研究将使我们能够评估L1 CAM作为对抗GBM干细胞侵袭的治疗靶点的潜力。我们希望这些研究的结果将为未来开发治疗GBM和其他脑肿瘤的新方案奠定坚实的基础。 公共卫生相关性：胶质母细胞瘤（GBM）是最具侵袭性和治疗最不成功的原发性脑肿瘤。大多数诊断为GBM的患者在两年内死亡，尽管有最佳治疗。这些致命癌症的标志是其高度浸润性和耐药性，导致肿瘤快速复发。我们已经证明GBM癌症干细胞显示出增强的侵袭能力并促进治疗抗性，这表明靶向GBM干细胞可能会显着改善治疗。我们已经确定了一个独特的表面蛋白（L1 CAM）介导GBM干细胞的侵袭。我们建议通过这种特异性靶点靶向GBM干细胞，以抑制肿瘤的侵袭和复发。这些研究将为开发新的，更有效的治疗GBM肿瘤进展和改善这些致命癌症的治疗奠定坚实的基础。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most invasive and lethal form of brain cancer. Despite aggressive multimodal therapy, the treatment of GBM remains largely ineffective. The fundamental problem of these fatal cancers is their highly infiltrative nature and resistant property that leads to rapid tumor recurrence. The development of improved therapies targeting the infiltrating GBM cells requires new insight into the cellular and molecular mechanisms underlying GBM invasion and dispersal. We have demonstrated that a sub-population of GBM cancer cells sharing similar properties with neural stem cells displays enhanced invasive capability, angiogenic potential and therapeutic resistance, suggesting that targeting these GBM stem cells may significantly improve the treatment. Our long-term goal is to develop novel strategies targeting GBM stem cells to inhibit GBM invasion, reduce therapeutic resistance and prevent tumor recurrence. In the search for potential targets specific for GBM stem cells, we identified L1CAM as a differentially elevated surface protein in GBM stem cells relative to non-stem tumor cells and neural progenitors. Targeting L1CAM by shRNA potently disrupts neurosphere formation, suppresses tumor growth and increases the survival of mice bearing intracranial GBM xenografts. In preliminary study, we found that L1CAM knockdown in vitro remarkably reduces invasive potential of GBM stem cells, and decreases expression of three invasion-associated proteins (RhoC, Cathepsin L2 and S100A4/Metastasin), suggesting that L1CAM plays a crucial role in mediating GBM stem cell invasion, and L1CAM may represent a critical target for developing a novel anti-invasion strategy. Based on our preliminary studies, we hypothesize that L1CAM promotes GBM stem cell invasion, and molecular targeting of L1CAM in GBM stem cells in vivo inhibits tumor invasion. We will test our hypothesis and accomplish the objectives by pursuing the following three specific aims: 1. Determine the role of L1CAM in GBM stem cell invasion in vivo through inducible knockdown in xenograft models; 2. Develop therapeutic targeting of GBM stem cells using L1CAM blocking peptides (L1HM06 and L1HT08) to suppress GBM invasion and growth; 3. Define the mechanisms by which L1CAM contributes to GBM stem cell invasion using downstream overexpression rescue experiments. We will pursue these specific aims using varied molecular and cellular approaches as well as in vivo techniques such as bioluminescent imaging technology. These studies will allow us to evaluate the potential of L1CAM as a therapeutic target against GBM stem cell invasion. We expect that the outcome from these proposed studies will lay a solid foundation for future development of new regimens for treatment of GBM and other brain tumors. PUBLIC HEALTH RELEVANCE: Glioblastomas (GBMs) are the most aggressive and least successfully treated primary brain tumors. Most patients diagnosed with GBM die within two years despite optimal therapy. The hallmark of these lethal cancers is their highly infiltrative nature and resistant property that leads to rapid tumor recurrence. We have demonstrated that GBM cancer stem cells displays enhanced invasive capacity and promote therapeutic resistance, indicating that targeting GBM stem cells may significantly improve the treatment. We have identified a unique surface protein (L1CAM) that mediates GBM stem cell invasion. We propose to target GBM stem cells through this specific target to inhibit tumor invasion and recurrence. These studies will lay a solid foundation for development of novel, more effective therapeutics against GBM tumor progression and improve the treatment of these lethal cancers.