Interaction between Tissue Factor, Junctional Adhesion Molecule-A, and Integrin B1 to drive self-renewal in glioblastoma

项目总结 胶质母细胞瘤(GBM)是最常见的发生在成人脑部的癌症，几乎在所有病例中都是致命的。一把钥匙 导致GBM患者预后不良的因素是一种称为癌症干细胞的细胞亚群。 (CSCs)，对常规使用的遗传毒性/细胞毒性疗法高度耐药，并最终表现为 复发性肿瘤。因此，抑制存活的CSCs的肿瘤复发将改善GBM 病人的结果。我们的初步和最近发表的工作表明，CSC的存活依赖于组织 因子(Tf)，一种保守的跨膜和分泌蛋白，参与血液凝结。转铁蛋白激活蛋白水解酶 激活受体2(PAR2)--基底膜细胞上的G蛋白偶联受体，促进CSC维持 通过对CSCs的标志物表达、自我更新能力和体内生长的分析，表明CSCs在体内的生长情况。 抑制转铁蛋白大大减少了CSC亚群，在某些情况下甚至导致肿瘤完全根除 在活体内。Tf-PAR2信号对CSCs的保护和增殖作用可能是通过激活 多类致癌受体酪氨酸激酶(RTK)，如EGFR，尽管其机制 Tf-PAR2-RTK对CSCs的刺激作用尚不清楚。我们的初步数据还表明，Tf与 通过稳定细胞间黏附促进细胞间黏附的JAM-A蛋白的表达 整合素β1。CSCs依赖于这种细胞间的黏附，我们的数据表明JAM-A是CSC所必需的 在GBM中的行为。由于Tf也可以通过整合素β1发出信号，我们的主要假设是Tf 上调JAM-A的表达，稳定整合素β1，增强TF作用于整合素β1的能力 并促进GBM的自我更新。这具有治疗意义，因为TF的促CSC作用是 与其止血作用无关，阻断JAM-A可能会降低TF的促肿瘤作用， 而不会导致因直接靶向转移因子而导致的出血。在具体目标1中，我们将检验假设 Tf通过PAR2-RTK信号驱动JAM-A的表达。我们将确定TF-PAR2的组件 对于TF-PAR2促肿瘤活性至关重要的复合体，并将测试TF触发JAM-A的能力 同时抑制RTK信号的各个主要下游通路的表达。在具体目标2中，我们将 确定JAM-A是否需要丝氨酸B3，我们已经证明它是一种与JAM-A结合的丝氨酸蛋白酶抑制剂， 对其支持CSC的活动表示感谢。在具体目标3中，我们将使用分子敲除来确定JAM-A是否是一种 对侵袭性、高TF表达的GBM有效的治疗靶点。这项工程的竣工将提前 我们对CSC亚群如何在GBM中保持的理解很可能适用于广泛的范围 癌症，并将在治疗许多恶性肿瘤方面展示一个引人注目的新治疗靶点， 包括GBM。

PROJECT SUMMARY Glioblastoma (GBM) is the most common cancer arising in the adult brain, and is lethal in nearly all cases. A key contributing factor to poor outcomes for GBM patients is a subpopulation of cells, known as cancer stem cells (CSCs), that are highly resistant to routinely used genotoxic/cytotoxic therapies, and ultimately manifest as recurrent tumor. Inhibiting tumor recurrence from CSCs that survive therapy would therefore improve GBM patient outcomes. Our preliminary and recently published work show that CSC survival is dependent on Tissue Factor (TF), a conserved transmembrane and secreted protein involved in blood clotting. TF activates protease- activated receptor 2 (PAR2), a G-protein-coupled receptor on GBM cells, which promotes CSC maintenance and expansion, as indicated by analysis of marker expression, self-renewal capacity, and in vivo growth of CSCs. TF suppression greatly reduces CSC subpopulations, in some cases even leading to complete tumor eradication in vivo. Protective and proliferative effects of TF-PAR2 signaling on CSCs appears to be through activation of multiple classes of oncogenic receptor tyrosine kinases (RTKs) like EGFR, although the mechanism by which TF-PAR2-RTKs stimulate CSCs is unclear. Our preliminary data also show that TF positively correlates with expression of Junctional Adhesion Molecule-A (JAM-A), a protein that promotes cell-cell adhesion by stabilizing integrin β1. CSCs depend on such cell-cell adhesion, and our data show that JAM-A is necessary for CSC behavior in GBM. Because TF can also signal through integrin β1, our overarching hypothesis is that TF upregulates JAM-A expression, which stabilizes integrin β1 and enhances the ability of TF to act on integrin β1 and promote self-renewal in GBM. This has therapeutic relevance, because the pro-CSC effects of TF are independent of its role in hemostasis, and blocking JAM-A could potentially reduce the pro-tumor effects of TF, without causing bleeding that would result from targeting TF directly. In Specific Aim 1, we will test the hypothesis that TF drives JAM-A expression through PAR2-RTK signaling. We will identify components of the TF-PAR2 complex that are essential for TF-PAR2 pro-tumor activities, and will test the ability of TF to trigger JAM-A expression while inhibiting each major downstream pathway of RTK signaling. In Specific Aim 2, we will determine whether JAM-A requires serpin B3, a serine-protease inhibitor that we have shown binds to JAM-A, for its pro-CSC activities. In Specific Aim 3, we will use molecular knockouts to determine whether JAM-A is an effective therapeutic target against aggressive, high TF-expressing GBM. Completion of this project will advance our understanding of how CSC subpopulations are maintained in GBM, could well be applicable to a wide range of cancers, and would demonstrate a compelling new therapeutic target in treating numerous malignancies, including GBM.