The unique roles of the GTP-binding/protein crosslinking enzyme transglutaminase-2 and signaling partners in aggressive cancers

摘要：我们的实验室专注于了解EGF受体（EGFR）及其信号传导 伴侣，GTP结合/蛋白质交联酶转谷氨酰胺酶-2（TGase-2），有助于 侵袭性癌症，并发现TGase-2通过阻断EGFR的降解来增强EGFR信号传导， c-Cbl。CA 201402的目的是检查TGase-2是否提供类似的保护作用。 对于胶质瘤干细胞（GSC）中的EGFR致癌变体EGFRvIII，以及这些蛋白质是否重要 细胞外囊泡（EV）的作用。我们的努力带来了许多新的发现， 这是本次更新申请的基础。这些发现包括TGase-2功能与 它能够采取两种不同的构象状态，一种是GTP结合的封闭状态， Cbl催化的降解，以及与大型EV结合的蛋白质交联能力开放状态，称为 微囊泡（MV），并且对于它们影响肿瘤微环境和刺激肿瘤微环境的能力是必要的。 血管生成然而，当TGase-2被诱导在癌细胞内采取开放状态时，它导致细胞凋亡。 死亡虽然我们发现EGFRvIII不需要TGase-2来保护它免受Cbl催化的 通过泛素化，我们发现EGFRvIII表达与谷氨酰胺代谢相关， 侵袭性癌细胞此外，我们发现侵袭性癌细胞产生大量的小EV （外泌体）含有免疫检查点配体PD-L1和细胞存活蛋白Survivin，由于 它们的谷氨酰胺代谢升高和它们的Sirtuin 1（SIRT 1）的下调， 脱乙酰酶我们现在将着手了解这些发现对增长的影响， 通过两个实验室在癌细胞信号传导方面的互补专业知识， （Cerione）和GSC的翻译研究（中野），如下：1）确定如何利用开放的 状态构象的TGase-2抑制癌细胞活力。我们将确定开放状态的TGase-2 导致癌细胞经历细胞死亡，并开发稳定这种状态的方法， 治疗策略2)了解EGFRvIII在侵袭性GSC中的表达如何与谷氨酰胺偶联 新陈代谢.我们将确定为什么抑制谷氨酰胺代谢会显著影响细胞水平， EGFRvIII表达，以及EGFRvIII表达和信号传导是否刺激MV的产生 含有这种致癌变体和TGase-2。3)理解表达之间的联系 EGFRvIII和TGase-2在侵袭性GSC中的表达以及大量具有独特免疫原性的外泌体的脱落 蛋白质货物。我们将确定GSC中SIRT 1的下调是否以及信号通路 涉及EGFRvIII和/或TGase-2的蛋白质，负责它们产生大量外泌体的能力 富含PD-L1和Survivin。这些研究将为攻击性行为的发展提供新的见解。 癌症，如高级别神经胶质瘤，以及突出了侵袭性疾病的新的治疗靶点。

Abstract - Our laboratories have focused on understanding how the EGF receptor (EGFR) and its signaling partner, the GTP-binding/protein crosslinking enzyme tranglutaminase-2 (TGase-2), contribute to aggressive cancers, and discovered that TGase-2 enhances EGFR signaling by blocking its degradation by c-Cbl. The goals of CA201402 were then to examine whether TGase-2 provides a similar protective effect for the EGFR oncogenic variant, EGFRvIII, in glioma stem cells (GSCs), and if these proteins are important for the actions of their extracellular vesicles (EVs). Our efforts have resulted in a number of new discoveries, which form the basis of this renewal application. These include the finding that TGase-2 function is coupled to its ability to adopt two distinct conformational states, a GTP-bound closed state that protects EGFRs from Cbl-catalyzed degradation, and a protein crosslinking-competent open-state that binds to large EVs, called microvesicles (MVs), and is necessary for their ability to influence the tumor microenvironment and stimulate angiogenesis. However, when TGase-2 is induced to adopt an open state within cancer cells, it causes cell death. Although we found that EGFRvIII does not require TGase-2 to protect it from Cbl-catalyzed ubiquitylation, we discovered EGFRvIII expression is coupled to glutamine metabolism which is elevated in aggressive cancer cells. Moreover, we found aggressive cancer cells generate large numbers of small EVs (exosomes) that contain the immune checkpoint ligand, PD-L1, and the cell survival protein Survivin, due to their elevated glutamine metabolism and their down-regulation of Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase. We will now set out to understand the consequences of these discoveries for the growth and survival of GSCs, through the efforts of two laboratories with complimentary expertise in cancer cell signaling (Cerione) and translational studies of GSCs (Nakano), as follows: 1) Determine how to exploit the open- state conformation of TGase-2 to inhibit cancer cell viability. We will establish how open-state TGase-2 causes cancer cells to undergo cell death and develop methods to stabilize that state as a potential therapeutic strategy. 2) Understand how EGFRvIII expression in aggressive GSCs is coupled to glutamine metabolism. We will determine why inhibiting glutamine metabolism markedly affects the cellular levels of EGFRvIII expression, and whether EGFRvIII expression and signaling stimulate the production of MVs containing this oncogenic variant and TGase-2. 3) Understand the connection between the expression of EGFRvIII and TGase-2 in aggressive GSCs and the shedding of large numbers of exosomes with unique protein cargo. We will establish whether down-regulation of SIRT1 in GSCs, and signaling pathways involving EGFRvIII and/or TGase-2, are responsible for their ability to produce large numbers of exosomes enriched in PD-L1 and Survivin. These studies will provide new insights into the development of aggressive cancers such as high-grade gliomas as well as highlight novel therapeutic targets for aggressive disease.