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)及其信号转导 合作伙伴，GTP结合/蛋白质交联酶-2(TGase-2)，有助于 侵袭性癌症，并发现TGase-2通过阻止EGFR的降解来增强其信号转导 C-Cbl.CA201402的目标是检查TGase-2是否提供了类似的保护作用 对于胶质瘤干细胞(GSCs)中的EGFR致癌变体EGFRvIII，以及这些蛋白质是否重要 细胞外小泡(EVS)的活动。我们的努力导致了一些新的发现， 这构成了这次续签申请的基础。其中包括发现TGase-2功能是偶联的 到它采用两种不同构象状态的能力，一种GTP结合的关闭状态，它保护EGFR CBL催化的降解，以及一种能够与大型电动汽车结合的蛋白质交联性开放状态，称为 微囊泡(MVS)，对于它们影响肿瘤微环境和刺激的能力是必要的 血管生成。然而，当TGase-2被诱导在癌细胞内处于开放状态时，它会导致细胞 死亡。虽然我们发现EGFRvIII不需要TGase-2来保护它免受Cbl催化的影响 泛素化，我们发现EGFRvIII的表达与谷氨酰胺代谢有关，谷氨酰胺代谢在 侵袭性癌细胞。此外，我们发现侵袭性的癌细胞会产生大量的小EV。 (外体)，含有免疫检查点配体PD-L1和细胞生存蛋白Survivin，由于 他们谷氨酰胺代谢的升高和对NAD依赖的Sirtuin 1(SIRT1)的下调 脱乙酰酶。我们现在将着手了解这些发现对经济增长和 通过两个在癌细胞信号传导方面具有互补专业知识的实验室的努力，GSCs的存活 (Cerione)和GSC的翻译研究(Nakano)，如下：1)确定如何利用开放的- TGase-2的状态构象抑制癌细胞活力。我们将确定开放状态TGASE-2如何 使癌细胞经历细胞死亡，并开发方法稳定这种状态作为潜在的 治疗策略。2)了解侵袭性GSC中EGFRvIII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显著影响细胞水平 EGFRvIII的表达，以及EGFRvIII的表达和信号是否刺激MVS的产生 含有该致癌变异体和TGase-2。3)理解的表达与 EGFRvIII和TGase-2在侵袭性GSCs中的表达及独有的外体大量脱落 蛋白质货物。我们将确定是否下调GSC中SIRT1的表达，以及信号通路 涉及EGFRvIII和/或TGase-2，是它们产生大量外切体的能力的原因 富含PD-L1和Survivin。这些研究将为攻击性的发展提供新的见解。 癌症，如高级别胶质瘤，以及突出侵袭性疾病的新治疗靶点。

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.