Metabolic regulation of exosome biogenesis as a determinant of cancer cell metastasis.

抽象的。该提案中概述的研究重点是侵袭性乳腺癌的机制， 细胞产生大量具有独特货物的外泌体，以及总分泌体， 显著增强了它们转移扩散的可能性。它们是基于令人兴奋的发展， 癌症生物学领域表明，外泌体，一种主要的细胞外囊泡（EV）， 在癌症进展的许多方面发挥重要作用。这些包括外来体的能力， 赋予肿瘤细胞显示对化疗试剂以及免疫应答的抗性的能力， 治疗，以及它们在促进转移扩散中的作用。我们最近发现- 侵袭性乳腺癌细胞对SIRT 1的调节对乳腺癌细胞的数量有重要影响。 它们产生的外泌体，外泌体货物的性质，以及它们的总组成， 分泌蛋白这是由于NAD+依赖性脱乙酰基酶/脱酰基酶Sirtuin（SIRT 1）在细胞凋亡中起关键作用。 通过一种新的机制来维持正常的溶酶体功能， 液泡ATP酶（v-ATP酶）的主要亚基。我们最近还发现， 外泌体的脱落似乎依赖于谷氨酰胺代谢的升高， 乳腺癌细胞（即它们的“谷氨酰胺成瘾”）。这些发现现在提出了重要的问题， 侵袭性乳腺癌细胞对谷氨酰胺代谢的依赖性如何影响和/或起作用 与SIRT 1表达/活化的下调一起调节溶酶体功能， 外泌体生物合成，从而产生刺激癌细胞侵袭并帮助驱动 转移过程。参与该提案的不同实验室将利用其 在生物化学和化学生物学方法中探测癌细胞的多学科专业知识 代谢和外来体生物发生，高分辨率成像，3D球体培养和肿瘤类器官， 以及使用小鼠模型来探索乳腺癌驱动机制的三个关键方面 转移这些是：1）检查SIRT 1下调、谷氨酰胺升高与SIRT 1表达之间的关系。 这可能与侵袭性乳腺癌细胞的代谢和具有独特货物的外泌体的产生有关。（二） 了解SIRT 1下调如何影响液泡ATP酶表达以产生分泌组 能够促进癌细胞侵袭。3)确定SIRT 1表达/活性如何影响 外泌体产生、细胞侵袭性和转移性扩散。人们期望 这些研究将导致确定令人兴奋的新的治疗策略， 侵袭性乳腺癌，最终，其他转移性疾病。

Abstract. The studies outlined in this proposal focus on the mechanisms by which aggressive breast cancer cells generate large numbers of exosomes with unique cargo, together with a total secretome that significantly enhances their potential for metastatic spread. They are based on exciting developments in the cancer biology field which show that exosomes, a major class of extracellular vesicles (EVs), play important roles in a number of aspects of cancer progression. These include the ability of exosomes to confer tumor cells with the capability to show resistance to chemotherapeutic reagents as well as to immune therapy, together with their roles in promoting metastatic spread. We recently discovered that the down- regulation of SIRT1 by aggressive breast cancer cells has an important influence on the numbers of exosomes that they generate, the nature of the exosome cargo, as well as the composition of their total secretome. This is due to the NAD+-dependent deacetylase/deacylase Sirtuin (SIRT1) playing a key role in maintaining normal lysosomal function through a novel mechanism that ensures the proper expression of a major subunit of the vacuolar ATPae (v-ATPase). We also have recently found that the formation and shedding of exosomes appear to be dependent on the elevations in glutamine metabolism characteristic of breast cancer cells (i.e. their ‘glutamine addiction’). These findings now raise important questions regarding how the dependence of aggressive breast cancer cells on glutamine metabolism influences and/or works together with the down-regulation of SIRT1 expression/activation to regulate lysosomal function and exosome biogenesis, thus producing a secretome that stimulates cancer cell invasiveness and helps drive the metastatic process. The different laboratories participating in this proposal will take advantage of their multi-disciplinary expertise in biochemical and chemical biology approaches in probing cancer cell metabolism and exosome biogenesis, high-resolution imaging, 3D spheroid culture and tumor organoids, and the use of mouse models, in probing three key aspects of the mechanisms driving breast cancer metastasis. These are: 1) Examining the relationship between SIRT1 down-regulation, elevated glutamine metabolism and the generation exosomes with unique cargo by aggressive breast cancer cells. 2) Understanding how SIRT1 down-regulation impacts vacuolar ATPase expression to generate a secretome capable of promoting cancer cell invasiveness. 3) Determining how SIRT1 expression/activity affects exosome production, cell invasiveness and metastatic spread in breast cancer models. The expectation is that these studies will lead to the identification of exciting new treatment strategies for the devastating effects of aggressive breats cancers, and ultimately, for other metastatic diseases.