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

抽象的。这项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体，连同一个总的分泌体， 极大地增强了它们转移扩散的可能性。它们的基础是令人兴奋的发展 癌症生物学领域表明，胞外小体(EV)的一大类--外切体发挥作用 在癌症进展的多个方面发挥重要作用。这些包括外显子的能力 赋予肿瘤细胞对化疗药物和免疫的抵抗力 治疗，以及它们在促进转移扩散方面的作用。我们最近发现羽绒- 侵袭性乳腺癌细胞对SIRT1的调节对SIRT1的数量有重要影响 它们产生的外体、外体货物的性质以及它们的总量的组成 秘密的。这是由于依赖NAD+的脱乙酰酶/脱乙酰酶Sirtuin(SIRT1)在 通过一种新的机制维持正常的溶酶体功能，以确保正确表达 空泡ATPae的主要亚单位(v-ATPase)。我们最近还发现，这个阵型和 外切体的脱落似乎依赖于谷氨酰胺代谢特征的升高。 乳腺癌细胞(即它们的“谷氨酰胺成瘾”)。这些发现现在提出了一些重要的问题 侵袭性乳腺癌细胞对谷氨酰胺代谢的依赖如何影响和/或起作用 同时下调SIRT1的表达/激活来调节溶酶体功能和 外体生物发生，从而产生一种分泌体，刺激癌细胞侵袭并帮助驱动 转移过程。参与这项提议的不同实验室将利用他们的 探索癌细胞的生化和化学生物学方法方面的多学科专业知识 代谢和外体生物发生，高分辨率成像，3D球体培养和肿瘤器官， 并使用小鼠模型，探索导致乳腺癌的三个关键方面的机制 转移。它们是：1)研究SIRT1下调和谷氨酰胺升高之间的关系 具有侵袭性的乳腺癌细胞的新陈代谢和产生独特的外切体。2) 了解SIRT1下调如何影响液泡ATPase表达以产生分泌组 能够促进癌细胞的侵袭性。3)确定SIRT1表达/活性如何影响 乳腺癌模型中外切体的产生、细胞侵袭和转移扩散。人们的期望是 这些研究将导致确定针对破坏性影响的令人兴奋的新治疗策略 侵袭性的呼吸系统癌症，并最终导致其他转移性疾病。

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.