Nuclear Transport of Extracellular Vesicle Biomaterials

项目摘要 靶向肿瘤微环境中癌症和基质细胞之间的相互作用， 治疗潜力最近，对这种类型的通信的兴趣已经扩展到信号分子之外 包括细胞外囊泡（EV），由癌细胞释放并由靶细胞局部或远距离吸收。 癌症EV含有负责促肿瘤发生和促转移作用的蛋白质和核酸。 我们对电动汽车世界的了解还处于起步阶段。EV货物分子一旦进入目标体内会发生什么 细胞，以及它们如何发挥其生物学效应仍然是模糊的。我们的长期目标是了解 细胞内途径和EV内容物在内化后的亚细胞命运，并将这些知识应用于 开发新的治疗策略，特别是在癌症方面。我们最近发现了一种新的EV核 其中包含VAP-A、ORP 3和Rab 7的三重VOR蛋白复合物协调 含有EV的晚期内体易位并对接到核膜内陷（NEI）中， 随后的EV货物核转移。初步证据表明，这种新的核生物相关性 通过发现ORP 3和VAP-A是促转移形态学变化所必需的，显示了该途径 的非转移性结肠癌细胞诱导的EV从转移性结肠癌细胞。我们的中央 一种假设是，这种细胞内途径介导了EV的许多作用，并且干扰 具有调节i）晚期内体与核膜之间的相互作用的机制，以及 ii）内吞的EV组分的核递送将损害细胞内的细胞间串扰， 癌症微环境，从而抑制肿瘤生长和转移的形成。我们建议 研究结肠癌源性EV生物材料的核递送对MSC的影响，并仔细研究 定义了参与这种新的核途径的蛋白质。 这一目标将通过两个具体目标来实现：（1）我们将研究这一核途径是否是 通过来源于转移性结肠癌细胞的EV转化MSC所需的，特别是 关注其对MSC增殖、迁移和侵袭力的影响;（2）详细解剖VOR 通过缺失或突变据报道涉及蛋白质-蛋白质和蛋白质-脂质的结构域和/或基序的复合物 相互作用，以及通过在相应的VAP-A、ORP 3和/或Rab 7突变体缺失的细胞中表达VAP-A、ORP 3和/或Rab 7突变体， 基因此外，我们还将研究是否涉及其他蛋白质和核孔成分。 这项研究是创新性的，因为（a）是基于我们发现的一种新的EV核途径和（B）， 核作为电动汽车货物的最终目的地尚未得到彻底调查。拟建项目 重要的是，它将（a）阐明一种新的和特征不佳的核途径，（B）加强 我们机构的研究环境，并让医学生参与拟议研究的所有阶段，（c） 为针对肿瘤微环境的癌症转移性疾病的创新疗法开辟了道路。

PROJECT SUMMARY Targeting the interaction between cancer and stromal cells in the tumor microenvironment holds great therapeutic potential. Recently, interest in this type of communication has expanded beyond signaling molecules to include extracellular vesicles (EVs), released by cancer cells and taken up by target cells locally or at distance. Cancer EVs contain proteins and nucleic acids responsible for pro-tumorigenic and pro-metastatic effects. Our knowledge of the EV world is in its infancy. What happens to EV cargo molecules once inside the target cells, and how they exert their biological effects is still obscure. Our long-term goal is to understand the intracellular route(s) and subcellular fate of EV content upon internalization and to apply this knowledge to developing novel therapeutic strategies, especially in cancer. We have recently identified a novel EV nuclear pathway in which a tripartite VOR protein complex, containing VAP-A, ORP3 and Rab7, orchestrates translocation and docking of EV-containing late endosomes into nuclear envelope invaginations (NEI) with subsequent nuclear transfer of EV cargo. Preliminary evidence of the biological relevance of this novel nuclear pathway was shown by the finding that ORP3 and VAP-A are required for pro-metastatic morphological changes of non-metastatic colon carcinoma cells induced by EVs from metastatic colon carcinoma cells. Our central hypothesis is that this intracellular pathway mediates many of the effects of EVs, and that interfering with mechanism/s that regulate i) the interaction between late endosomes and nuclear membrane, and ii) the nuclear delivery of the endocytosed EV components will impair the intercellular crosstalk in the cancer microenvironment, and thereby inhibit tumor growth and formation of metastases. We propose to investigate the effects of nuclear delivery of colon cancer-derived EV biomaterials on MSCs and to carefully define the proteins involved in this novel nuclear pathway. This objective will be accomplished by two specific aims: (1) we will investigate whether this nuclear pathway is required for the transformation of MSCs by EVs derived from metastatic colon carcinoma cells, paying particular attention to its impact on MSC proliferation, migration and invasiveness; (2) we will dissect in detail the VOR complex by deletion or mutation of domains and/or motifs reportedly involved in protein-protein and protein-lipid interactions, and by expression of VAP-A, ORP3 and/or Rab7 mutants in cells depleted of the corresponding gene. In addition, we will investigate whether other proteins and nuclear pore components are involved. This study is innovative because (a) is based on our discovery of a novel EV nuclear pathway and (b) the nucleus as a final destination of EV cargo has not yet been thoroughly investigated. The proposed project is significant because it will (a) clarify a novel and poorly characterized nuclear pathway, (b) strengthen the research environment of our Institution and involve medical students in all phases of the proposed research, (c) open the way to innovative therapies for cancer metastatic disease targeting the tumor microenvironment.