Nuclear Transport of Extracellular Vesicle Biomaterials

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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.