Exosomes are secreted extracellular vesicles corresponding to intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) that deliver bioactive compounds to the cytoplasm of target cells. Exosomes play numerous roles in cancer metastasis by transferring oncogenes, inducing myofibroblast differentiation, promoting invadopodia formation and preventing immune surveillance of tumor cells. Pro-metastatic behavior is dependent on cells undergoing epithelial to mesenchmyal transition (EMT) where a number of Ca2+ channels are aberrantly expressed resulting in inappropriate Ca2+-stimulations. The mechanisms of Ca2+-regulated exocytosis are conserved across all metazoan cell types where SNAREs promote lipid bilayer fusion. Members of the Munc13 family of proteins enhance SNARE interactions and therefore greatly accelerate vesicle fusion. Fusion of MVB with the plasma membrane causes exosome release and my preliminary data are the first to show that acute Ca2+ elevation stimulates exosome release and further implicates Munc13-4 as a major Ca2+- sensor. Induction of EMT increases Munc13-4 levels in pancreatic (Panc-1) and lung (A549) carcinoma cell lines by immunoblot. Moreover, acquisition of a mesenchymal phenotype by use of TGF-1 shifts exosome release from exclusively basal to Ca2+-sensitive. The primary hypothesis of this proposal is that Ca2+-stimulated exosome release is regulated by Ca2+-binding to Munc13-4.
AIM 1 will determine if Munc13-4-Ca2+-binding regulates a unique population of exosomes and determine the secretory dynamics of Ca2+-stimulated exosome release for the first time using super-resolution microscopy. Basal or Ca2+-stimulated exosomes will be purified to identify if a unique set of RNA and protein is released following stimulation.
AIM2 will determine the fusion event regulated by Munc13-4 that controls exosome release. Panc-1 and A549 cells undergoing EMT following shRNA downregulation of Munc13-4 or use of a novel Munc13-4 Ca2+-dependent inhibitor will be imaged using super-resolution microscopy to determine the endosomal trafficking steps in exosome secretion regulated by Ca2+ and Munc13-4. Understanding the molecular mechanisms that regulate Ca2+-stimulated exosome secretion and intracellular trafficking may provide therapeutic targets to block tumor progression and metastasis. This proposal will also provide training on cutting edge molecular biology and super resolution microscopy from experts in the field. Imaging of live cells in super resolution is now a requirement for high end cell biological studies. Training in the Martin laboratory which has extensive experience in uncovering the basic mechanisms of secretion will allow me to generate novel ideas on the mechanisms of exosome secretion. In addition, the Martin laboratory has a strong track record of producing scientists at the independent investigator level preparing the applicant for a career leading an academic laboratory.

Public Health Relevance

Mortality in cancer is usually not caused by the initial tumor itself but rather dissemination of the tumor to other tissues in a process termed metastasis. This proposal will elucidate the mechanisms cancer cells use to communicate with surrounding healthy tissue to promote metastasis. Understanding the mechanisms of cancer cell communication will allow better treatment of cancer metastasis and this laboratory has identified a specific inhibitor that may be used to block cancer cell communication to prevent metastasis.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Jakowlew, Sonia B
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University of Wisconsin Madison
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United States
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Messenger, Scott W; Woo, Sang Su; Sun, Zhongze et al. (2018) A Ca2+-stimulated exosome release pathway in cancer cells is regulated by Munc13-4. J Cell Biol 217:2877-2890