MicroRNAs (miRNAs) fine-tune cellular signaling by regulating expression of signaling proteins, and aberrant expression of miRNAs is observed in many cancers. The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. In previous investigations of miRNA-mediated control of c-Src-related oncogenic pathways, we identified miRNAs that were downregulated in association with c-Src transformation and uncovered the signaling networks by predicting their target genes, which might act cooperatively to control tumor progression. Here, to further elucidate the process of cell transformation driven by c-Src, we analyzed the expression profiles of miRNAs in a doxycycline-inducible Src expression system. We found that miRNA (miR)-129-1-3p was downregulated in the early phase of c-Src-induced cell transformation, and that reexpression of miR-129-1-3p disrupted c-Src-induced cell transformation. In addition, miR-129-1-3p downregulation was tightly associated with tumor progression in human colon cancer cells/tissues. Expression of miR-129-1-3p in human colon cancer cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasion. We also identified c-Src and its critical substrate Fer, and c-Yes, a member of the Src family of kinases, as novel targets of miR-129-1-3p. Furthermore, we found that miR-129-1-3p-mediated regulation of c-Src/Fer and c-Yes is important for controlling cell adhesion and invasion. Downregulation of miR-129-1-3p by early activation of c-Src increases expression of these target genes and synergistically promotes c-Src-related oncogenic signaling. Thus, c-Src-miR-129-1-3p circuits serve as critical triggers for tumor progression in many human cancers that harbor upregulation of c-Src. K E Y W O R D Scolon cancer, malignancy, microRNA, signaling, Src | 419 OKUZAKI et Al.

Cancer cells aberrantly secrete large amounts of extracellular vesicles (EVs) with cargo molecules different from those secreted by healthy cells 1,2 ; therefore, EVs have attracted widespread attention as a novel diagnostic marker that can be easily obtained through liquid biopsy. 3 EVs produced by cancer cells induce phenotypic changes in other cells in both the local and distant tumor microenvironment via the activity of their cargo, thereby supporting the establishment of the premetastatic niche and promoting organspecific metastasis. [4][5][6] Furthermore, EVs play a crucial role in the maintenance of cellular homeostasis as an additional mechanism for

Cancer cells secrete aberrantly large amounts of extracellular vesicles (EVs) including exosomes, which originate from multivesicular bodies (MVBs). Because EVs potentially contribute to tumor progression, EV inhibitors are of interest as novel therapeutics. We screened a fungal natural product library. Using cancer cells engineered to secrete luciferase-labeled EVs, we identified asteltoxin, which inhibits mitochondrial ATP synthase, as an EV inhibitor. Low concentrations of asteltoxin inhibited EV secretion without inducing mitochondrial damage. Asteltoxin attenuated cellular ATP levels and induced AMPK-mediated mTORC1 inactivation. Consequently, MiT/TFE transcription factors are translocated into the nucleus, promoting transcription of lysosomal genes and lysosome activation. Electron microscopy analysis revealed that the number of lysosomes increased relative to that of MVBs and the level of EVs decreased after treatment with asteltoxin or rapamycin, an mTORC1 inhibitor. These findings suggest that asteltoxin represents a new type of EV inhibitor that controls MVB fate.