Mechanistic Studies of CapZβ Signaling in the Maturation of Early Endosomes
DescriptionMetastasis is the fundamental cause of the high level of cancer mortality in the world today. Whereas <10% of cancer patients die of primary tumors, up to 90% die of metastasis. Endosomal trafficking is an evolutionarily-conserved cellular process, which is known to play an important role in metastasis; however, the underlying molecular mechanisms remain poorly understood. We previously demonstrated that a small chemical, vacuolin-1 (V1), prevents the transition of earlyto- late endosomes via the activation of RAB5, and this results in the formation of enlarged early endosomes. We also showed that V1 significantly inhibits metastasis of various cancer cells in both xenograft and transgenic mouse models, which indicates that V1 is a potential anti-metastasis drug. In our current study, we found that knockout of capping protein Z beta (CapZβ) by CRISPR/Cas9 abolished the ability of V1 to induce the enlargement of early endosomes in cells. We showed that CapZβ is associated with early endosomes, and the treatment of cells with V1 induced an accumulation of CapZβ on the surface of enlarged early endosomes. However, CapZβ knockout significantly inhibited both the maturation of early endosomes and cell migration, and these defects could be rescued by adding back sgRNA-resistant CapZβ (rCapZβ). We also demonstrated that RAB5 is less active in CapZβ knockout cells. Moreover, CapZβ knockout significantly inhibited metastasis of breast cancer cells in an orthotopic metastatic mouse model. We hypothesize that CapZβ is essential for the maturation of early endosomes by regulating RAB5. Therefore, here we propose to: (1) further determine the function of CapZβ in early endosome maturation; and (2) dissect the molecular mechanisms underlying CapZβ-mediated early endosome maturation. Given that dysfunctional endosomal trafficking has been associated with many human diseases, including cancer, the success of this project will shed light on the mechanisms underlying endosomal trafficking, and reveal additional drug targets for cancer therapy.
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