CapZβ在早期内体成熟中的功能及分子机制研究

内体运输在癌症转移中起重要作用;然而，其潜在的分子机制仍然知之甚少。我们发现小分子化合物V1是一个有效的内体运输抑制剂，并在多种小鼠模型中均能显著抑制癌细胞的转移。我们随后发现CapZβ敲除消除了V1对内体运输的调控。CapZβ敲除还导致早期内体成熟及细胞迁移的缺陷，并抑制了RAB5的活性。此外，在原位转移小鼠模型中，CapZβ基因敲除显著抑制乳腺癌细胞的转移。我们假设CapZβ通过调节RAB5来调控早期内体的成熟。因此,我们在本次研究中将:(1)进一步确定CapZβ在早期内体成熟中的功能;(2)同时进一步研究CapZβ在早期内体成熟中的分子机制。因为内体运输与包括癌症在内的许多人类疾病有关，该项目的成功将不仅有助于阐明内体运输的分子机制，还能揭示癌症治疗的其他药物靶点。

Metastasis 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 early-to-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.