Annexin A6诱导肿瘤细胞自噬及其分子机制

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some non-essential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Our previous study showed that there are a large number of newly synthesized proteins involved in starvation-induced autophagy and annexin A6 (ANXA6) is one member of them. But the role of ANXA6 in the regulation of autophagy is still not known. Our preliminary data showed that knockdown of ANXA6 decreases the autophagic flux level, attenuates the fusion of autophagosome with lysosome and inhibits lysosomal degradation ability. The related studies indicate that ANXA6 may regulate autophagosome formation by enabling appropriate ATG9A or ATG16L trafficking from endosomes to autophagosomes. But it still needs to be investigated. In this proposal, we aim to systematically study the effect of ANXA6 on autophagy via knockdown of ANXA6 level in Hela cells and to understand the biological function of such autophagy in human cervical cancer, using both cell culture and nude mice xenograft model. Moreover, we will investigate the molecular mechanisms underlying ANXA6-mediated autophagy by focusing on the roles of ATG9A or ATG16L. Finally, we will determine the ANXA6 expression level in human cervical cancer tissues and analyse the association of ANXA6 with the autophagic markers and tumor development stages. Taken together, results from this study will provide new evidence in understanding the important function of ANXA6 in tumor suppression and support the development of novel anti-cancer approaches by targeting ANXA6-mediated autophagic process.

自噬是指细胞在自噬相关基因的调控下通过溶酶体降解自身受损的细胞质蛋白或细胞器，它在转录水平的调控越来越被关注。我们前期的研究发现在饥饿诱导的Hela细胞自噬过程中，有大量新合成的蛋白分子，annexin A6（ANXA6）便是其中之一，但ANXA6在细胞自噬过程中的调控作用并不清楚。我们的预实验结果表明，干扰Hela细胞中ANXA6表达可抑制细胞自噬流，减少自噬泡与溶酶体的融合，减弱溶酶体的降解功能，提示ANXA6诱导细胞自噬发生。与其它膜联蛋白一样，ANXA6促进自噬小体的形成可能与循环内体中含有ATG9A或ATG16L的囊泡被转运至自噬泡有关，但具体机制有待于进一步的研究。本项目拟在此基础上，通过干扰Hela细胞中ANXA6表达、建立荷瘤动物模型、宫颈癌病理组织分析等方法，分析ANXA6对细胞自噬的诱导作用及其相关分子机制，明确ANXA6诱导的细胞自噬在宫颈癌发生发展中的作用。

膜联蛋白ANXA6是细胞自噬过程中新合成的蛋白分子，但ANXA6调控自噬的作用机制并不清楚。本课题中，我们研究了ANXA6调控自噬的作用，分析了ANXA6调控自噬的分子机制，探讨了自噬在宫颈癌发生过程中的作用。研究结果显示，干扰宫颈癌细胞中ANXA6表达可抑制细胞自噬流，减少自噬泡与溶酶体的融合，减弱溶酶体的降解功能，提示ANXA6诱导自噬发生。蛋白组学分析ANXA6靶蛋白显示ANXA6与许多RAB蛋白相互作用并影响细胞内吞功能，提示ANXA6可能通过影响蛋白转运而发挥作用。跨膜蛋白ATG9A参与细胞内吞过程以及自噬泡形成。研究中发现，ANXA6通过RAB或F-actin蛋白将含ATG9A的囊泡从循环内体转运至自噬泡，并参与自噬泡形成。该过程受mTOR信号通路调控，ANXA6通过影响PI3K-AKT和ERK信号通路而抑制mTOR活性。体外细胞和体内动物实验结果都证明ANXA6通过诱导自噬而抑制宫颈癌细胞生长增殖及体内成瘤，宫颈癌组织免疫组化结果也显示ANXA6与自噬蛋白LC3表达水平具有相关性。以上研究结果揭示了ANXA6诱导自噬的分子机制，明确了自噬在宫颈癌发生过程中的作用及重要性，ANXA6有望被发展为判断宫颈癌发生及预后的分子标志物，并为宫颈癌治疗提供了实验依据和研究基础。