NAT10在肿瘤细胞能量代谢中的作用及分子机制研究

We previously found that the nucleolar acetylase NAT10 translocated from the nucleolus into nucleus under energy stress and chemotherapy. NAT10 is acetylated in cell and autoacetylated in vitro. Importantly, NAT10 was deacetylated by energy deprivation. Protein mass spectrometry analysis was performed to define the acetylation site on NAT10 after in vitro acetylation. The result showed that NAT10 was acetylated at lysine426. We mutated lysine426 to arginine to get NAT10KR mutant. As expected, NAT10KR failed to be acetylated in cell. In addition, NAT10KR lost the capability of activating rRNA transcription. We performed in vitro deacetylation experiments and found that NAT10 was deacetylated by SIRT1 rather than SIRT6 or SIRT7. Importantly, we found that NAT10 interacted with SIRT1 in the nucleus and this interaction was enhanced under energy stress. It was of our interest that NAT10 interacted with AATF in response to chemotherapy. Thus, we are to uncover the mechanism by which NAT10 translocated into the nucleus in response to energy stress and chemotherapy. We are also going to study the NAT10-regulated genes in response to energy stress and chemotherapy. We will study the effect of using inhibitors of glucolysis in combination with chemotherapy and knockdown of NAT10 in nude mice model aiming to discover a novel strategy for tumor therapy. Another phenomena caused our attention is that NAT10 was over-expressed in the nucleus and cell membrane in human tumor tissues including hepatocellular (HCC) and colorectal (CRC) cancers. In this study, we will study the mechanism of NAT10 translocation in human tumors aiming to provide a target for bio-therapy of HCC and CRC.

我们发现：1.核仁蛋白NAT10在低糖状态下从核仁转位到细胞核，并发生去乙酰化；NAT10去乙酰化抑制rRNA的转录；NAT10可被SIRT1去乙酰化；低糖条件下，NAT10与SIRT1结合增强。2. NAT10在人肿瘤组织中转位于细胞核或细胞膜上，其定位的改变与肿瘤患者预后相关。3. NAT10在细胞受到化疗药损伤时转位到细胞核，与AATF结合。本研究将阐明肿瘤细胞在低糖和化疗药物的损伤下NAT10抑制细胞生长、促进细胞生存的分子机制；通过小鼠动物模型阐明NAT10在抑制糖酵解、化疗药处理时对肿瘤细胞的成瘤、转移和对化疗药敏感性的影响；阐明NAT10在肿瘤组织中转位到细胞核或细胞膜的机制、探讨导致患者预后差的作用机制。旨在揭示肿瘤细胞在低糖条件下调节生长与生存的机制、及对化疗药抵抗的机制，为控制肿瘤进展和治疗肿瘤探索新的途径。

当肿瘤细胞遇到能量危机时，需要通过自噬 “补给ATP 产生”或降低 ATP 消耗。在增殖的细胞中核糖体合成消耗总能量的80%，肿瘤细胞在能量应激条件下如何调节核糖体合成和自噬是肿瘤细胞生存的重大问题。本研究发现：在正常条件下，NAT10通过乙酰化Che-1、抑制Che-1对下游基因的转录，抑制自噬。在能量应激时，NAT10被SIRT1去乙酰化，失去对Che-1的抑制，释放自噬，促进细胞在能量应激条件下的存活。这一发现为肿瘤细胞在能量应激条件下的存活提供了一个新的机制。在有丝分裂中，Eg5通过维持纺锤体的正常形成发挥作用。本项目发现，NAT10可能通过结合并乙酰化Eg5，稳定Eg5蛋白，维护细胞有丝分裂，在阻断NAT10乙酰化酶活性或沉默NAT10表达后，细胞发生有丝分裂灾难而导致死亡。为使用NAT10抑制剂治疗肿瘤提供了科学依据。对NAT10的自身蛋白修饰研究发现，NAT10的第426位赖氨酸（K426）被其自身乙酰化，NAT10 K426乙酰化是其在rRNA的加工中所必需。本研究发现，在肝癌肿瘤组织中，NAT10定位于细胞质和细胞膜时，肿瘤侵袭性强、患者的预后差，我们发现NAT10有两个核仁定位序列，当这两个核仁定位序列都发生突变后，NAT10定位到细胞质和细胞膜上，细胞膜上的NAT10与E-cadherin结合，促进Tubulin乙酰化，促进肿瘤转移。同时，NAT10在肝癌中通过结合并稳定突变的p53促进肝癌转移。为NAT10在肿瘤中的作用提供了科学依据。由于NAT10的表达在肿瘤中发挥重要作用，对NAT10的表达调控进行研究，发现NAT10的表达受miR-6717-5p的抑制。本项目的研究结果为NAT10在肿瘤发展和治疗中的作用提供了重要科学依据。