乙酰化酶NAT10激活RhoGTPase促进结肠癌侵袭与转移机制研究

Metastasis, a characteristic of cancer cells, is a fatal threat to cancer patients. Actually, anti-metastasis is still a pivotal challenge to oncologists in clinical. And therefore, the elucidation of mechanism on metastasis has long been pursued by lots of researchers in life science. Owing to great efforts made in the latest decade, many issues regarding to metastatic process of cancer cells have become clearer ever than before. As one of those, the molecular basis of cell motility or migration has been extensively revealed and remodeling or assembly of cytoskeleton has emerged at the central stage. The activation of RhoGTPases, a group of factors that belongs to small GTPases, has been identified a crucial step for initiation of cytoskeletal remodeling to result in formation of protrusions for cell migration such as filopodia, lamellipodia, focal adhesions and stress fibers. In the precess, a vast of proteins sequentially are activated or inactivated through direct interaction and protein modification such as phosphorylation and acetylation/or deactylation. However, in contrast to phosphorylation, the regulation by protein actylation has been less documented. In previous research, we have found that NAT10, an acetyltransferase, could take a translocation from nucleolus to cytoplasm or membrane of colorectal cancer cells under the activation of Wnt/beta-catenin signaling. The membrane translocation of NAT10 dominantly distributes in the front of cancer invasion,as the same as the nuclear accumulation of beta-catenin,correlating with the advanced stage and spread of colorectal cancers. The proposed investigation will further focus the mechanism on the regulation of NAT10 on the motility and migration of cancer cells, including: 1) the activation of RhoGTPases such as RhoA, RAC1 and CDC42 by NAT10 through acetylation of GD1-alpha and p120-catenin;2)the remodeling or assembly of cellular cytoskeleton, actins and microtubules; 3) the interactive effects between NAT10 and deacetylase of HDAC6 in the regulation process; 4) the genomic alterations of NAT10 in colorectal cancers. The research is believed to provide more clues for uncovering the mechanism of cancer metastasis and more targets for anti-metastasis strategy.

转移是恶性肿瘤的根本特性，也是肿瘤治疗的最大难点。阐明侵袭、转移机制是抗肿瘤的关键之一。以RhoGTPases激活所诱导的细胞骨架蛋白重构或组装是促进肿瘤细胞侵袭、转移的核心环节。而其中蛋白质磷酸化、乙酰化修饰起着重要的调节作用。在前期研究我们发现乙酰化酶NAT10（N-acetyltransferase 10）与结肠癌Wnt通路激活，以及侵袭、转移密切相关，并可能通过激活细胞骨架重构而调节细胞迁移与侵袭。本研究拟在此基础上，运用分子生物学、细胞生物学、分子病理学等技术，进一步确定NAT10通过乙酰化GD1α及p120-catenin激活RhoGTPases、调节细胞骨架重构而促进癌细胞侵袭的作用；并确定在此作用过程中去乙酰化酶HDAC6的作用；确定NAT10基因改变和相关作用分子在结肠癌侵袭、转移中的关系与作用；为深入揭示NAT10调节肿瘤侵袭、转移分子机制、寻找新的抗肿瘤转移靶点提供线索。

转移是结肠癌治疗的最大难点，阐明其侵袭转移机制是抗肿瘤的关键之一。在本研究中，我们对乙酰化酶NAT10(N-acetyltransferase 10)与结肠癌侵袭转移相关性、作用，NAT10上游相关信号通路激活的调节，NAT10对细胞骨架重构的调节及机制，抑制NAT10对结肠癌细胞生物学特性的影响进行了全面、多层次的实验研究。结果表明：1)NAT10蛋白表达易位与结直肠癌侵袭转移、临床分期及预后相关。NAT10基因过表达及胞浆再分布增强结直肠癌细胞迁移和侵袭能力。2)NAT10基因过表达及再分布并非由基因突变，而主要受到糖原合成酶激酶GSK-3β直接调节：即GSK-3β活性抑制可以增加NAT10蛋白稳定性，提高其蛋白水平表达与亚细胞分布。而GSK-3β调节NAT10的作用受上游Wnt信号通路的调节，其中LRP6的激活起着重要的作用，并且也受到与EGFR/AKT信号通路的cross talk作用调节。磷酸化LRP6 以及磷酸化AKT均是结肠癌预后不良的标志。3)NAT10具有乙酰化α-tubulin提高其稳定性的作用，并促进细胞微触须(Microtentacles) 形成而增强癌细胞间或与间质的粘附性。4)NAT10可诱导微丝重构，促进细胞伪足形成。NAT10可以提高RhoGTPases活性，但不是直接通过对 RhoGDIα、p120-catenin、Tiam1乙酰化作用而实现，而可能与Wnt等上游信号通路对细胞骨架的调节有关。5)NAT10与去乙酰化酶HDAC6具有相互作用，共同完成对细胞骨架的动态平衡调节。6)NAT10抑制剂Remodelin可降低NAT10水平，抑制其相应生物学功能：降低乙酰化微管水平，抑制细胞周期演进，细胞有丝分裂，细胞迁移，从而抑制结肠癌细胞生物学行为。另外，研究还提示NAT10可能通过其它机制调节癌细胞侵袭转移，如：1)NAT10诱导和促进癌细胞衰老相关分泌表型(SASP, senescence-associated secretory phenotype)，通过调节肿瘤微环境而影响侵袭转移；2)NAT10可能参与癌细胞侵袭性伪足(Invadosome)的形成；3)NAT10通过修饰tRNA前体，促进细胞外泌体RNA生成的作用。综上，本研究深入揭示了NAT10调节结肠癌侵袭、转移的作用与机制，为临床诊治提供了潜在的新标志物和新治疗靶点。

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