抑癌蛋白PTEN的去泛素化酶鉴定及蛋白稳定性调控研究

The tumor suppressor PTEN (phosphatase and tension homologue) is physiologically relevant to the development of various types of human cancers and governs a plethora of cellular processes by suppressing the PI3K-Akt pathway through its lipid phosphatase activity. The tumor suppressor activity of PTEN is tightly controlled by the subtle variation of its dosage. Notably, the half-life of PTEN protein is relatively long, suggesting that the mechanism controlling the PTEN protein stability is pivotal for the PTEN activity. So far, although the E3 ubiquitin ligase which promotes the ubiquitination of PTEN and the deubiquitinase (DUB) responsible for de-monoubiquitination of PTEN have been reported, the deubiquitinase for de-polyubiquitination of PTEN and then prevents its degradation has not been characterized. We recently identified the deubiquitinase OTUD3 (OTU domain containing protein 3) as a specific member of human DUB family which can interacts with and stabilizes PTEN protein. OTUD3 prevents the proteasomal degradation of PTEN through removing the K48-linked polyubiquitination of PTEN. Depletion of OTUD3 by RNA interference resulted in the dramatic degradation of PTEN, the increased activation of Akt kinase and the accelerated tumor cell proliferation and invasion. These data suggest that OTUD3 is a critical stabilizer protein of PTEN. In this program, we ought to establish the OTUD3 gene knockout and transgenic mice models and investigate the functional relevance of the OTUD3-PTEN interaction in animal development, metabolic regulation and tumorigenesis. We will also characterize the key sites responsible for the interaction between PTEN and OTUD3, and explore the effect of tumor mutation on their interaction and function. Through tumor formation assays in nude mice and specimen analysis of cancer patients, we expect to obtain the evidence to support OTUD3 as a novel tumor suppressor gene. Taken together, this program will contribute to elucidate the regulatory mechanisms of PTEN tumor suppressor.

抑癌蛋白PTEN与许多肿瘤发生发展关系密切，它作为磷酸酶抑制PI3K-Akt通路激活，其抑癌活性与剂量的精细水平密切相关，加之PTEN蛋白半衰期相对较长，揭示PTEN蛋白稳定性的维持机制具有重要意义。虽已知促PTEN降解的泛素连接酶和去单泛素化的去泛素化酶（DUB），但抑制PTEN降解、促其去多聚泛素化的DUB仍未知。我们从DUB的90个成员中鉴定到OTUD3特异结合并增强PTEN稳定性，它通过去除PTEN的K48型泛素链阻止其降解；其敲低导致PTEN泛素化降解加快、Akt过度激活、细胞增殖与侵袭增加。因此这是一个重要的PTEN稳定因子。本项目拟建立OTUD3基因敲除与转基因小鼠模型，确证OTUD3调控PTEN稳定性的重要性及其在代谢调控中的功能；确定二者的结合位点，筛查肿瘤突变；通过动物成瘤与临床病例研究，获取OTUD3作为抑癌基因的证据。项目对于深入理解PTEN的调控机理具有重要意义。

抑癌蛋白PTEN的稳态调控机制包括蛋白量与亚细胞定位的调节机制是肿瘤研究领域的重要科学问题。项目研究内容包括鉴定维持PTEN蛋白稳定性的去泛素化酶、鉴定PTEN的生理性泛素连接酶、揭示PTEN亚细胞定位调控机制。项目进展如下：（1）通过全库筛选，鉴定到维持PTEN蛋白稳定性的去泛素化酶OTUD3，它通过去除PTEN的多聚泛素链、维持其蛋白稳定性、抑制乳腺癌发生发展。OTUD3在乳腺癌组织中低表达，伴随PTEN低表达，与肿瘤进程相关。OTUD3在部分患者中存在功能失活突变，使得PTEN过度降解、失去抑癌功能。（2）建立了OTUD3基因敲除与转基因小鼠模型，开展了乳腺癌与肺癌成瘤研究，发现其敲除导致乳腺癌成瘤加快、而转基因则成瘤减慢；进一步在肝癌、结直肠癌、宫颈癌等验证了OTUD3的抑癌作用。令人吃惊的是，发现OTUD3敲除导致肺癌成瘤减慢、而转基因则加快成瘤，提示促肺癌的作用。机制研究发现OTUD3通过去泛素化并稳定GRP78促进肺癌增殖。这表明在不同微环境下，OTUD3通过调控不同底物发挥抑癌或促癌的功能。（3）利用小鼠模型揭示泛素连接酶WWP2（而非CHIP）是促进PTEN降解的主要E3，去泛素化酶OTUD3和USP13均是阻止PTEN降解的主要DUB，二者单/双敲除均可诱发小鼠自发成瘤。这些体内证据明确了究竟哪些酶在生理条件下控制了PTEN稳定性。（4）发现高葡萄糖条件下PTEN可被Nedd8修饰，XIAP和NEDP1主导修饰形成和去除。该修饰不影响PTEN稳定性但可促进其入核并结合FASN，加快脂肪酸合成，促进肿瘤形成，这些结果揭示了PTEN的一种新修饰类型及核内PTEN的促癌功能。综上，项目取得了系列原创性发现，加深了对PTEN稳态调控机制的理解。论文发表在Nature Cell Biology、JBC等，另有2篇被Nature Communications回修，获2项发明专利授权，培养7名研究生。项目负责人入选国家万人计划、国家百千万人才工程，获得谈家桢生命科学奖创新奖、树兰医学奖青年奖等。