EGLN3的非羟化酶功能对LUBAC在血管生成中作用的影响

Lung carcinoma is the most common human cancer and is the leading cause of cancer-related deaths in China. The morbidity and mortality of lung cancer continue to rise. There is an unmet need for the effective therapeutics to treat lung carcinoma. Angiogenesis has crucial roles in the development, progression and metastasis of lung cancer. Numerous evidence demonstrates that angiogenesis is predominantly driven by the transcription factor hypoxia-inducible factor 1 (HIF1), which is composed of the labile α and the constitutively expressed β subunits. The expression of the HIF1α protein is tightly controlled by different mechanisms. Ubiquitination of HIF1α is critically important for its homeostasis. Ubiquitination enhances HIF1α protein stability or promotes its degradation, which depends on the type of ubiquitination. The newly identified linear ubiquitination is catalyzed by linear ubiquitin chain assembly complex (LUBAC), the only known E3 ubiquitin ligase that is capable of generating linear ubiquitin chain. The role for LUBAC in HIF1 signaling remains undefined. We are the first to discover the linear ubiquitination of HIF1α in cells. We also found that: 1) HIF1α is a novel binding protein for LUBAC; and 2) LUBAC enhanced the expression of HIF1α protein and VEGF, a well known downstream target of HIF1. Through characterization of a novel LUBAC-HIF1α-VEGF signaling pathway, we will elucidate the role for the linear ubiquitination in regulating the expression, nuclear translocation and transcriptional activity of HIF1α. This study will demonstrate the importance of the linear ubiquitination in driving angiogenesis, growth and metastasis of lung cancer. We also discovered that EGLN3 antagonized the interaction of LUBAC with HIF1α and attenuated HIF1α expression induced by LUBAC. Remarkably, EGLN3 performed the aforementioned functions in a manner independent of its hydroxylase activity. We will investigate the non-catalytic role for EGLN3 in regulating the linear ubiquitination of HIF1α and angiogenesis mediated by LUBAC. Successful completion of this proposal will provide novel insights into the mechanisms by which LUBAC regulates the homeostasis of the HIF1α protein, angiogenesis, cancer growth and metastasis. Our study has a potential translational relevance in the treatment of patients with cancer.

血管生成在肺癌的发生、发展和转移中发挥重要作用。HIF1a在血管生成中发挥着极为关键的作用,其表达受到严格调控。泛素化对HIF1a表达至关重要。HIF1a泛素化抑制或促进HIF1a降解，这取决于泛素化类型。线性泛素化是一种新型泛素化。我们首次发现HIF1a线性泛素化。泛素连接酶LUBAC催化线性泛素化。我们发现LUBAC结合HIF1a和增强HIF1a及VEGF表达。我们将通过研究LUBAC-HIF1a-VEGF通路阐明LUBAC和线性泛素化对HIF1a表达及核转位和血管生成等的作用。EGLN3抑制LUBAC-HIF1a相互作用和LUBAC诱导的HIF1a表达。EGLN3上述作用不依赖其羟化酶活性。我们研究EGLN3非羟化酶功能对LUBAC诱导的HIF1a线性泛素化、血管生成等的影响。该研究将阐明LUBAC在血管生成，肿瘤生长和转移中的作用和调控，具有重要的理论价值及临床转化前景。

LUBAC在肿瘤发生发展中具有重要作用，但作用机制仍模糊不清。HIF1a蛋白表达调控是肿瘤领域的重大课题。现已公认，细胞O2浓度是调节HIF1a蛋白表达的关键机制。我们发现，LUBAC显著增强HIF1a蛋白稳定性，但这不依赖细胞O2浓度。我们发现，LUBAC与HIF1a相互作用；LUBAC诱导HIF1a线性泛素化，HIF1a线性泛素化增强其稳定性及活性。我们发现，LUBAC通过拮抗CMA关键分子HSC70和LAMP2A与HIF1a的相互作用和CMA降解HIF1a的作用而稳定HIF1a。因此，我们阐明了调控HIF1a蛋白稳定性的新机制。LUBAC促进HIF1a活性和增强VEGF表达与产生，我们建立了一个全新的LUBAC-HIF1a-VEGF-血管生成通路。LUBAC通过调控HIF1a促进肿瘤血管生成、增殖、侵袭和迁移从而促进肿瘤发生发展。我们的研究揭示了LUBAC调控肿瘤的新机制。我们发现，EGLN3通过拮抗LUBAC的作用抑制HIF1a表达和活性，阐明了EGLN3调控HIF1a 的新机制。我们也发现EGLN3羟化酶通过羟化Erk3抑制CMA降解Erk3的作用。这加深了我们对EGLN3生物学作用及其机制的理解。EGLN3在肿瘤中起着重要作用，我们对EGLN3羟化酶活性在肿瘤中的作用及其机制知之甚少。利用本课题组构建的EGLN3羟化酶活性丧失的小鼠，我们首次阐明了肺癌间质细胞（特别是巨噬细胞）EGLN3羟化酶活性对肺癌生长的影响及机理；我们也探讨了肺癌细胞EGLN3羟化酶活性对肺癌生长的影响及其机制。我们首次揭示了EGLN3羟化酶活性对肺癌生长的作用及机制。总之，我们阐明了EGLN3在肿瘤发生发展中的新功能和新机制。NLRP3炎症小体是LUBAC的底物，NLRP3炎症小体在诸多疾病中发挥关键作用。动脉粥样硬化是人类心血管疾病的罪魁祸首。越来越多的证据显示，NLRP3炎症小体在动脉粥样硬化发生发展中发挥关键作用，但机制不详。我们阐明了NLRP3炎症小体活化对细胞线粒体损伤、巨噬细胞泡沫细胞形成及胞葬作用的影响，极大地拓宽了NLRP3炎症小体在动脉粥样硬化发生发展中的作用机制。我们的研究表明：LUBAC抑制剂和EGLN3抑制剂具有潜在的肿瘤治疗价值；开发特异的NLRP3抑制剂将有利于动脉粥样硬化及相关疾病（如冠心病和中风）的防治。我们的研究具有重要的理论价值和临床转化前景。