中国科学院昆明动物研究所机构知识库

肿瘤转移是大多数癌症患者死亡的重要原因，肿瘤微环境在转移中扮演重要角色。原位肿瘤中的肿瘤浸润淋巴细胞，巨噬细胞，成纤维细胞以及血管内皮细胞等与肿瘤的生长和转移相关，而远端器官的血管内皮细胞对肿瘤细胞的粘附能力，决定了循环肿瘤细胞是否转移并定居至远端器官。我们在MMTV-Neu乳腺癌自发小鼠模型和尾静脉注射肿瘤细胞肺转移小鼠模型中发现，敲除E3泛素连接酶Hectd3基因能显著降低肿瘤的肺转移。用PyMT-GFP肿瘤细胞尾静脉注射短时间（20 h）取肺观察发现，Hectd3 KO小鼠肺部粘附的肿瘤细胞数量明显降低。小鼠肺部血管内皮细胞（mEC）对肿瘤细胞的粘附是肿瘤细胞进入肺实质组织的一个先决条件，而内皮细胞中粘附分子的上调能促进这种粘附作用。用炎症因子LPS和TNFα刺激分离的原代小鼠肺部血管内皮细胞发现，Hectd3 KO能降低粘附分子VCAM-1，ICAM-1和E-selectin的表达水平。在人脐静脉内皮细胞（HUVEC）中，敲低HECTD3能抑制炎症因子LPS，TNFα和IL-1β对粘附分子VCAM-1，ICAM-1和E-selectin的诱导表达，并且降低HUVEC细胞对多种人肿瘤细胞，如HCC1937，MDA-MB231，MDA-MB468和Jurkat，的体外粘附能力。进一步研究发现， HECTD3能与IKK复合物中的激酶IKKα相互作用并泛素化稳定IKKα蛋白水平，促进经典NF-κB信号通路活化；另一方面，HECTD3促进IKKα入核，入核的IKKα修饰靶基因启动子处Histone3 Ser10的磷酸化，促进NF-κB对粘附分子的转录调控。总而言之，在内皮细胞中，HECTD3与NF-κB信号通路关键分子IKKα相互作用，可以通过非降解型泛素化修饰增加其稳定性和核定位，从而诱导下游粘附分子表达，增强肿瘤细胞的远端器官转移。Hectd3 KO小鼠生长发育及生殖能力均无明显异常，这可能提示抑制HECTD3没有明显的毒副作用。IKKα的抑制剂在小鼠模型明显抑制肿瘤转移。筛选针对HECTD3的特异性小分子抑制剂，可能成为一种防治肿瘤转移的新策略。 TRAF3的K63链多聚泛素化修饰对于TBK1参与的Ⅰ型干扰素诱导起关键作用，多种模式识别受体信号通路都能促进TRAF3的K63链多聚泛素化，激活TBK1。然而，TRAF3本身作为一个E3泛素连接酶能发生自我泛素化修饰，但TRAF3的K63链泛素化修饰形式是其自身泛素化的结果还是由其他E3介导仍待进一步研究。我们发现，E3泛素连接酶Hectd3的基因敲除小鼠通过限制细菌体内播散强烈地抵抗胞内菌，如弗兰西斯菌（F. novicida）分枝杆菌（Mycobacterium）和李斯特菌（Listeria），的感染。体内外实验均证明，在细菌感染过程中，HECTD3缺失会破坏宿主细胞Ⅰ型干扰素通路的响应。HECTD3能介导TRAF3 138位赖氨酸的K63链类型的泛素化修饰，促进TRAF3-TBK1复合物的形成，继而促进Ⅰ型干扰素的产生。我们的研究为TRAF3的功能调节提供了新的机制，也为抗击胞内菌感染和炎症疾病提供了新的潜在治疗靶标。

Tumor metastasis, which commonly occurs at the late stage, is responsible for the majority of cancer deaths. Tumor microenvironment plays a critical role in metastasis. Many types of stromal cells can affect the growth and development of tumor, including tumor infiltrating lymphocytes (TILs), tumor-associated macrophages(TAMs), tumor-associated fibroblasts and endothelial cells, while the colonization of circulating tumor cells in distant organs depends on adhesion by vascular endothelium. Here we demonstrated that Hectd3 KO mice significantly reduced breast cancer cell lung metastasis in the MMTV-Neu mice model and the experimental metastasis model transplanted tumor cells through tail vein injection. In the experimental metastasis model, we found that less PyMT-GFP tumor cells were in the lung of Hectd3 KO mice with pretreatment by LPS. Hectd3 KO downregulated the expression of adhesion moleculs, such as VCAM-1,ICAM-1 and E-selectin, in the mouse primary endothelial cells (mEC) isolated from pulmonary vessels，when the mEC were treated with LPS or TNFα. When HECTD3 was knocked down in HUVEC (human umbilical vein endothelial cell), the expression of adhesion molecules (VCAM-1, ICAM-1 and E-selectin) induded by inflammation factors, including LPS, TNFα and IL-1β, was inhibited, and the fuction of HUVEC adhering to human breast cancer cell lines, including HCC1937, MDA-MB231 and MDA-MB468 and acute T cell leukemia cell line Jurkat. Subsequently, we found that HECTD3 can interact with and stabilize IKKα，a component of IKK kinase complex which plays a crucial role in canonical NF-κB pathway, which increased the nuclear localization of p65 and the activation of NF-κB pathway. On the other hand, HECTD3 may promote the nuclear translocation of IKKα through non-degradative ubiquitin modification. In the nucleus, IKKα can phosphorylate Histone3 Ser10 at the of target genes of NF-κB pathway and upregulate the expression of these genes. In summary, HECTD3 interacts with IKKα and ubiquitinates IKKα with non-degradative poly-ubiquitin chains, which stabilize IKKα and promote expression of adhesion molecules and cancer metastasis. HECTD3 may be a safety drug target, because the Hectd3 KO mice were born at expected Mendelian ratios and had comparable survival rate as the WT mice. HECTD3 small molecule inhibitors may provide a novel strategy for tumor metastasis prevention and treatment. Lysine-63–linked polyubiquitination of TRAF3 coordinates the engagement of pattern recognition receptors to recruited adaptor proteins and downstream activator TBK1 in pathways that induce type I interferon (IFN). Whether autoubiquitination or other E3 ligases mediate K63-linked TRAF3 polyubiquitination remains unclear. We demonstrated that mice deficient in E3 ligase gene Hectd3 remarkably increased host defense against infection by intracellular bacteria F. novicida, Mycobacterium, and Listeria by limiting bacterial dissemination. In the absence of HECTD3, type I IFN response was impaired during bacterial infection both in vivo and in vitro. HECTD3 promotes type I IFN production by mediating K63-linked polyubiquitination of TRAF3 at residue K138. Our study offers novel insights into mechanisms of TRAF3 modulation and provides potential therapeutic targets against infections byintracellular bacteria and inflammatory diseases.