Zhu Jun, Lv Yongzhi, Hao Jun, Shi Tingyu, Wang Shuai, Wang Ke, Fan Xiaoyan, Guo Yuan, Zhang Jian, Li Jipeng
State Key Laboratory of Cancer Biology, Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.
The First Affiliated Hospital of Xi'an Medical University, Xi'an, China.
J Gastrointest Oncol. 2020 Dec;11(6):1200-1213. doi: 10.21037/jgo-20-557.
N-myc downstream-regulated gene 2 (NDRG2) and estrogen receptor beta (ERβ) both play key roles in cellular differentiation in colorectal cancer (CRC). Previous studies have demonstrated that ERβ co-locates with and directly transactivates NDRG2. However, the effect of NDRG2 on ERβ and its underlying mechanism remain largely unknown. Our aim of the study is to explore the effect of NDRG2 on ERβ and their contributions to progression of CRC.
The Cancer Genome Atlas (TCGA) database was first utilized to validate the clinical significance of ERβ and NDRG2 in CRC. MTT and scratch migration assays were carried out to verify the role of ERβ and NDRG2 in CRC cells. Western blotting and polymerase chain reaction were performed to analyze the effect of NDRG2 on ERβ, and an immunoprecipitation assay was conducted to explore the protein-protein interaction.
ERβ and NDRG2 were both found to be significantly down-regulated in tumor tissues from the TCGA-CRC database. NDRG2 was also observed to enhance the protein stability of ERβ while could not change messenger RNA (mRNA) level of ESR2 (encoding ERβ). A positive relationship was found to exist between the two proteins in CRC cells, with NDRG2 prolonging the half-life of ERβ and improving its nuclear translocation. Through detecting expression of ERβ downstream genes (such as TP53 and JNK) and performing related function experiment, we demonstrated that NDRG2 could promote transcriptional activation of ERβ target genes and enhance the function of tumor suppressors when the ERβ agonist diarylpropionitrile (DPN). The immunoprecipitation assay showed that NDRG2 could affect the complex components of ubiquitin-protein ligase E3A (UBE3A, known as E6AP) and ERβ, reducing the ubiquitin-mediated proteasome degradation of ERβ.
In the current study, we found that NDRG2 could bind with UBE3A to hinder the binding of UBE3A with ERβ. Moreover, a positive feedback loop was discovered between NDRG2 and ERβ, which provides a novel insight and therapeutic target for CRC.
N-myc下游调控基因2（NDRG2）和雌激素受体β（ERβ）在结直肠癌（CRC）的细胞分化中均发挥关键作用。既往研究表明，ERβ与NDRG2共定位并直接反式激活NDRG2。然而，NDRG2对ERβ的影响及其潜在机制仍 largely未知。本研究的目的是探讨NDRG2对ERβ的影响及其对CRC进展的作用。
首先利用癌症基因组图谱（TCGA）数据库验证ERβ和NDRG2在CRC中的临床意义。进行MTT和划痕迁移试验以验证ERβ和NDRG2在CRC细胞中的作用。进行蛋白质印迹和聚合酶链反应以分析NDRG2对ERβ的影响，并进行免疫沉淀试验以探索蛋白质-蛋白质相互作用。
在TCGA-CRC数据库的肿瘤组织中发现ERβ和NDRG2均显著下调。还观察到NDRG2可增强ERβ的蛋白质稳定性，但不能改变ESR2（编码ERβ）的信使核糖核酸（mRNA）水平。在CRC细胞中发现这两种蛋白质之间存在正相关关系，NDRG2延长了ERβ的半衰期并改善了其核转位。通过检测ERβ下游基因（如TP53和JNK）的表达并进行相关功能实验，我们证明当使用ERβ激动剂二芳基丙腈（DPN）时，NDRG2可促进ERβ靶基因的转录激活并增强肿瘤抑制因子的功能。免疫沉淀试验表明，NDRG2可影响泛素-蛋白连接酶E3A（UBE3A，也称为E6AP）和ERβ的复合物成分，减少泛素介导的ERβ蛋白酶体降解。
在本研究中，我们发现NDRG2可与UBE3A结合以阻碍UBE3A与ERβ的结合。此外，在NDRG2和ERβ之间发现了一个正反馈环，这为CRC提供了新的见解和治疗靶点。
