AKT 의존적 MEL-18 인산화의 역할에 대한 연구

Abstract

MEL-18은 polycomb repressive complex (PRC)-1의 구성 요소로서, AKT 인산화를 음성적으로 조절하는 것을 통해 암 억제 유전자로서의 역할을 한다. MEL-18의 homolog인 BMI-1을 포함하고 있는 몇몇의 polycomb group 단백질들은 AKT의 새로운 기질이라는 것이 밝혀졌다. AKT 조절인자들은 AKT에 의해 조절될 수 있으므로, MEL-18과 AKT간의 상호적인 피드백 가능성을 확인하였다. 따라서 본 실험에서는 AKT 의존적인 MEL-18 인산화가 MEL-18의 암억제 기능에 중요한지 밝히고자 하였다. AKT는 MEL-18 단백질과 직접적으로 상호작용하고, AKT 활성에 의존적으로 트레오닌 334 (T334) 부위를 인산화 시킨다. 정상적인 MEL-18과 T334A (촉매 비활성화 돌연변이)를 각각 발현하는 세포주를 구축하여 RNA-seq 분석을 수행하였고, 여러 유전자들의 달라진 발현 패턴을 증명하였다. 이 효과는 MEL-18 인산화에 의한 PRC-1과 PRC-2 간의 변화된 상호작용에 기인한다. EZH2는 Histone H3 lysine-27 삼중 메틸화 (H3K27me3) 를 유도하는 PRC-2의 구성요소이다. MEL-18 인산화가 존재할 때, MEL-18은 EZH2와 결합함으로써 H3K27me3 의존적인 ITGB4 유전자 억제를 유도한다. ITGB4 유전자는 EMT와 암줄기세포 조절인자로서 역할을 한다. 또한 MEL-18의 EMT와 암줄기세포 유사 특징을 억제하는 효과는 MEL-18 T334A 발현 세포주에서는 영향을 미치지 않는다. in vivo 이종 이식 실험에서 인산화의 돌연변이 형태에 의해 MEL-18의 암억제 활성화는 나타나지 않는다. 따라서 본 연구에서는 AKT 의존적인 MEL-18 T334 부위의 인산화는 인체 유방암에서 EMT와 CSC을 억제하는 것을 통해 암 억제 기능을 수행한다는 것을 제시하였다.| MEL-18, a component of polycomb repressive complex (PRC)-1, acts as a tumor suppressor in human cancer. However, the regulatory mechanism of its protein activity and epigenetic functions remain largely unknown. Here, I found that AKT-dependent MEL-18 phosphorylation enhances the tumor suppressive function of MEL-18 via altering polycomb group (PcG)-mediated epigenetic mechanisms. AKT directly interacted with MEL-18 protein and phosphorylated it at threonine 334 (T334) in a dependent manner of AKT activity. RNA-seq analysis identified the differentially expressed genes between MEL-18 wild type‒ and T334A (catalytic inactive mutant) expressing cells that were functionally involved in the epithelial -mesenchymal transition (EMT)-related phenotypes including tumor migration and metastasis. In the PcG-mediated gene silencing mechanism, MEL-18 interacted with EZH2, a main component of PRC2, in addition to RING1B in a dependent manner of its phosphorylation at T334, and facilitated histone H3 lysine-27 tri-methylation (H3K27me3)-mediated transcriptional repression of several EMT and cancer stem cell regulators including ITGB4 and TCF4. The inhibitory effect of MEL-18 on EMT/CSC was abolished in MEL-18 T334A (catalytic inactive mutant)-expressing cells. An in vivo xenograft assay also exhibited the reversed tumor suppressive activity of MEL-18 by its phosphorylation mutant form. Taken together, these findings suggest that AKT-dependent MEL-18 phosphorylation at T334 plays a role in maintaining tumor suppressive function of MEL-18 by contributing to the inhibition of EMT and CSC in breast cancer.; MEL-18, a component of polycomb repressive complex (PRC)-1, acts as a tumor suppressor in human cancer. However, the regulatory mechanism of its protein activity and epigenetic functions remain largely unknown. Here, I found that AKT-dependent MEL-18 phosphorylation enhances the tumor suppressive function of MEL-18 via altering polycomb group (PcG)-mediated epigenetic mechanisms. AKT directly interacted with MEL-18 protein and phosphorylated it at threonine 334 (T334) in a dependent manner of AKT activity. RNA-seq analysis identified the differentially expressed genes between MEL-18 wild type‒ and T334A (catalytic inactive mutant) expressing cells that were functionally involved in the epithelial -mesenchymal transition (EMT)-related phenotypes including tumor migration and metastasis. In the PcG-mediated gene silencing mechanism, MEL-18 interacted with EZH2, a main component of PRC2, in addition to RING1B in a dependent manner of its phosphorylation at T334, and facilitated histone H3 lysine-27 tri-methylation (H3K27me3)-mediated transcriptional repression of several EMT and cancer stem cell regulators including ITGB4 and TCF4. The inhibitory effect of MEL-18 on EMT/CSC was abolished in MEL-18 T334A (catalytic inactive mutant)-expressing cells. An in vivo xenograft assay also exhibited the reversed tumor suppressive activity of MEL-18 by its phosphorylation mutant form. Taken together, these findings suggest that AKT-dependent MEL-18 phosphorylation at T334 plays a role in maintaining tumor suppressive function of MEL-18 by contributing to the inhibition of EMT and CSC in breast cancer.