Perturbation of MLKL-mediated endosomal trafficking enhances TRAIL signal to increase cancer cell death

Abstract

Mixed Lineage Kinase Domain-like (MLKL) is the essential molecule of programmed necrotic cell death, Necroptosis. To induce necroptosis, MLKL is phosphorylated by upstream partner, Receptor Interacting Protein Kinase-3 (RIPK3 or RIP3) and then translocate cellular membrane to disrupt membrane integrity. Recent data suggests that function of MLKL in RIP3-indendent manner in the effective generation of intraluminal and extracellular vesicles as well as in myelin sheath breakdown to promotes sciatic nerve regeneration. In this study, we investigated that whether MLKL play a role in link between TRAIL receptor endocytosis and apoptosis. Depletion of MLKL enhances TRAIL-induced cell death in various cancer cell lines and enhancement of cell death is RIP3-independent manner. TRAIL-induced cytotoxic signals are further increased by preventing endocytosis and MLKL depletion facilitates degradation of TRAIL-DR5. TRAIL-induced intracellular downstream signals were prolonged by depletion of MLKL and then prolonged cytotoxic signals promote cell death. Our data indicate that defect of the intracellular trafficking of TRAIL-DR5 by depletion of MLKL enhances cancer cell death. Based on our findings, it is tempting to speculate that to promote death receptor signals, it may effective to reduced MLKL expression and to prevent survival receptor signal, upregulated MLKL expression contribute to abolish the intracellular signal through the MLKL-dependent extracellular vesicles secretion. Although the processes that regulate these pathways require further elucidation for detail molecular mechanisms, both ways will be the potent therapeutic strategies to target cancer.

현재까지 보고에 의하면, MLKL이라는 단백질은 주로 세포사멸기작 중 하나인 Necroptosis에서 필수적인 작동인자의 하나로써 상위의 RIP3 단백질에 의해 인산화되어 조절되는 것으로 잘 알려져 있다. 최근 보고에 의하면 RIP3-비의존적인 MLKL의 기능으로써 관내막(intraluminal vesicle)과 관외막(extraluminal vesicle)을 형성하여 내막수송 (endosomal trafficking)에 기여한다는 논문이 보고되었다. 하지만 이런 MLKL의 기능과 세포사멸에 대한 직접적인 연관이 보고된 바 없어 본 연구에서는 TRAIL(TNF-related apoptosis-induced ligand) 신호에서의 MLKL 기능에 대해서 연구하고자 한다. TRAIL은 암세포사멸을 일으키는 대표적인 리간드로 이의 수용체인 Death receptor 4/5(DR 4/5)에 결합하여 내포작용(endocytosis)이 일어나 신호전달이 유도되고, 신호전달 이상 이 세포사멸에 영향을 미친다는 것이 보고된 바 있다. 이러한 내용을 토대로 본 연구에서는 MLKL이 결실됨으로써 TRAIL에 의한 세포사멸과 세포신호가 증가됨을 확인하였고, 이는 MLKL이 TRAIL과 TRAIL 수용체의 막 수송에 관여하여 나타나는 결과라는 것을 제시하고 있다.