PO.MCB03.03 · 分子与细胞生物学
Cyclin F在代谢应激过程中于非小细胞肺癌中的新兴作用
Emerging roles of Cyclin F in Non-small lung cell carcinoma during metabolic stress
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：Cyclin F作为孤儿Cyclin，与其他Cyclin不同之处在于：1）它不结合任何细胞周期蛋白依赖性激酶（cdk）；2）它拥有一个结构域，将Cyclin F归类为一类独特蛋白——F-box蛋白的成员。诸如Cyclin F这类蛋白由此促进对底物蛋白的识别，并介导Skp1-Cul1-F box（SCF）复合体对蛋白的泛素化和降解。Cyclin F在肿瘤发生中的作用尚不明确，但鉴于其作为基因组守护者和营养感应分子的作用，我们在肺癌模型中探究了其在肿瘤中的作用。
目的：阐明Cyclin F驱动的泛素化及对细胞蛋白调控的独特功能，这些改变了癌症的致瘤能力。
方法：鉴定Cyclin F相互作用组——使用腺病毒系统异位表达Cyclin F；免疫沉淀Cyclin F及其推定的相互作用蛋白，用LC-MS/MS检测蛋白。研究生理条件下相互作用的动态。理解Cyclin F介导的蛋白调控对癌细胞（非小细胞肺癌；NSCLC）细胞通路的影响。
结果：我们构建了一种表达Cyclin F并带有HA和FLAG标签的腺病毒载体（Ad-CCNFHF-GFP）。表达Cyclin F及标签的腺病毒由HEK293A细胞产生。病毒滴度用于在H1299细胞中异位表达Cyclin F，随后免疫沉淀Cyclin F并鉴定与Cyclin F相关的蛋白质组。我们在筛选中鉴定出一种RNA结合蛋白，称为PTBP1。PTBP1与肿瘤细胞中异常的剪接活性相关，其表达与不良预后相关。我们证实，饥饿期间Cyclin F的上调导致PTBP1蛋白水平相应下降。PTBP1下调导致PKM mRNA第10外显子的排除，并改变了PKM2/PKM1比值。我们观察到Cyclin F介导的PTBP1和PKM2耗竭降低了肿瘤细胞的糖酵解能力，并进一步导致癌细胞在饥饿时增殖、侵袭和迁移率下降。小鼠数据也印证了类似结果。
结论：我们的研究为代谢应激过程中蛋白相互作用如何改变癌细胞命运提供了线索。它确立了Cyclin F负向调控PTBP1/PKM2水平，并在体外/体内模型中降低NSCLC细胞的致瘤潜能。阐明分子相互作用将为NSCLC进展和治疗抵抗提供见解，这是设计靶点的第一步。
关键词：非小细胞肺癌、Cyclin F、致瘤能力、腺病毒表达、RNA结合蛋白
查看英文原文 English abstract
Introduction : Cyclin F, the orphan Cyclin, is unique from other Cyclins- 1) it does not bind to any cyclin dependent kinase (cdk) and 2) It has a domain which categorizes Cyclin F as a member of a distinct class of proteins the F-box proteins. Proteins such as Cyclin F, thereby facilitates the recognition of substrate proteins and mediates the ubiquitylation and degradation of the protein by the Skp1-Cul1- F box (SCF) complex. Role of Cyclin F in tumorigenesis is obscure but owing to its role as a genome guardian and nutrient sensing molecule, we explored its role in tumor in a lung cancer model
Aim : To delineate the unique function of Cyclin F driven ubiquitylation and regulation of cellular proteins which alters the tumorigenic capacity of cancers.
Methodology : Identification of Cyclin F interactome- ectopic expression of Cyclin F using adenoviral system; immunoprecipitation of Cyclin F and its putative interacting proteins, detection of proteins with LC-MS/MS. Investigate the dynamics of the interaction under physiological conditions. Understanding the effect of Cyclin F mediated regulation of proteins in cellular pathways in cancer cells (Non-small lung cell carcinoma ; NSCLC).
Results : We developed an adenoviral vector expressing Cyclin F along with HA and FLAG tags (Ad-CCNFHF-GFP). Adenovirus expressing Cyclin F and the tags were produced from HEK293A cells. The viral titer was used to ectopically express Cyclin F in H1299 cells, followed by immunoprecipitation of Cyclin F and identification of the proteome associated with Cyclin F. We identified an RNA binding protein in our screen, called PTBP1. PTBP1, is associated with aberrant splicing activity in tumor cells and its expression is associated with poor prognosis. We verified that Cyclin F upregulation during a period of starvation led to concomitant decrease in PTBP1 protein levels. PTBP1 downregulation led to exclusion of exon 10 of PKM mRNA and altered the PKM2/PKM1 ratio. We observed that Cyclin F mediated depletion of PTBP1 and PKM2, lowering the glycolytic capacity in tumor cells. It further led to decrease in proliferation and invasion and migration rates of cancer cells upon starvation. Mice data also corroborate similar results.
Conclusion : Our study provides clues on how protein interaction during metabolic stress can shift the fate of cancer cells. It establishes that Cyclin F negatively regulates PTBP1/PKM2 levels and reduces the tumorigenic potential of NSCLC cells in in-vitro/in-vivo models. Elucidating molecular interactions will provide insight into NSCLC progression and therapeutic resistance, which is the first step towards designing targets.
Key words: Non-small lung cell carcinoma, Cyclin F, tumorigenic capacity, adenoviral expression, RNA binding proteins
利益披露 Disclosure
R. Tamang, None..
S. Das, None..
S. Choudhury, None.