Emerging roles of Cyclin F in Non-small lung cell carcinoma during metabolic stress

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