| Lung cancer is one of the fastest growing malignant tumors with morbidity and mortality.In 2018,about 2.1 million people worldwide were diagnosed with lung cancer,and 1.8 million people died as a result of the disease.The incidence of lung cancer is increasing year by year along with industrialization and environmental pollution around the world.The occurrence of lung cancer is closely related to the environment.Xuanwei and Gejiu in Yunnan province have been among the towns with highest lung cancer mortality in China.Tobacco,the use of smoky coal and mining of tin mines could be the main risk factors for lung cancer in in these areas.Non-small cell lung cancer(NSCLC)accounts for about 80% of all lung cancer.The most difficult problems in the treatment of non-small cell lung cancer are tumor metastasis and tumor recurrence.At present,most patients with lung cancer have progressed to the advanced stage at the time of diagnosis,and the overall prognosis of the patients are not very good.Targeted therapies and immunotherapies have become the main directions of future lung cancer treatment.Abnormalities in intracellular signal transduction can lead to diseases such as cancer.It is highly necessitous to investigate the complexity of signalling network of lung cancer cells and to identify new therapeutic targets.The mitogen-activated protein kinase(MAPK)pathway has been widely studied and is recognized as the central signaling network controlling cell proliferation,differentiation,and survival.Over-expression or aberrant activation of the pathway components is a common indicator for cancer and contributes to tumor initiation and progression.Rapidly accelerated fibrosarcoma(Raf)kinases(A-Raf,B-Raf,and C-Raf)are key components of the MAPK pathway and are downstream effectors of epidermal growth factor receptor(EGFR)and human oncogenic Ras.The basal kinase activity of A-Raf is much lower than the other Raf kinases.The expression of A-Raf is less ubiquitous than the other Raf kinases.This resulted in a rapid increase in our knowledge of the biological functions of the B-Raf and C-Raf isoforms,which may in turn be contrasted with the little that is known about A-Raf.Therefore,the comprehensive analysis for the function of A-Raf is particularly important.Protein-protein interactions are fundamental to the biological functions of a protein.There is no systematic identification and analysis of A-Raf interacting proteins,which also hinders further research on the function of A-Raf kinase.Therefore,based on the analysis of B-Raf and C-Raf interacting proteins in the early stage of our laboratory,we identified the binding partners of A-Raf under resting condition by proteomics such as co-immunoprecipitation and mass spectrometry.(1)We found 132 proteins that interact with A-Raf,and 126 proteins were novel A-Raf interacting partners.These proteins were 92% positive in binding verification by western blotting.(2)Our data suggested that A-Raf regulates apoptosis,RNA catabolism and cell adhesion by interacting with proteins.(3)We identified all ten hallmarks of cancer in these interacting proteins,suggesting that A-Raf is involved in carcinogenesis.These findings have identified potential regulators of A-Raf and provide a systemic insight into its biological functions.In order to further explore how interacting proteins contribute to differences in the signaling functions of the three Raf kinases,we report the comparative interactomes of the three Raf kinases under serum-starved and epidermal growth factor(EGF)-stimulated conditions.We identified nearly 400 novel interacting proteins;some interacted with all three isoforms while others interacted exclusively with one or two.Comparing the interactomes of the three Raf kinases under serum-starved conditions revealed 163 shared proteins,suggesting that Raf proteins share common functions in their inactive state.In contrast,only 87 common interacting proteins were identified under EGF-stimulated conditions,suggesting that Raf proteins perform distinct functions through specific interactions when they are activated.Several A-Raf interacting proteins were involved in single fertilization and sperm-egg recognition,suggesting that A-Raf plays a role in reproduction by interacting with other proteins.Our interactome data help define the differences between the three Raf kinases and may uncover new functions and regulatory mechanisms.Acyl-coenzyme A binding domain containing 3(ACBD3)is a multi-functional Golgi protein,which has been associated with a diverse array of cellular functions,including steroidogenesis,virus replication,apoptosis and Huntington's disease.Our experiments found that ACBD3 interacted with the three Raf kinases.We selected ACBD3 as a specific research object and explored its role in the development of non-small cell lung cancer.The results showed that ACBD3 promoted cell proliferation,migration and epithelial-mesenchymal transition(EMT)in H1299 cells.Knocked out ACBD3 significantly reduced tumor formation in nude mice.The sensitivities of cells to cisplatin(CDDP)and 5-Fluorouracil(5-FU)were positively correlated with the expression of ACBD3.Cell signaling pathway studies showed that as the expression of ACBD3 increased,the phosphorylation of B-Raf activated site S445 increased,and the phosphorylation of ERK also increased significantly.In conclusion,we explored the function of A-Raf interacting proteins,and identified 126 novel A-Raf interacting partners.Comparative interactome analysis under different culture conditions revealed distinct and overlapping properties of Raf family kinases.Then we explored the role of ACBD3 in the development of lung cancer.We preliminarily determined that ACBD3 can promote the development of tumors.It will provide new research targets for the clinical treatment of lung cancer. |
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