Roles Of RAF Kinase Binding Protein RUVBL1 And HAX1 In Environment Related Lung Cancer Development

Lung cancer remains the leading cause of cancer-related deaths in the world and in China.Also,lung cancer is kind of “endemic disease” in Yunnan Province.There is no effective treatment of lung cancer until now.Non-small cell lung cancer(NSCLC)is the main kind of lung cancer.NSCLC has poor prognosis compared to other kinds of lung cancer.Molecularly targeted therapies is a significant subject of the development of advanced therapies for lung cancers.Therefore,it is especially important to explore new drugs targeting at non-small cell lung cancer.It is generally believed that lung cancer is associated with environmental degradation and genetic factors.Studies have shown that the pollution in Xuanwei,Yunnan province is more severe than that in other regions,and the expression of EGFR and Ras genes is different from that of other regions.Whether genetic or environmental factors,the intracellular signal transduction must changed ultimately then lead to cell and tissue carcinogenesis.The RAF/MEK/ERK pathway controls many fundamental cellular functions and plays key roles in lung carcinogenesis.The pathway is activated in a large number of human cancers,including lung adenocarcinoma,and small-cell lung cancer.However,the proteins that regulate this pathway remain largely unknown.The pathway is activated by sequential phosphorylation of RAS,RAF kinase,the serine/threonine protein kinase MEK,and ERK.ERK1 and ERK2 are key effector kinases of this signaling pathway and are phosphorylated by upstream MAPK/ERK(MEK1/2).Phosphorylated ERK1/2 proteins(p-ERK1/2)form dimers and translocate from the cytoplasm to the nucleus.ERK1/2 and p-ERK1/2 expression is associated with the poor survival of lung cancer patients and high level phosphoralation of ERK1/2 is an independent prognostic factor of poor survival in NSCLC patients.RAF kinase family have three numbers,A-RAF,B-RAF,C-RAF(RAF1).They plays important roles in cancer tumorigenesis.RAF kinases are regulated by a network of protein-protein interactions and phosphorylation-dephosphorylation events.The binding partners of the RAF kinases,RKIP,and C-RAF kinase negatively regulate the activation of major survival pathways.We previously demonstrated that subunit a of eukaryotic translation initiation factor 3(eIF3a)binds to C-RAF kinase and prevents C-RAF activation.Downregulation of eIF3 a by small interfering RNA enhanced ERK activation.In addition,we revealed that the RAF kinase-binding protein ?-arrestin2 connects the G-protein-coupled receptor and the epidermal growth factor receptor pathways.B-RAF(V600E)mutant isassociated with many kinds of cacncer.There are a number of rrugs targetd at B-BRAF.However,some problems come with it.Therefore,it is meaningful to find new regulation mechnism and new interacting proteins of B-RAF.In our study,we analyed the interaction proteomics results.and find out that B-RAF can bind 85 proteins in a steady state,9 of which were previously reported.And the remaining 79 proteins are the new binding proteins of B-RAF.Among the 79 proteins,we choose two proteins RUVBL1 and HAX1.They are the commom interacting protein of A-RAF and C-RAF.In order to study new function and mechnism of RUVBL1 and HAX1,we constructed the RUVBL1 knockout A549 cell line and HAX1 knockout A549 cell line.And we performed transcriptome analysis of the two proteins.The data of the transcriptome revealed that after RUVBL1 was knocked out,the expression of many genes changed.The expression of 703 genes increased and 581 genes decreased.About 30% differential expressd genes enrichedin MAPK pathway.It can be concluded that RUVBL1 might paly important role in MAPK pathway.And next,we carried out a series of experimens related to cancer functions such as growth,migration,stemness and so on.Also,we performed experiments related to the mechanism of RUVBL1.When RUVBL1 was knocked out,we found the phosphorlation of ERK decreased and phosphorlation of C-RAF(serine 259)increased.And phosphorlation of C-RAF(serine 259)can inhibit the activation of ERK.This might be the reason why RUVBL1 knockout can inhibit the lung cancer tumorigenesis.We use the same methds to study HAX1.The data of the transcriptome revealed that after HAX1 was knocked out,the expression of more genes changed.The expression of 1272 genes increased and 1798 genes decreased.Nearly 30% differential expressd genes enriched in MAPK pathway.About 10% genens enriched in apoptosis pathway It can be concluded that HAX1 might paly important role in MAPK pathway.And next,we carried out a series of experimens related to cancer functions such as growth,migration,stemness and so on.Also,we performed experiments related to the mechanism of HAX1.When HAX1 was knocked out,we found the phosphorlation of ERK decreased and phosphorlation of C-RAF(serine 259)increased.And phosphorlation of C-RAF(serine 259)can inhibit the activation of ERK.This might be the reason why HAX1 knockout can inhibit the lung cancer tumorigenesis.In conclusion,we explored the function of B-RAF interacting proteins and successfully identified 79 new B-RAF binding proteins.We also explored the role of two RAF interacting proteins,RUVBL1 and HAX1,from the microscopic level in the development of tumors,and identified enhancement role of RUVBL1 and HAX1 in the occurrence of lung cancer.Even we traced back to its expression level in human tumor samples which shown the strong individual difference of RUVBL1 and HAX1 expression in lung cancer tissues.These will provide a theoretical basis for the exploration of RAF's new functions and mechanism of non-small cell lung cancer;and will provide new targets for the clinical treatment researches of non-small cell lung cancer.