The Role Of ERK5 In Tumor Development,Metastasis And Radioresistance

Mitogen-activated protein kinases(MAPKs)are important signal transducing enzymes,involved in various facets of cellular regulation,such as gene expression,cell proliferation,cell motility,cell survival and death.Aberrations of MAPK pathways are frequent occurrences in cancer,and have a significant part in the tumorigenesis and the tumor progression.There are three well-characterized subfamilies of MAPKs:extracellular signal-regulated kinase(ERK1/2),Jun N-terminal kinase(JNK)and p38 MAPK.The fourth and least studied mammalian MAPK pathway,extracellular signal regulated kinase 5(ERK5),also known as big MAP kinase 1(BMK1),is stimulated by an extensive range of mitogens and cellular stresses,and contributes to the regulation of cell survival,proliferation,and differentiation.The links between abnormal levels of ERK5 expression and cancers have been established by the analyses of human tumors in recent years.Nevertheless,the precise mechanisms and specific targets,by which ERK5 mediates the processes leading to the survival,proliferation,or metastasis in cancer,have remained elusive.Therefore,this study aims to elucidate what kind of role ERK5 plays in cancer with respect to lung cancer development,radioresistance,cell migration and tumor metastasis.The investigation started by the detection that both the protein level and the phosphorylation status of ERK5 were considerably elevated in solid lung tumor growth process,which suggested that ERK5 pathway was activated by the stress stimulation in tumor micro-environment,and its ectopic activation was probably linked to the development of lung cancer.Thus,the constructs were generated to allow the stable over-expression of ERK5 in A549 lung cancer cells,and it was observed that the ectopic expression of ERK5 promoted the cell proliferation as well as the transition of G2/M cell cycle,which were related to the higher cyclin B1 expression.The experiments in vitro and in vivo further supported that the ability of malignant transformation and xenografts tumor growth was enhanced by ERK5 over-expression in A549 cells.These findings indicated that ERK5 signaling pathway contributed to the malignant nature of lung cancer.Since ERK5 pathway might be stimulated by a wide range of cellular stresses,the investigation subsequently scrutinized whether ERK5 activation is related to the production of radioresistance in cancer cells.It was observed that the activity of ERK5 was significantly intensified in A549 cells when exposed to the ionizing radiation(IR)stress.Activated ERK5 could affect DNA damage checkpoint pathway,such as the repression of the protein level of p53 which exerts a crucial influence over IR response,the drop of the early response of CHK1 phosphorylation,and the promotion of the expression of cyclin B1-one of the important downstream effector proteins.Consequently,ERK5 over-expression could increase DNA damage repair response,suppress cell cycle arrest,and protect cell from apoptosis.Therefore,activated ERK5 pathway could improve cancer cell survival in IR stress,and enhance the radioresistance in lung cancer therapy.On account of these findings,the strategy was taken that target ERK5 shRNA knockdown combined with radiotherapy(RT)was applied to the animal experiment.The Lewis lung carcinoma bearing-mice experimental results displayed an obvious increased anti-tumor effect in ERK5 knockdown combined with low or high doses RT.ERK5 knockdown could considerably improve the radiation sensitivity of lung cancer therapy.It was warranted that ERK5 signaling assumed an essential role in reversing IR responsiveness in lung cancer cell,and raised the possibility of a potent adjuvant therapeutic approach to the improvement of the efficacy of RT for lung cancer with lower drug doses.During the investigation into ERK5,it was observed that there were morphological changes in ERK5 over-expression A549 cells.Through the detection of cytoskeleton by F-actin staining,it was perceived that ERK5 over-expression motivated the disruption of the actin cytoskeleton,while the stress fibers around the nuclear were almost invisible,and more lamellipodium-like structures appeared.Moreover,the cytoskeleton exhibited higher reorganization rate in ERK5 over-expression A549 cells by serum stimulation.The results of wound-healing assay and transwell assay showed that ERK5 over-expression dramatically promoted the migration and invasion of A549 cells in vitro.Moreover,ERK5 activity was also associated with the high motility in mouse melanoma cell lines B16F1/B16F10 and human lung giant cancer cell lines 95C/95D,which were respectively cell sublines with different spontaneous metastatic potentials.In addition,the transplantable animal model of metastatic melanoma was adopted in this study,the results of which also revealed that the inhibition of ERK5 activity suppressed the primary tumor growth as well as the metastasis of B16F10 cells into lung and lymph nodes.Taken together,these results indicated the important role of ERK5 in cell migration and tumor metastasis.As ERK5 might be involved in numerous processes of cell migration,this study held a preliminary discussion of the mechanism from the perspective of its influence on FAK.The focal-adhesion kinase(FAK)is a non-receptor protein tyrosine kinase that is localized to the cytoplasm side of focal adhesion,and has a profound effect on signal transduction pathways that are initiated at sides of integrin-mediated cell adhesions and by growth factor receptors.High FAK levels or deregulation of its signaling may contribute to tumor progression,invasiveness,and metastasis.It was observed that both the protein level and phosphorylation status of FAK could be affected by ERK5.The activation of the MEK5-ERK5 pathway caused the phosphorylation and stabilization of the transcription factor USF1,and resulted in the promotion of its transcriptional activities on FAK gene expression.Furthermore,activated ERK5 could phosphorylate FAK S910 and induce FAK Y397 dephosphorylation.The alternate phosphorylation between Y397 and S910 which are important phosphorylation sites of FAK might be related to the disassembly and turnover of focal contacts.Thus,ERK5 signaling probably took part in the regulation of FAK expression and the temporal regulation of FAK activity that might contribute to the dynamics control of focal contacts.