| Objective:It is reported that PEAK1(pesudopodium-enriched atypical kinase 1)is a non receptor tyrosine kinase with catalytic activity and is widely expressed in many tissues and organs.We intend to investigate the expressions of PEAK1 in non-small cell lung cancer(NSCLC)tissues,and the effects of PEAK1 on the NSCLC cells during the development were investigated.Then,the downstream prominent effectors were also measured to explored the possible signaling pathway that PEAK1 participated in.Finally,Our research provides a solid evidence to assess PEAK1 as a candidate for tumor therapy.Methods:(1)The relationship between the expressions of PEAK1 and clinical characteristics of NSCLC patientsIn our research,immunohistochemistry was utilized to detect the expressions of PEAK1 in 102 cases of paraffin-embedded lung cancer tissues from Zhongda Hospital Affiliated to Southeast University.We next analyzed the association of PEAK1 expressions with the clinicopathologic characteristics of NSCLC patients.The 102 samples included 68 cases of NSCLC tissues and 34 cases of adjacent normal tissues.(2)The effects of PEAK1 in NSCLC cells on the ability of proliferation,migration,invasion and metastasis1.The expressions of PEAK1 in five NSCLC cells were detected at the levels of both gene and protein by qRT-PCR and Western blot.Then,95 D and H1299 cell lines were chosen for the further studies.2.Lentivirus vectors were utilized to infect 95 D cells and H1299 cells,then the 95 D and H1299 cell lines with PEAK1 stably overexpressing and knocked out as well as the corresponding control cell lines were constructed.Then,the successfully infected cells were confirmed by Western bot.3.CCK-8,scratch wound healing and transwell assay were utilized to evaluate the effects of PEAK1 on the cell proliferation,migration and invasion ability,respectively.To produce peritoneal dissemination metastatic models,PEAK1 stably overexpressing and knocked out cell lines as well as the corresponding control cell lines were injected into mice abdominal cavity.Then,the effects of overexpressing PEAK1 and knocking out PEAK1 on the peritoneal metastasis were evaluated in vivo.(3)To explore the key signaling pathway of PEAK1 involved in the migration,invasion,metastasis and epithelial-to-mesenchymal transition(EMT)in NSCLC cellsThe morphology of the cell models described above were compared under a light microscope;The expression levels of E-cadherin and N-cadherin in each experimental group and control group described above were measured by Western blot and Immunofluorescence.The protein levels of matrix metalloproteinase-2(MMP2)and MMP9 were also detected in cell cultures mentioned above and in mice models;To explore the key factors regulated by PEAK1,the protein phosphorylation levels of its key downstream signaling effectors were measured.Then the pharmacological inhibitors targeted these signaling were employed to assure their roles in the migration,invasion,metastasis and EMT of NSCLC promoted by PEAK1.Results:1.The results show that PEAK1 was significantly overexpressed in non-small cell lung cancer tissues compared to paracancerous tissues based on the data of immunohistochemical staining(P<0.01).Overexpression of PEAK1 did not correlate with age,tumor size,histology,tumor differentiation or TNM stage(P>0.05),but was significantly associated with gender and LN metastasis(P<0.05).2.The abilities of migration and invasion were significantly promoted in 95 D cells and H1299 cells stably overexpressing PEAK1(P<0.05).Whereas,knocking out PEAK1 in 95 D cells and H1299 cells resulted in a suppressed role in migration and invasion(P<0.05).The group of mice injected with PEAK1 overexpressing cells bore more metastatic nodules in the peritoneal cavity than the control group(P<0.05).On the Contrary,knocking out PEAK1 had the opposite effects on tumor metastasis in animal models.3.Overexpression of PEAK1 in 95 D cells and H1299 cells induced the conversion of polarized epithelial cells to spindle-shaped,fibroblast-like mesenchymal cells with decreased cell-cell contact.The expression levels of E-cadherin and N-cadherin were significantly decreased and increased(P<0.05).Furthermore,overexpression of PEAK1 could induce the expressions of MMP2,MMP9(P<0.05).Metastatic colonies from PEAK1 overexpressing group in vivo showed stronger N-cadherin,MMP2 and MMP9 staining,but weaker E-cadherin staining compared to controls(P<0.05).What's more,the mRNA and protein levels of transcription factor ZEB2 or Twist2 were found higher in PEAK1 overexpressing 95 D or PEAK1 overexpressing H1299 than those in the corresponding control cells respectively(P<0.05).All the results above suggest that PEAK1 makes the NSCLC cells acquisite EMT characteristics.On the contray,knocking out PEAK1 in 95 D cells and H1299 cells induced the conversion of epithelial cells to polarized epithelial cells with tighter organization.The expression levels of E-cadherin and N-cadherin were significantly increased and decreased(P<0.05).In addition,knocking out PEAK1 could reduce the expressions of MMP2 and MMP9(P<0.05).The same results were also observed in vivo(P<0.05).The mRNA and protein levels of transcription factor Zeb2 or Twist2 were found lower in 95 D with PEAK1 knocked out or H1299 with PEAK1 knocked out than those in the corresponding control cells respectively(P<0.05).The results of knocking out PEAK1 suggest that PEAK1 really contributes to the acquisition of EMT characteristics in NSCLC cells.During exploring the signaling pathway,upregulation of PEAK1 obviously stimulated the phosphorylation of extracellular signal-regulated kinase-1/2(ERK1/2)and janus kinase-2(JAK2)in 95 D cells and H1299 cells(P<0.05).Downregulation of PEAK1 remarkably decreased the activation of ERK1/2 and JAK2 in 95 D cells and H1299 cells(P<0.05).To assure the roles of JAK2 and ERK1/2 in regulating PEAK1 to promote the abilities of migration,invasion,metastasis and EMT in NSCLC cells,the pharmacological inhibitors targeted these signaling were employed.The results show that the expression of E-cadherin was restored(P<0.05),and the expressions of N-cadherin,MMP2 and MMP9 were reduced upon using PD98059 and/or AZD1480 in 95 D cells stably overexpressing PEAK1(P<0.05).When PD98059 and/or AZD1480 was employed,the migration and invasion of these cells were also inhibited(P<0.05).All the resuls above suggest that JAK2 and ERK1/2 could regulate PEAK1 to promote the abilities of migration,invasion,metastasis and EMT of NSCLC cells.Conclusions:PEAK1 was upregulated in NSCLC tissues and significantly associated with gender and LN metastasis.The proliferation ability of NSCLC was not regulated by PEAK1,but PEAK1 stimulates migration,invasion and metastasis of NSCLC cells both in vitro and in vivo.The mechanism was that,PEAK1 may regulate different EMT transcription factors through JAK2 and ERK1/2 signaling pathways in different NSCLC cells,thus making NSCLC acquisitie EMT characteristics and enhancing the migration,invasion and metastasis of NSCLC. |
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