| Lung cancer is a leading cause of cancer incidence and cancer-related mortality in China and worldwide.Non-small cell lung cancer(NSCLC)represents approximately 80% of all lung cancers and is mainly classified as squamous cell carcinoma or adenocarcinoma.Multiple oncogenic events are often detected in the majority of advanced stage lung cancers.Therefore,it is important to address the potential pathological drivers of disease progression to better control this deadly cancer.To clarify the key-genes related to NSCLC progression,human microarray V4.0 was performed to screen the global profiling of proteincoding transcripts in eight NSCLC tissues and matched normal lung tissues.ETV4 was found to be upregulated significantly in tumor tissues.ETV4,together with ETV1 and ETV5,belongs to the PEA3 subfamily of the ETS transcription factor family,plays important roles in the regulation of gene expression during early embryogenesis,diabetes and oncogenesis.The dysregulation of target genes of ETV4,a DNA-binding transcriptional factor,has significant effects on ETV4-driven tumors.It was recently reported that ETV4 is associated with the prognosis and EGFR-TKI resistance of lung adenocarcinomas.However,the clinicopathologic significance and molecular events regulated by ETV4 in lung cancer progression are still poorly understood.Based on the screening and validation of the microarray assay,ETV4 was confirmed to be the most preponderant PEA3 factors.The aim of this study was to detect the role of ETV4 in lung cancer progression,identify functional targets involved in ETV4-driven tumorigenesis,and to explore the clinicopathologic significance of ETV4-downstrean target genes across distinct cancer subtypes.Three parts are involved in the study: 1.Expression and biological effects of ETV4 in non-small cell lung cancer;2.The mechanism of ETV4 involved in the process of NSCLC invasion and metastasis;3.The clinical significance of ETV4-PXN/MMP1 expression in other four kinds of tumors.Our results may throw new lights on tumor progression in NSCLCs,and provide common molecular biomark across distinct cancer subtypes.Part one Expression and biological effects of ETV4 in non-small cell lung cancerObjective: To analysis the clinicopathologic significance of ETV4 in human NSCLC tissues,and explore its role in the process of tumor growth and metastasis in NSCLC.Methods:Human mRNA expression profiling microarray was used to screen the expression of differentially expressed molecules in 8 pairs of NSCLC tumors and their matched normal lung tissues;KEGG was used to analyze the signaling pathways involved in differential genes;q RT-PCR was used to verify ETV4 mRNA expression in 22 pairs of tumors and their matched normal lung tissues;GEO datasets was used to analysis ETV4 mRNA expression in NSCLC;Immunohistochemical staining(IHC)was used to detect the expression of ETV4 protein in paraffin tissues of NSCLC patients,and its correlation with clinicopathological features was analyzed further.In addition,the effects of ETV4 knockdown on NSCLC cell proliferation,migration and invasion were detected in vitro;Stably ETV4 knockdown cells were selected after lentivirus infection,and the role of ETV4 in tumor growth and metastasis was determined through subcutaneous tumor injection and tail vein injection in nude mice.Results:1 The expression of ETV4 mRNA in NSCLC is significantly higher than that of normal lung tissues.Microarray analysis indicated that ETV4 expression is significantly overexpressed in tumor tissues compared with matched normal lung tissues(FC=7.14,P=0.007).In 22 patients with NSCLC,ETV4 mRNA was significantly upregulated in 90.91%(20/22)of tumor tissues as compared with the matched normal lung tissues.Furthermore,three microarray datasets downloaded from GEO dataset confirmed that ETV4 mRNA expression in primary lung cancers was obviously increased as compared with that in normal lung tissues.2 The expression of ETV4 protein and its clinicopathological significance in human NSCLCsTo understand the clinical significance of ETV4 protein in NSCLCs,we performed IHC analysis of FFPE sections of 131 patients.Positive nuclear ETV4 staining was observed in 66 of 131(50.38%)tumor tissues that was positively associated with tumor size,lymph node metastasis,metastasis,and TNM stage(all P < 0.001).In addition,ETV4 expression was significantly correlated with advanced TNM stage in 80 lung adenocarcinomas(ACs)(P <0.001),but no relationship with the histology and EGFR mutation of lung ACs.Kaplan-Meier analysis indicated that the overall survival rates of 81 patients with NSCLC were inversely correlated with ETV4 protein expression(P <0.0001).Taken together,the elevated ETV4 expression may play an important role in lung tumorigenesis.3 The biological effects of ETV4 in NSCLC cells in vitro.3.1 The efficiency of ETV4 knockdown:Compared to the control groups,ETV4 expressions were significantlyinhibited both at mRNA and protein level in H1703,H1299,and H358 T cellsusing specific siRNA against ETV4(P<0.05).3.2 Effects of ETV4 on cell proliferationETV4 knockdown resulted in reduced proliferation of H1703(48-96 h),H1299(48-96 h),and H358T(72-96 h)cells,as observed with the MTT assay(all P < 0.001)and real-time cellular analysis for up to 96 h.3.3 Effect of ETV4 on cell invasion and migration abilityTranswell assay indicated that the migration and/or invasive abilities was significantly decreased following the siRNA-mediated downregulation of ETV4 expression in H1703,H1299 and H358 T cells(P<0.05),suggesting that knockdown of ETV4 can inhibit cell invasion and migration.4 The role of ETV4 on tumor growth and metastasis in vivo4.1 The efficiency of stably ETV4 knockdown:To determine whether ETV4 was required for tumor cell growth in vivo,we stably knocked down ETV4 expression in A549 cells using a lentiviral shRNA system.The efficiency of ETV4 knockdown was confirmed by using q RT-PCR and western Blot assays.In addition,silencing of ETV4 significantly reduced cell proliferation at 72-96 h in MTT assay(P < 0.05),as well as the invasion ability in transwell experiments(P < 0.05).4.2 Effects of ETV4 on tumor growth in nude mice of subcutaneously implanted modelCompared to the contro grop,ETV4 knockdown significantly reduced tumor formation as compared with control A549 cells(P < 0.001).The size and weight of the tumors derived from ETV4 knockdown A549 cells were significantly lower than those derived from the control cells(P < 0.001).4.3 Effects of ETV4 on lung metastasis in metastatic nude mouse modelIn metastatic nude mouse model,the average number of lung metastasis per mouse was significantly reduced in shETV4-transfected A549 cells as compared with the control cells(P<0.05).Histological analysis demonstrated that the metastatic nodules formed by shETV4-transfected cells were smaller than those formed by the control cells.Summary:1.Combined the results of microarray and validation test,ETV4 was proved to be the most preponderant PEA3 factor that was significantly related to advanced stage,lymph node metastasis,and poor prognosis of NSCLCs2.SiRNA-mediated downregulation of ETV4 in NSCLC cells could suppress the ability of cell proliferation,migration and invasion in vitro.3.Stably knocked down ETV4 expression significantly reduced tumor formation and lung metastasis in nude mice.Taken together,ETV4 has oncogenetic roles and participates in the formation and progression of lung cancers.Part two The molecular mechanism of ETV4 involved in the process of NSCLC invasion and metastasisObjective: To identify the molecular mechanism underlying the ETV4-mediated increase in tumor growth and progression of NSCLCs.Methods:Human microarray analysis was performed to compare protein-coding transcripts levels in H1703,H1299,and H358 T cells treated with or without si-ETV4(PXN and MMP1 was significantly downregulated);q RT-PCR and Western blot assay was used to verify the PXN and MMP1 expressions upon gain or loss of ETV4;Dual luciferase reporter assay was performed to detect the regulation of ETV4 on the promoter regions of PXN and MMP1;The effects of PXN and MMP1 knockdown on NSCLC cell proliferation and invasion were detected in vitro;Recovery experiment was desigened to observe the role of PXN and MMP1 involved in ETV4-drived tumor progression;IHC was used to detect the expression of PXN and MMP1 protein in paraffin tissues of NSCLC patients,and its correlation with clinicopathological features was analyzed further;In addition,the regulation role of CIC on ETV4-PXN/MMP1 axis was analyzed.Results:1.Effects of ETV4 knockdown on genes expression in NNSCLC cells by Human microarray analysis.KEGG pathway analysis revealed that the downregulated genes in ETV4 knockdown cells were significantly involved in Wnt signaling,pathways in cancer,cell cycle,DNA replication,MAPK signaling,and focal adhesion pathway.With P < 0.05 and FC > 3.5 as cutoff values,24 genes were found to be upregulated and 49 genes were downregulated in si-ETV4-transfected cells.2.PXN is a important downstream target gene regulated by ETV4Among the 49 downregulated genes,PXN was significantly inhibited after the ETV4 knockdown(FC = 4.02,P = 0.019).We performed RT-q PCR validation in NSCLC cells after the knockdown or overexpression ETV4.In H1703,H1299,and H358 T cells transfected with si-ETV4,PXN mRNA level was significantly downregulated,consistent with the microarray result(P <0.05).In ETV4-overexpressing H358 and HEK293 T cells,PXN mRNA was significantly overexpressed by ETV4(P < 0.05).Western blot assay confirmed that the expression of PXN protein was consistent with the change of ETV4 expression.Furthermore,Luciferase assay confirmed that ETV4 enhanced PXN promoter activity in HEK293 T cells after co-transfection with Luc-PXN vector and an ETV4-expressing vector or empty vector.3.MMP1 was the most significantly downstream target MMP genes by ETV4 in NSCLCs.Among the 23 members of MMP family,only MMP1,MMP12,MMP13,and MMP14 were significantly upregulated in NSCLC tissues.RT-q PCR validation showed that MMP1 and MMP14 mRNA levels were significantly higher in the tumors than in the 22 paired normal lung tissues(both P < 0.05).Through gain or loss of function experiments,as well as luciferase assay,MMP1 was confirmed to be the the most significantly downstream target MMP genes by ETV4 in NSCLCs.4.Effects of PXN and MMP1 on NSCLC cell proliferation,migration and invasion in vitro.To investigate whether PXN and MMP1 are functionally linked with the ETV4-mediated tumor progression,we examined the effect of PXN and MMP1 on cell phenotypes.SiRNA-mediated silencing of PXN or MMP1 expression significantly reduced H1299 and H1703 cell proliferation at 48-96 h,as observed in the MTT assay(P < 0.0001),as well as their migration and invasion abilities,as observed in transwell experiments(P < 0.001).5.PXN and/or MMP1 knockdown reversed the role of ETV4 on NSCLC cell proliferation and migrationH358 cells stably overexpressing ETV4 was selected after transfection.The overexpression of ETV4 in H358 cells significantly promoted cell proliferation and migration,which were partially abolished after the inhibition of PXN and MMP1 alone or in combination(P < 0.05).Altogether,these results demonstrated that PXN and MMP1 are important downstream targets that contribute to ETV4-induced cell proliferation and migration in NSCLC cells.6.Elevated ETV4-PXN/MMP1 protein expressions are associated with poor prognosis of NSCLCsTo explore the clinical significance of ETV4-PXN/MMP1 in NSCLC,we performed IHC test on tissues from the 131 patients with NSCLC that were used for ETV4 detection.PXN and MMP1 staining was positive in 40.46%(53of 131)and 45.04%(59 of 131)tumor tissues,respectively,and both were significantly associated with tumor size,lymph node metastasis,metastasis,and TNM stage(all P < 0.01).Moreover,a positive correlation was observed between ETV4 and PXN expressions(P < 0.001),as well as between ETV4 and MMP1 expressions(P < 0.001)in 131 human NSCLC specimens.Kaplan-Meier analysis indicated that the overall survival rates of patients with NSCLC were inversely correlated with the expression of PXN,MMP1,and ETV4-PXN/MMP1 protein.7.The expression of transcription repressor CIC and its clinicopathological significance in human NSCLCsIHC test was used to detect CIC expression in the 131 NSCLCs.Positive nuclear CIC expression was observed in 77 of 131(58.78%)tumor tissues,negatively associated with lymph node metastasis,metastasis,and TNM stage(all P < 0.001).CIC expression was negatively correlated with ETV4(r=-0.583,P < 0.001),PXN(r =-0.271,P = 0.002),and MMP1(r =-0.355,P <0.001)expressions.8.CIC knockdown leading to de-repression of ETV4-PXN/MMP1 axis in NSCLCCIC expression was knocked down using specific siRNA against CIC in A549 cells that expressed endogenous CIC.CIC knockdown significantly relieved the expression of ETV4,PXN,and MMP1 both at mRNA and protein levels in A549 cells.These data suggested that CIC inactivation might be associated with the promotion of lung metastases through the depression of the ETV4-PXN/MMP1 axis in NSCLC.Summary:1.ETV4,a DNA-binding transcriptional factor,could regulate a series of downstream genes that involved in Wnt signaling,cell cycle,DNA replication,focal adhesion,and mitogen-activated protein kinase(MAPK)signaling pathway and reinforced the oncogenic functions of ETV4 in lung tumorigenesis.2.PXN and MMP1 are the key downstream target genes of ETV4,and both were significantly associated with tumor size,lymph node metastasis,metastasis,and TNM stage of NSCLCs.PXN and/or MMP1 inhibition partially abolished cell proliferation and migration induced by ETV4.3.Transcription repressor CIC expression was negatively correlated with ETV4,PXN,and MMP1 expressions in NSCLCs.CIC knockdown leads to increased expression of ETV4-PXN/MMP1 axis,which promotes tumor progression.4.Low CIC and high ETV4-PXN/MMP1 expression was implicated in the clinical progression and worse prognosis of NSCLCs.Part three The clinical significance of ETV4-PXN/MMP1 expression in other four kinds of tumorsObjective: To explore the clinicopathologic significance of ETV4 and the downstream target genes PXN and MMP1 across distinct cancer subtypes.Methods:FFPE specimens from surgical tissues of hepatocellular carcinoma(68cases),colorectal AC(47 cases),invasive breast cancer(60 cases),and thyroid papillary cancer(61 cases)were collected.IHC was used to detect the expression of ETV4,PXN,and MMP1 protein in the above tumor paraffin tissues,and the correlations with clinicopathological features was analyzed further.Results:1.The relationship between ETV4 and PXN or MMP1 protein expression in human hepatocarcinomaIn 68 hepatocarcinomas tissues,the positive rate of ETV4,PXN,and MMP1 protein was 8.24%,48.53%,and 48.53%,respectively.The coexpression rates of ETV4-PXN and ETV4-MMP1 were 25.00% and 27.94%.ETV4 expression was associated with tumor size,intravascular carcinoma thrombus,and TNM stage(P<0.05).ETV4-MMP1 co-expression was associated with intravascular carcinoma thrombus and TNM stage(P<0.05).Spearman analysis indicated that ETV4 expression positively correlated with PXN(r = 0.271,P = 0.026)and MMP1(r=0.367,P=0.002)level in 68 hepatocarcinoma specimens.2.The relationship between ETV4 and PXN or MMP1 protein expression in human colorectal adenocarcinomaIn 47 colorectal adenocarcinomas tissues,the positive rate of ETV4,PXN,and MMP1 protein was 46.81%,55.32%,and 70.21%,respectively.ETV4,PXN expression or ETV4-PXN co-expression(40.43%)was associated with Dukes stage,lymph node metastasis,and TNM stage(P<0.05).ETV4-MMP1co-expression(42.55%)was associated with Dukes stage and TNM stage(P<0.05).Spearman analysis indicated that ETV4 expression positively correlated with PXN(r=0.586,P<0.001)and MMP1(r=0.331,P=0.023)level in 47 colorectal adenocarcinomas specimens.3.The relationship between ETV4 and PXN or MMP1 protein expression in human invasive breast cancerIn 60 invasive breast cance tissues,the positive rates of ETV4,PXN,and MMP1 protein were 55.00%,50.00%,and 56.67%,which all associated with tumor size,lymph node metastasis,and TNM stage(P<0.05).ETV4-PXN(50.00%)and ETV4-MMP1(48.33%)co-expression was associated with tumor size and TNM stage(P<0.05).Spearman analysis indicated that ETV4 expression positively correlated with PXN(r=0.905,P<0.001)and MMP1(r=0.696,P<0.001)level in 60 invasive breast cance specimens.4.The relationship between ETV4 and PXN or MMP1 protein expression in human thyroid papillary cancerIn 61 thyroid papillary cancer tissues,the positive rates of ETV4,PXN,and MMP1 protein were 50.82%,60.66%,and 63.93%.The co-expression rates of ETV4-PXN and ETV4-MMP1 were 39.34% and 37.70%,both were associated with lymph node metastasis.Spearman analysis indicated that ETV4 expression only positively correlated with PXN(r=0.349,P=0.006)expression in 61 thyroid papillary cancer specimens.Summary:1.ETV4 was positively correlated with PXN and MMP1 expression in hepatocarcinoma,and ETV4-MMP1 co-expression was associated with intravascular carcinoma thrombus and TNM stage.2.ETV4 expression was positively correlated with PXN and MMP1 expression in colorectal adenocarcinoma,and ETV4-PXN/MMP1 coexpression was associated with Dukes stage and lymph node metastasis of patients.3.High-positive correlation between ETV4 and PXN or MMP1 expression was observed in human invasive breast cancer.ETV4-PXN co-expression was associated with tumor size,TNM stage,and HER2 expression,while ETV4-MMP1 co-expression was associated with tumor size,lymph node metastasis,and TNM stage.4.ETV4 was only positively correlated with PXN expression in thyroid papillary cancer,and ETV4-PXN co-expression was associated with lymph node metastasis of patients.Conclusion:1.Combined the results of microarray and validation test,ETV4 was proved to be the most preponderant PEA3 factor that was significantly related to advanced stage,lymph node metastasis,and poor prognosis of NSCLCs.Reduced ETV4 expression suppressed the growth and metastasis of NSCLC both in vivo and in vitro.Taken together,ETV4 has oncogenetic roles and participates in the formation and progression of lung cancers.2.ETV4,a DNA-binding transcriptional factor,could regulate a series of downstream genes that involved in Wnt signaling,cell cycle,DNA replication,focal adhesion,and mitogen-activated protein kinase(MAPK)signaling pathway and reinforced the oncogenic functions of ETV4 in lung tumorigenesis.3.PXN and MMP1 are the key downstream target genes of ETV4,and both were significantly associated with tumor size,lymph node metastasis,metastasis,and TNM stage of NSCLCs.PXN and/or MMP1 inhibition partially abolished cell proliferation and migration induced by ETV4,suggested that ETV4-PXN/MMP1 axis is an important biomarker of tumor progression and worse outcomes of NSCLCs.4.Transcription repressor CIC expression was negatively correlated with ETV4,PXN,and MMP1 expressions in NSCLCs.CIC knockdown leads to increased expression of ETV4-PXN/MMP1 axis,which promotes tumor progression.Low CIC and high ETV4-PXN/MMP1 expression was implicated in the clinical progression and worse prognosis of NSCLCs.5.ETV4-PXN/MMP1 axis may serve as a common diagnostic or therapeutic molecular biomark across distinct cancer subtypes,including NSCLC,hepatocarcinoma,colorectal cancer,breast cancer,and thyroid tumor. |
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