Preliminary Study On The Function And Its Mechanism Of ENO1 In Lung Cancer

Background and objectiveLung cancer arises from the bronchial mucosal epithelium and it is the leading cause of cancer mortality worldwide.Non-small cell lung cancer(NSCLC)is the most commonly diagnosed type of lung cancer,accounting for approximately 85%of all cases.Although the continuous progress has been made for surgical resection,chemotherapy,and radiation therapy,prognoses have not significant improved.In recent years,molecular targeted therapy has become the most prevalent approach.Therefore,the understanding of the molecular alterations in NSCLC.SCLC and their pathways is significant for molecular targeted therapy.During tumor formation and expansion,increasing glucose metabolism is necessary for the unrestricted growth of tumor cells.Enolases are glycolytic enzymes responsible for the ATP-generating conversion of 2-phosphoglycerate to phosphoenolpyruvate.In mammals,three different enolase isoforms exist:alpha-,beta,-and gamma-enolase.Each are encoded by three distinct genes and expressed in a tissue and development-specific manner.Distributed in a variety of tissues,α-enolase(ENO1)was originally described as an enzyme responsible for the glycolytic pathway.In addition to its glycolytic function,accumulating evidence has demonstrated that ENO1 is a multifunctional protein involved in several biological and pathophysiological processes depending on its cellular localization.The molecular weight of ENO1 protein is 48 kDa.It is expressed in the cytoplasm and considered as an oncogene in tumor pathogenesis.However,another transcript of ENO1 can be translated into a 37-kDa c-Myc promoter-binding protein(MBP-1),which represses transcription and is localized in the nucleus.Although early researches have supported ENO1 could inhibited the growth of tumor cell,the present studies confirmed that ENO1 displays carcinogenesis in cancers.Overexpression of ENO1 has been previously demonstrated in several types of tumors including NSCLC.However,investigators have reported conflicting results.Some researchers have shown that the expression of ENO1 was upregulated in NSCLC tissues and was associated with poorer clinical outcomes.On the contrary,Chang Y.S.et al.demonstrated that the levels of ENO1 protein were significantly decreased in NSCLC and overexpression of ENO1 inhibited epithelial-mesenchymal transition(EMT)in the A549 cell line.Therefore,neither expression nor the functional mechanisms of ENO1 in NSCLC have been clearly established.The purpose of the study was to explore the expression and the function of ENO1 and CD 133 in lung cancer.Investigators have reported conflicting results in NSCLC.Some researchers have shown that the expression of ENO1 was upregulated in NSCLC tissues and was associated with poorer clinical outcomes.On the contrary,Chang Y.S.et al.demonstrated that the levels of ENO1 protein were significantly decreased in NSCLC and overexpression of ENO1 inhibited epithelial-mesenchymal transition(EMT)in the A549 cell line.Based on the conflicting results of ENO1 in NSCLC,our purpose of the study was to explore the expression and the function of ENO1 in NSCLC,and further explore its involvement of signal transduction pathways,and provide a molecular mechanism of the occurrence and development of NSCLC.We performed a Meta-analysis to determine the relationship between ENO1 expression and small cell lung cancer;We performed a Meta-analysis to determine the relationship between CD133 expression and NSCLC.At the same time new ideas for the treatment of lung cancer were provided.Content and methods1 Identification of expression of ENO1 in NSCLC samples(1)Twenty-six(26)surgical resected fresh primary NSCLC tissues and paired para-cancer lung tissues as well as non-cancerous lung tissues(5 cm away from tumor edge),55 paraffin-embedded primary NSCLC specimens,and 17 paraffin-embedded non-cancerous lung specimens were obtained from the Third Affiliated Hospital of Kunming Medical University(Yunnan,China).Patients with a diagnosis of relapse and who had received preoperative radiation,chemotherapy,or biotherapy were excluded from the study to avoid any changes in tumor marker determination due to the effect of the treatment.The clinical processes were approved by the Ethics Committees of the Third Affiliated Hospital of Kunming Medical University,and patients provided informed consent.(2)RNA was extracted from26 fresh primary NSCLC tissues and paired para-cancer lung tissues as well as non-cancerous lung tissues,and was transcribed into cDNA.Then cDNA was applied to quatititive fluorescent PCR to detect the mRNA level of ENO1.(3)Immunohistochemistry was performed in 55 paraffin-embedded primary NSCLC specimens and 17 paraffin-embedded non-cancerous lung specimens to detect the expression level of ENO1.(4)Quatititive fluorescent PCR and Western blot were used to detect the expression level of ENO1 mRNA and protein in NSCLC cells and immortalized normal bronchial epithelial cell line(HBE).(5)Immunofluorescence was performed to analyze the location of ENO1 in NSCLC cells.2 Functional research of ENO1 in NSCLC cell lines(1)We firstly used lentivirus-mediated full-length ENO1-GFP(ENO1)to constitutively overexpress ENO1 in A549 cells in order to assess its role in NSCLC.Quatititive fluorescent PCR and Western blot were used to detect the expression level of ENO1 mRNA and protein.(2)Three lentiviral short hairpin RNA(shRNA)vectors were used to specifically and stably knock down the expression of ENO1 in the SPCA-1 cell line,and the expression levels of ENO1 mRNA and protein were determined by qRT-PCR and Western blot.(3)In vitro viability of stably overexpressed or suppressed NSCLC cells was detected by MTT.(4)In vitro clone formation ability of stably overexpressed or suppressed NSCLC cells was detected by Clone Formation Assay.(5)The change of invasion and migration ability of stably overexpressed or suppressed NSCLC cells was identified by Transwell and Boyden experiment.(6)Nude mices were subcutaneous injection with stably overexpressed or suppressed NSCLC cells to identified the change of oncogenicity and proliferation ability in NSCLC cell lines.3 Priliminary investigation of molecμlar basis mediated by ENOl in NSCLC(1)The protein levels of cell cycle and EMT-associated genes were examined in A549 and SPCA-1 cells with stably overexpressed or suppressed ENO1.(2)The protein levels of FAK/PI3K/AKT pathway were examined in A549 and SPCA-1 cells with stably overexpressed or suppressed ENO1.(3)The protein levels of FAK/PI3K/AKT pathway,cell cycle and EMT-associated genes were examined after Ang Ⅱ treatment of ENO1-suppressed SPCA-1 cells.4 Meta-analysis of the Correlation between ENO1 and SCLC.5 Meta-analysis of the Correlation between CD133 and NSCLC.Resμlts1.ENO1 is highly expressed in NSCLCQuantitative real-time reverse transcription PCR(qRT-PCR)was used to measure the expression of ENO1 mRNA in 26 fresh primary NSCLC tissues(T),their corresponding para-cancer lung tissues(P),and their corresponding non-cancerous lung tissues(N).The ENO1 mRNA expression level was increased in NSCLC tissues in comparison to non-cancerous lung tissues(P<0.05).The expression levels and subcellular localization of ENO1 protein in 55 paraffin-embedded primary NSCLC specimens and 17 paraffin-embedded non-cancerous lung specimens were measured by immunohistochemical staining.Expression of ENO1 both in the cytoplasm and nucleus(MBP-1)were observed in NSCLC tissue,but as ENO1 is only known to localize in the cytoplasm,only this specific staining was evaluated.ENO1 protein was highly expressed in NSCLC tissues compared to non-cancerous lung samples(P<0.05).Further,ENO1 was observed to express in the cytoplasm but not in the nucleus in NSCLC A549 and SPCA-1 cells by immunofluorescence assay,and its upregulated expression levels in mRNA and protein were also found in both two cells compared to immortalized human bronchial epithelial cell line HBE.2.Stable ENO1-overexpressed and ENO1-suppressed NSCLC cells were constructedSince ENO1 expression is higher in SPCA-1 than in A549,we firstly used lentivirus-mediated full-length ENO1-GFP(ENO1)to constitutively overexpress ENO1 in A549 cells in order to assess its role in NSCLC.The result showed that ENO1 expression was obviously upregulated in A549-ENO1 cells compared to its control PLV-Ctr cells,and the expression of MBP-1 was not observed.Further,three lentiviral short hairpin RNA(shRNA)vectors were used to specifically and stably knock down the expression of ENO1 in the SPCA-1 cell line,and the expression levels of ENO1 and MBP-1 were determined by qRT-PCR and Western blot.The result indicated that ENO1 expression was obviously downregulated in shENO1-B and shENO1-C cells compared to their respective control PLV-scrambled control shRNA(shCtr)cells.3.ENO1 promotes cell proliferation,clone formation in vitro,and tumorigenicity in vivoWe assessed the effect of ENO1 expression on A549 cell growth in vitro.The growth curves determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays showed that overexpressed ENO1 significantly elevated cell viability compared to its control cells.Colony formation assays showed that overexpressed ENO1 significantly increased cell proliferation compared to its control cells.On the contrary,suppressed ENO1 expression in SPCA-1 cells significantly inhibited cell viability and clone formation.To confirm the growth effect of ENO1 in vivo,we performed an in vivo tumorigenesis study by inoculating A549 with or without ENO1 overexpression and SPCA-1 cells with or without ENO1 knockdown into nude mice.Mice were sacrificed 15 days after inoculation,with average tumor weights of PLV-Ctr vs A549-ENO1 group and PLV-shCtr vs shENO1-B group,respectively(P<0.05).These results suggest that ENO1 significantly promotes cell growth in vitro and in vivo.4.ENO1 promotes cell migration and invasionTo examine the effect of ENO1 on cell migration and invasion,a transwell apparatus and Boyden chamber coated with Matrigel were used.After 10-h incubation,an elevated number of migrated cells were observed in A549-ENO1 compared to its control cells(P<0.05).On the contrary,stably suppressed ENO1 expression in SPCA-1 cells inhibited cell migration and invasion in both shENO1-B and shENO1-C cell groups compared to their respective control cells(P<0.05).5.ENO1 regulates the expression of cell cycle associated genes in NSCLCTo further study the mechanism by which ENO1 regulates cell proliferation,migration,and invasion,the protein levels of cell cycle and EMT-associated genes were examined in A549 and SPCA-1 cells with stably overexpressed or suppressed ENO1.In ENO1 stably overexpressing A549 cells,activation of p-Rb(ser 780)was increased as well as the elevated expression of cyclin D1,cyclin El,and c-Myc.In contrast,the expression of p21 was inhibited.Stably knocking down endogenous ENO1 expression in SPCA-1 inhibited the activation of p-Rb(ser 780),and the expression of cyclin D1,cyclin El,and c-Myc were decreased,while levels of p21 were upregulated.6.ENO1 regulates the expression of EMT-associated genes in NSCLCWe also found that upregulated ENO1 expression elevated the expression of EMT marker genes including snail,vimentin,and N-cadherin,yet inhibited E-cadherin in A549 cells.Conversely,downregulated ENO1 expression in SPCA-1 cells inhibited the expression of these proteins and elevated E-cadherin expression.However,stable downregulated or upregulated ENO1 did not induce any epithelial to mesenchymal morphology transition changes in A549 or SPCA-1 cells.7.ENO1 regulates FAK-mediated PI3K/AKT pathway to promote cell proliferation,migration,and invasionPI3K/AKT has been reported to be a key signal pathway promoting cell proliferation and EMT and can be modulated by FAK.We found that overexpression of ENO1 significantly increased levels of(3-catenin and phosphorylated FAK,PI3K,and AKT,but not their total protein levels.Suppression of ENO1 had the opposite effect on the FAK/PI3K/AKT pathway.To further study the mechanism by which ENO1 regulates cell proliferation,migration,and invasion,ENO1-suppressed SPCA-1 cells were treated with human angiotensin Ⅱ(Ang Ⅱ)to induce the phosphorylation of FAK.We observed a consistent effect on the FAK/PI3K/AKT pathway after Ang Ⅱ treatment of ENO1-suppressed SPCA-1 cells whereby levels of p-AKT,cyclin D1,c-Myc,p21,and β-catenin were restored.These results implied that ENO1 is an upstream signal factor modulating the FAK/PI3K/AKT pathway in NSCLC,and ENO1 regulates FAK/PI3K/AKT pathway to promote cell proliferation,migration,and invasion.8.Meta-analysis of the Correlation between ENO1 and SCLC.Meta analysis of the correlation between ENO1 and SCLC were applicated,then results showed that the expression of ENO1 had nothing to do with in SCLC.9.Meta-analysis of the Correlation between CD133 and NSCLC.CD133 is one of markers of CSC,Meta-analysis were applicated to explore the relationship between the expression of CD 133 and NSCLC,the result of the analysis showed that there was significantly associated between the positive expression of CD 133 and cells with low or middle differentiation and lymph node metastasis in NSCLC.ConclusionENO1 protein was highly expressed in NSCLC tissues compared to non-cancerous lung samples by quantitative real-time reverse transcription PCR(qRT-PCR)and immunohistochemical staining.ENO1 was observed to express in the cytoplasm in NSCLC cells by immunohistochemical staining and immunofluorescence assay,and its upregulated expression levels in mRNA and protein were also found in both two cells compared to immortalized human bronchial epithelial cell line HBE.In this study,we found that overexpressed ENO1 significantly elevated cell proliferation and clone formation in vitro as well as tumorigenesis in vivo.Furthermore,we also observed that overexpressed ENO1 induced cell migration,invasion,and metastasis in NSCLC.Meanwhile,we also observed that suppressed ENO1 significantly inhibited cell proliferation,clone formation,migration,invasion,and metastasisin in vitro as well as tumorigenesis in vivo.The biological functions of ENO1 found in this study provide a mechanistic basis for the pathological and clinical observations.When we examined the key regulators of cell cycle at the G1-S phase transition,we discovered that suppression of ENO1 inhibited the expression of c-Myc,cyclin D1,p-Rb,and cyclin E1 while elevating the expression of p21.EMT is regarded as a key event in tumor migration and invasion progression.In this study,we further examined the expression of EMT marker genes and found that knocking down ENO1 expression induced the protein levels of E-cadherin while suppressing the expression of snail,vimentin,and N-cadherin in NSCLC cells.In addition,we provide compelling evidence that ENO1 promotes cell proliferation,migration,invasion,and tumorigenesis by activating the FAK-mediated PI3K/AKT pathway and further modulating their downstream signal molecules.The results the Meta-analysis showed that the expression of ENO1 had nothing to do with in SCLC.The result of the Meta-analysis showed that there was significantly associated between the positive expression of CD 133 and cells with low and middle differentiation and lymph node metastasisin in NSCLC;Our study demonstrates that ENO1 and CD 133 may be a potential therapeutic target for NSCLC treatment.