Galectin-4 Involves In The Modulation Of Non-small Cell Lung Cancer By Binding To K-Ras And Disrupting Its Downstream Pathways

Background and objectivesNon small cell lung cancer(NSCLC)remains one of the most malignant diseases and the leading cause of cancer-related deaths worldwide.Because of the short survival time,high risk of treatment and poor prognosis,it is paramount to uncover the mechanisms under NSCLC and find new targets or biomarkers for the treatment of NSCLC.The gene profile analysis of NSCLC patients showed that epidermal growth factor(EGFR)and K-Ras are common carcinogens,and their expressions are closely related to the metastasis of NSCLC cells.Galectin-4(Gal-4)is one of the tandem type galectins.It is up-regulated in a variety of tumors and regulates the proliferation,apoptosis and metastasis of tumor cells.In previous researches,our research group uncovered that gal-4 has an opposite effect of the metastasis in different NSCLCs for the first time,in detail,gal-4 restrained the migration and invasion of A549 and H460 cells,and promoted the invasion and migration of H1299 and Calu-1 cells,which may be related to the Ras protein.But the underlying mechanism of the opposite regulatory effects of gal-4 on different NSCLCs and the relationship between gal-4 and Ras still remains unclear.In the present study,we explored the interactions between gal-4 and K-Ras which is one of the most important pro-tumor factors for lung cancer and effects of gal-4 on K-Ras downstream signaling pathways,elucidating the detailed regulatory mechanism of gal-4 on NSCLCs.The results will explore the possibility of gal-4 to be a biomarker in NSCLC cells and provide a new therapeutic strategy in the treatment of NSCLC.Methods and ResultsMethods:1.Regulation of intracellular gal-4 on the biological activity of K-Ras in NSCLCs.Four NSCLC cells(A549,H460,H1299,Calu-1 cells)were used for investigation the regulatory effects of gal-4 in the present study.Firstly,PCR-RFLP analysis was applied to detect K-Ras mutant type and wild type in four NSCLC cells.Later,siRNA and plasmid transfection technology are used to decrease or increase the expression of gal-4 in four NSCLC cells respectively,and the expression of gal-4 and K-Ras were determined by Western blot.Next,in order to uncover the effects of gal-4 on the cellular localization of K-Ras in NSCLC,the expression of K-Ras in cell membrane and cytoplasm respectively,after silencing the expression of gal-4 using siRNA technology,was revealed via Western blot and immunofluorescence methods.Subsequently,the location of K-Ras and gal-4 in NSCLC was shown by immunofluorescence assay,and the formation of complexes between gal-4 and K-Ras was detected by co-immunoprecipitation experiment.In order to further study the regulation of gal-4 on K-Ras,the expression of K-Ras,gal-4,EGFR,and p-EGFR were detected by Western blot after treatments with siRNA of gal-4 and EGFR activator EGF.2.The opposite regulation mechanism of gal-4 on the invasion and migration of NSCLCs is related to the K-Ras/c-raf/MEK/ERK signaling pathway.Computer simulation-assisted modeling and co-immunoprecipitation method were used to detect the effect of gal-4 on the K-Ras/c-raf complex after overexpression of gal-4.After siRNA interfering with gal-4,the expression and phosphorylation of c-raf,MEK1/2 and ERK were shown via Western blot.Wound scratch assay and transwell invasion assay were used to explore the migration and invasion of NSCLC after treatments with MEK1/2 inhibitor Pimasertib;the expression of adhesion molecules were detected after treatment with MEK1/2 inhibitor Pimasertib by Western blot.After interfering with gal-4 and adding MEK1/2 inhibitor Pimasertib,changes of the migration and invasion of NSCLC cells were explored by wound scratch assay and transwell invasion assay.3.The opposite regulation effects of gal-4 on the invasion and migration of NSCLC is related to the expression and activation of K-Ras/JNK/c-Jun.After interfering with gal-4 in four NSCLC cells,the modulation effects of gal-4 on the expression and activation of JNK pathway were uncovered by Western blot;then the effect of gal-4 on the translocation of p-c-Jun into nucleus was detected by immunofluorescence experiment;wound scratch assay and transwell invasion assay were used to explore the effects of JNK on the invasion and migration of NSCLC cells after treatment with JNK inhibitor SP600125.Changes of expression of adhesion molecules were uncovered by Western blot after treatment with JNK inhibitor;Finally,after interfering of intracellular gal-4,the effect of JNK on the migration and invasion of NSCLC cells were investigated by wound scratch assay and transwell invasion assay after adding JNK inhibitor.4.Effects of intracellular gal-4 on lung metastasis of H460 cells or Calu-1 cells in nude mice.H460 cells and Calu-1 cells,which were silenced of gal-4,were injected into the tail vein of nude mice.The aim of this assay is to explore the opposed effects of gal-4 on the invasion and migration of H460 cells and Calu-1 cells in vivo.The nude mice were weighed every three days and their growth statues were observed.When the lung metastasis of the nude mice was obvious,the lungs of the nude mice were dissected and the lung tumor metastasis of H460 and Calu-1 cells was determined by observing the lung nodules via stereomicroscope.Then,PCR-RFLP analysis was applied to detect the gene type of K-Ras of lung tissue in H460 and Calu-1 cell control group.In addition,representative H&E staining was of frozen lung sections from nude mice and the expressions of adhesion molecules(N-cadherin and E-cadherin)were detected by immunofluorescence.Results:1.Gal-4 inhibits the expression and the location of K-Ras on the cell membrane of K-Ras in A549 and H460 cells,and promotes the expression and the location of K-Ras on the cell membrane in H1299 and Calu-1 cells.PCR-RFLP results showed that both A549 and H460 were K-Ras mutant type cells,and H1299 as well as Calu-1 were K-Ras wild type cells.Western blot results showed that the expression of gal-4 can be reduced after transfection of siRNA in the four cells,while the expression of gal-4 can be increased after transfection of plasmid.After interfering with gal-4,the expression of K-Ras was increased in A549 and H460 cells,while decreased in H1299 and Calu-1 cells.In order to detect the effect of gal-4 on K-Ras expression,EGFR,one of the important activators of K-Ras,was chosen and its activator EGF along with siRNA of gal-4 was added.The results of Western blot experiments showed that after interfering with si-gal-4,the expression of EGFR in NSCLC cells did not change significantly,while the activation of EGFR was up-regulated in A549 and H460 cells,and was down-regulated in H1299 and Calu-1 cells.And the expression of K-Ras in the four cells did not change significantly after treatment with EGFR activator by Western blot assay,indicating that gal-4 regulate K-Ras expression is not through activation of EGFR.Later,both immunofluorescence assay and Western blot experiment uncovered that,after silencing of gal-4,the expression of K-Ras was increased on the cell membrane,and decreased in the cytoplasm in A549 and H460 cells.On the contrary,in H1299 and Calu-1 cells,the expression of K-Ras was decreased on the cell membrane,and increased in the cytoplasm.Results indicated that gal-4 regulated oppositely the expression of K-Ras in different NSCLC cells and the accumulation of K-Ras on the cell membrane.2.In NSCLCs,endogenous gal-4 directly binds to K-Ras and affects its binding to downstream effector c-raf.Immunofluorescence experiments showed that gal-4 and K-Ras co-localized in four NSCLC cells,suggesting that there may be a direct interaction between the two molecules.After the interaction of gal-4 and K-Ras with different gene types was simulated by PyMOL,co-immunoprecipitation experiments were performed to find that gal-4 and K-Ras formed a complex in NSCLCs.In A549 and H460 cells,gal-4/K-Ras complex increased after over-expression of gal-4,while in H1299 and Calu-1 cell,the expression of gal-4/K-Ras complex decreased.In order to investigate the role of the gal-4/K-Ras complex on the downstream effectors of K-Ras to a greater extent,firstly,gal-4 molecule was added to the K-Ras/c-raf model as shown in PyMOL,and the results of immunofluorescence showed that co-binding domain of gal-4 and K-Ras,c-raf existed.After treatment with si-gal-4 in NSCLCs,the expression of c-raf was increased in A549 and H460 cells,but decreased in H1299 and Calu-1 cells.Moreover,the results of co-immunoprecipitation experiment showed that gal-4 inhibited the interaction between K-Ras and c-raf in A549 and H460 cells,while promoted in H1299 and Calu-1 cells.These above results indicated that gal-4 could directly bind to K-Ras to regulate the expression and activation of its downstream effector c-raf by affecting the complex formations in four NSCLC cells.3.Intracellular gal-4 regulates the activation of K-Ras/c-raf/MEK/ERK to regulate the migration and invasion of NSCLCs.After interfering with gal-4,the results of Western blot shown,the increasing phosphorylation levels of MEK1/2 and ERK were shown in A549 and H460 cells,and the decreasing level were in H1299 and Calu-1 cells.Wound scratch assay and transwell invasion assay exhibited that MEK1/2 inhibitor suppressed the migration and invasion of four NSCLC cells.Furthermore,the MEK1/2 inhibitor Pimasertib could increase the expression of E-cadherin,and decrease the expression of MMP-2,β-catenin,CD44,N-cadherin and vimentin.After silencing of gal-4,Pimasertib could inhibit the migration and invasion of NSCLC,indicating that gal-4 regulates the migration and invasion of NSCLC via modulating the phosphorylation of K-Ras/c-raf/MEK/ERK.At the same time,the regulation of gal-4 on the expression of EMT related molecules was also related to the activation or inhibition on the phosphorylation of K-Ras/c-raf/MEK/ERK in four NSCLCs.4.Intracellular gal-4 regulates cell invasion and migration through the K-Ras/JNK/c-Jun pathway.After interfering with gal-4,the phosphorylation levels of JNK and c-Jun were increased in A549 and H460 cells,while were decreased in H1299 and Calu-1 cells by Western blot.The results of immunofluorescence experiments showed that,after interfering with si-gal-4,the entrance of p-c-Jun into nucleus was up-regulated in A549 and H460 cells,but was down-regulated in H1299 and Calu-1 cells.Subsequently,when the JNK inhibitor SP600125 was introduced,wound scratch assay and transwell invasion assay showed that SP600125 inhibited the invasion and migration of NSCLC.And SP600125 could increase the expression of E-cadherin and down-regulate the expression of CD44,β-catenin,MMP-2,N-cadherin.After interference with gal-4,the JNK inhibitor SP600125 decreased the migration and invasion of NSCLC.These results suggested that gal-4 regulated the migration and invasion by regulating the activation of K-Ras/JNK/c-Jun pathway.5.Suppression of gal-4 expression increases lung metastasis of H460 cells and decreases lung metastasis of Calu-1 cells in vivo.The greater number of lung metastasis nodules were in the group which were injected with si-gal-4 H460 cells,comparing to the control H460 cells group.However,the less number of lung metastasis nodules of Calu-1 cells were in nude mice by injection with si-gal-4 Calu-1 cells,comparing to the control mice by injection with control Calu-1 cells.Moreover,the tumor cells of lung tissues showed an increase via H&E staining,and the expression of N-cadherin increased,while the expression of E-cadherin decreased,in H460 si-gal-4 group comparing to H460 control group.On the contrary,the tumor cells of lung tissues showed a decrease by H&E staining,and the expression of N-cadherin decreased in Calu-1 si-gal-4 group comparing to Calu-1 control group.The results indicated that intracellular gal-4 could suppress the lung metastasis of H460 cells,but increase the lung metastasis of Calu-1 cells in nude mice.Conclusion1.In A549 and H460 cells,gal-4 can down-regulate the expression of K-Ras,inhibit the location of K-Ras on the cell membrane,directly bind to K-Ras and inhibit its binding to the downstream effector c-raf;while in H1299 and Calu-1 cells,gal-4 can up-regulate the expression of K-Ras,promote the location of K-Ras on cell membrane and the formation of K-Ras/c-raf complexes.2.In A549 and H460 cells,gal-4 can inhibit the activation of K-Ras/c-raf/MEK/ERK,thereby enhancing the expression of E-cadherin,lessening the expression of MMP-2,N-cadherin,CD44,vimentin and β-catenin,leading to inhibit its invasion and migration.In H1299 and Calu-1 cells,gal-4 activates K-Ras/c-raf/MEK/ERK,thereby decreasing the expression of E-cadherin and raising the expression of CD44,vimentin,β-catenin,N-cadherin and MMP-2,thereby promoting invasion and migration of two cells.3.Gal-4 inhibits the activation of JNK and c-Jun in A549 and H460 cells,and then inhibits the entry of p-c-Jun into the nucleus,thereby enhancing the expression of E-cadherin and lessening the expression of N-cadherin,β-catenin,CD44,MMP-2,leading to the inhibition of invasion and migration.By contRast,gal-4 can promote the activation of JNK and c-Jun and the entrance of p-c-Jun,thereby decreasing the expression of E-cadherin,raising the expression of N-cadherin,β-catenin,CD44,MMP-2 to promote EMT,thereby promoting invasion and migration in H1299 and Calu-1 cells.4.Intracellular gal-4 inhibits the metastasis of K-Ras mutant type NSCLC,while promotes the metastasis of K-Ras wild type NSCLC in nude mice.