The Mechanism Of Galectin-3 Promoting Metastasis Of Tumor Cells Via Regulating E/N-cadherin And CD44

BackgroundGalactose lectins(galectins)belonging to the lectin super family have high affinity for binding to ?-galactose glucoside.So far,a total of 15 galactose lectins were found,and all of the galactose lectins contain one or two highly conserved carbohydrate recognition domain(CRD).Galectin-3(Gal-3)is the only one chimeric type of galectin family.It contains of a CRD and a tandem repeat sequence.Gal-3 is widely expressed both in normal tissue and tumor cells.It is synthesized in cytoplasm and has the ability to shuttle between the cytoplasm and nucleus,and can also transport to the cell membrane.Gal-3 is involved in tumor proliferation,migration,apoptosis,adhesion and blood vessel formation.The high expression level of gal-3 in many tumor cells is closely associated with the malignant degree of tumor.According to researches in recent years,serum level of gal-3 of many patients with cancer was obviously higher than that of healthy people,such as breast cancer,lung cancer,oral cancer,stomach cancer,colon cancer,and pancreatic cancer patients,etc.What's more,a research showed that gal-3 in serum could induce polarization distribution of MUC1 on the surface of tumor cell,resulting in exposing cell adhesion molecules(CAMs),thus promoting adhesion between tumor cells and vascular endothelial cells.Another research also reported that the expression of gal-3 could induce the integrin activation,so as to promote the tumor cells and extracellular matrix of fibronectin and laminin adhesion.CAMs can regulate adhesion between cell-cell and cell-matrix,which plays an important role in tumor invasion and metastasis.E-cadherin can combine with another homotypic type E-cadherin,which plays an important role in maintaining epithelial cell polarity and integrity.E-cadherin can also participate in the regulating tumor cells homotypic aggregation.Thus,E-cadherin is widely considered as a tumor suppressor and has the ability to inhibit tumor metastasis.However,N-cadherin and CD44 can promote tumor cell metastasis and adhesion between tumor cells and endothelial cells.Especially,the expression of N-cadherin can inhibit E-cadherin induced adhesion,and promote the metastasis of the tumor regardless of the expression of E-cadherin.However,whether gal-3 promotes metastasis of tumor cells by regulating the expression of E/N-cadherin and CD44?What's the difference of extracellular and intracellular gal-3 in regulating the three adhesion molecules and metastasis of tumor cells?The specific regulatory mechanism has not yet been reported.In our research,we chosed a non-small cell lung cancer A549 cell line that expressed gal-3 to investigated the relationship between intracellular gal-3 E-cadherin,N-cadherin and CD44.Correspondingly,we chosed another prostate cancer PC3 cell line that didn't express gal-3 to research the relationship between extracellular gal-3 and these three CAMs.And MUC1 were lower expressed in the two cell lines,which avoiding the interaction between MUC1 and gal-3.Methods and Results1.Effects of intracellular gal-3 on regulating adhesion molecular and tumor metastasis were determined in A549 cells.(1)Gal-3 siRNA was used to downregulate the expression of gal-3 in A549 cells,and the expression of the three CAMs was detected via Western blotting method.According to the results,the expression of the three CAMs were decreased by gal-3,wich indicated that intracellular gal-3 could upregulate the expression of E-cadherin,N-cadherin and CD44.(2)But we found that intracellular gal-3 didn't affect the homotypic aggregation of A549 cells via flow cytometry.(3)We also found thatdownregulation of gal-3 by siRNA reduced the adhesion between A549 cells and HUVECs.In order to further confirm that gal-3 regulated the adhesion by promoting?-catenin entering into nucleus,we utilized the cytoplasm and nucleus extraction method,and Western Blotting assay to observe the distribution of ?-catenin in cytoplasm and nucleus.Results showed that intracellular gal-3 promoted ?-catenin entering into nucleus.And in the presence of ICG-001,an inhibitor of P-catenin,the adhesion induced by gal-3 was significantly inhibited.Therefore,intracellular gal-3 promoted the adhesion probablely through increasing the accumulation of P-catenin in nuleus.However,E-cadherin was not detected in HUVECs cells.So,E-cadherin didn't involve in the adhesion between tumor cells and HUVECs.Then we investigated the mechanism that intracellular gal-3 regulating the expression of N-cadherin and CD44.(4)Confirming which adhesion molecule involved in gal-3 induced adhesion between A549 cells and HUVECs,and the mechanism that gal-3 regulating the adhesion molecules.Our results showed that gal-3 could upregulate the expression of E/N-cadherin and CD44.So,if these three adhesion molecules involve in the adhesion?First of all,we checked whether E/N-cadherin was expressed in HUVECs.We found that N-cadherin,but not E-cadherin,was expressed in HUVECs.The ligands of E/N-cadherin are the isomorphic cadherin molecules,while the ligand of CD44 is hyaluronic acid which exists in almost all cells.Thus,intracellular gal-3 promoted A549 cells adhesion to HUVECs probably through regulating the expression of N-cadherin and CD44.Next step,we detected the mechanism that gal-3 regulating the expression of N-cadherin and CD44.When the ?-catenin/TCF gene transcription was blocked by ICG-001,the expression of N-cadherin was downregulated while the expression of CD44 had no significant change.But in the presence of ICG-001,intracellular gal-3 didn't affect the expression of these two CAMs.Therefore,gal-3 regulated the expression of N-cadherin and CD44 through increasing the accumulation of(3-catenin in nuleus.(5)Effects of intracellular gal-3 on migration and invasion of A549 cells were respectively measured by wound scratch and transwell assay.In our research,we respectively detected effects of gal-3 siRNA alone,ICG-001 alone and their combination on migration and invasion of A549 cells.The gal-3 interference or ICG-001 group showed a lower migration or invasion ability.However,their combination group almost losed the migration or invasion ability.Thus,blocking ?-catenin/TCF gene transcription could enhance the inhibition on migration or invasion induced by gal-3 interference.Therefore,promoting the accumulation of ?-catenin in nucleus is one of the important way for intracellular gal-3 promoting migration and invasion.2.Effects of extracellular gal-3 on regulating adhesion molecular and tumor metastasis were investigated.(1)Gal-3 could downregulate the expression of E-cadherin mediated by EGFR.Because our previous results had confirmeded that extracellular gal-3 could continuously activate epidermal growth factor receptor(EGFR),and then downregulate the expression of E-cadherin.So,we would further investigate the mechanism that extracellular gal-3 downregulated the expression of E-cadherin.Fistly,we investigated the effect of conexist of gal-3 and EGF on P-EGFR.We found that P-EGFR was high expressed within 1h.Thus,1h was chosed for the action duration for follow-on researches.Western blotting results showed that extracellular gal-3 upregulated the expression of P-EGFR,P-Akt,NF-?B and ZEB1.We speculated that extracellular gal-3 downregulated the expression of E-cadherin through PI3K/P-Akt/NF-?B/ZEB1 signaling pathway in the presence of EGF.(2)The level of E-cadherin/?-catenin complex was detected by co-immunoprecipitation assay.Results showed that extracellular gal-3 could decrease the level of E-cadherin/?-catenin complex.(3)And,we found that extracellular gal-3 inhibited the homotypic aggregation of PC3 depending on the presence of EGF.(4)In addition,extracellular gal-3 could enter into tumor cells and regulte the expression of N-cadherin and CD44 but not E-cadherin in PC3 cells.After PC3 cells treated with gal-3 for 0,1,2,4,8 h,we found the maximum expression of gal-3 in PC3 cells at 2h,and then gradually declined.At the same time,we found that the expression of E-cadherin had no significant change,but the expression of N-cadherin and CD44 was highest at 8h.Because HUVECs cells did not express E-cadherin,and extracellular gal-3 didn't regulate the expression of E-cadherin without EGF,therefore we no longer utilize EGF in the follow-on experiments.(5)We invetigated whether extracellular gal-3 influenced the adhesion of either A549 cells or PC3 cells to HUVECs.Results showed that extracellular gal-3 promoted the adhesion of both A549 cells and PC3 cells to HUVECs.Western blotting results showed that gal-3 increased the expression of N-cadherin and CD44 in A549 cells and PC3 cells at 8h.(6)Then,whether extracellular gal-3 could internalize into cells and promote ?-catenin entering into the nucleus was investigated.Extraction of cytoplasmic and nuclear proteins assay showed that extracellular gal-3 could enter into cytoplasma and nucleus,and at the same time the accumulation of ?-catenin was increased in nucleus.Therefore,extracellular gal-3 could promote the accumulation of P-catenin in nucleus via entering into tumor cells,and then increased the expression of N-cadherin and CD44.Furthermore,we confirmed the effect of the expression of N-cadherin or CD44 on the adhesion between A549 cells and HUVECs by siRNA transfection.Results showed that the adhesion was reduced after the expression of N-cadherin or CD44 was decreased by siRNA transfection.The N-cadherin siRNA showed stronger reduction than CD44 siRNA.By adding gal-3 to recover the intercellular adhesion,the CD44 siRNA treated A549 cells showed more adhesion to HUVECs than N-cadherin siRNA treated cells.This indicated that the expression of N-cadherin played a more important role in gal-3 induced adhesion between tumor cells and HUVECs than CD44.Conclusion1.Intracellular gal-3 regulated the expression of N-cadherin and CD44 through increasing the accumulation of ?-catenin in nuleus,then promoted A549 cells adhesion to HUVECs.2.Intracellular gal-3 had no effect on the homotypic aggregation of A549 cells.3.Intracellular gal-3 promoted miration and invasion through increasing the accumulation of p-catenin in nuleus of A549 cells.4.In the presence of EGF,extracellular gal-3 downregulated the expression of E-cadherin and upregulated the expression of N-cadherin through PI3K/P-Akt/NF-KB/ZEB1 signaling pathway in PC3 cells,then inhibited the homotypic aggregation of PC3 cells.5.Extracellular gal-3 upregulated the expression of N-cadherin and CD44,but not E-cadherin,through entering into PC3 cells to increase the accumulation of p-catenin in nuleus.6.Extracellular gal-3 could promote the adhesion of both A549 cells and PC3 cells to HUVECs by increasing accumulation of ?-catenin in nucleus.7.N-cadherin was more important than CD44 in gal-3 induced adhesion.