The Role And Molecular Mechanism Of Lipocalin2 In Epithelial Mesenchymal Transition Of Colorectal Cancer

Colorectal cancer(CRC)is the third most common cancer and accounts for 8%of all incident cases.In China,due to the changes of western-like lifestyle,dietary pattern,and growth of aging population,CRC incidence grows rapidly and converges on the 60-74 elderly people.Despite diagnostic and therapeutic advances,the survival rate of CRC patients is still unsatisfactory because of metastasis and recurrence.The five-year relative survival rate of patients with distant metastasis is only 13%according to cancer statistics.Thus,further investigation of molecular mechanisms of metastasis is crucial and more novel,effective and easy targets of therapies are needed.Epithelial mesenchymal transition(EMT)is a highly conserved cellular program that allows epithelial cells to convert into mesenchymal cells.During this process,non-motile,polarized epithelial cells are losing their epithelial charecters,like cell-cell junctions and expression of epithelial markers,and convert into individual,non-polarized,motile and invasive mesenchymal phenotypes.There is a growing understanding that EMT contributes to CRC invasion and metastasis.Several complex networks and interactions between multiple factors and signaling pathways are involved in EMT.Among all of them,nuclear factor-kappa B(NF-κB)signaling pathway plays essential roles in both inflammation and cancer progression.Activation of NF-κB is not only the central mediator of inflammatory responses of host defense against infection and stress,but also potentiates tumor progression via regulating a series of genes that were responsible for survival,proliferation,migration and invasion of cancer cell,like oncogene p53.Snail,which is the critical transcription factor involved in EMT and induced by NF-κB in cancer process,contributes to EMT and cancer via inhibiting expression of E-cadherin,which plays a key role in EMT and metastasis.In addition to control the cell architecture of both epithelial tumor cells and mesenchymal host cells,snail also regulates the paracrine and mechanical signaling between tumor and host cells,thereby modulating metastasis formation.Thus,in tumor cells,expression of snail can promote partial EMT and thus be the crutial metastatic property of sternness and motility.Lipocalin2(LCN2),also called neutrophil gelatinase-associated lipocalin,was originally identified as an innate immunity response protein for its capacity of limiting bacterial growth by sequestrating the iron-laden siderophore.Although recent studies have demonstrated that LCN2 was upregulated in various cancers and participated in tumorigenesis and metastasis,the underlying mechanism is still poorly understood.For example,some studies have reported that LCN2 suppresses EMT and metastasis of hepatocellular cancer,and inhibits invasion and angiopoiesis of pancreatic cancer.Conversely,other studiers demonstrated that LCN2 promotes EMT progression via modulating different signaling pathways in hepatocelluar cancer and prostate cancer,suggesting the complex roles of LCN2 in cancer progression.In addition,LCN2 could also transport retinol,its major metabolite,all-trans retinoic acid(ATRA)was involved in several tumor progress,but the cellular mechanism is unclear.In this study,we aimed to investigate the roles of LCN2 and ATRA play in EMT progression of CRC5 follow by exploring the underlying mechanisms and the interations between LCN2 and ATRA in EMT of CRC.To explore the function of LCN2 during EMT and metastasis in CRC,we established CRC cell lines of LCN2 knockdown and overexpression,then detected the changes of EMT markers and biological behaviors both in vitro and in vivo,and analized the relationship of LCN2 and EMT markers and its clinical significance.SW480 and HT29 cell lines,which had highly endogenous expression of LCN2,were selected to performe assay of knockdown of LCN2 by lentiviral vector containing sh-RNA-LCN2,SW620 and RKO cell lines,which expressed little LCN2,were chosen for stable expression of LCN2.We first employed real-time qPCR,western blot,immunofluorescennce,wound healing,CCK8,clone formation and transwell assays to assess the role of LCN2 in EMT of CRC cells;second,we established orthotopic transplantation tumor and lung metastasis of mouse model to explore LCN2 function in tumorigenesis and metastasis in vivo;third,we also analyzed the correlation between LCN2 and EMT markers,and the clinical significance of LCN2 on CRC patients.The results showed that knockdown of LCN2 significantly downregulated E-cadherin expression,while upregulated expression of vimentin,and enhanced proliferation,migration and invasion ability.Conversely,overexpression of LCN2 reversed the above functions of LCN2 in CRC cells.Furthermore,LCN2 could also suppress tumorigenesis and metastasis in vivo,suggesting that LCN2 plays a negative role in regulating EMT of CRC.Next,we detected changes of NF-κB/snail pathway in LCN2 knockdown and overexpression cell lines,then changed NF-κB activity or snail expression to further explore whether NF-κB/snail signaling pathway was involved in LCN2-modulated EMT in CRC.We found that phosphorylated,nucleus expression of NF-κB and snail expression were remarkably elevated in LCN2 knockdown cell lines.Immunofluorescence results also showed that the fulorescenece intensity and nuclear accumulation of snail were also remarkably increased in LCN2 knockdown cell lines.The opposite results were observed in LCN2 overexpression cell lines.Moreover,the luciferase reporter assay revealed that LCN2 attenuated the promoter activity of NF-κB.By using Leptomycin B(LMB,the activator of NF-κB)and Bay11-7082(the specific inhibitor of NF-κB),we confirmed that NF-κB increased both vimentin and snail expression,while decreased E-cadherin expression in CRC cells.These results implied that LCN2 may inhibit NF-κB/snail signaling pathway by targeting NF-κB promoter activity in CRC cells.We next blocked the enhanced NF-kB expression with specific siRNA in SW480-sh-LCN2 cells by using LMB and restored the decreased NF-κB or snail expression by snail overexpression vector in SW620-LCN2 cells,respectively,then assessed the changes of EMT markers.As expected,transfection of siRNA for NF-κB significantly restored E-cadherin,ZO-1 and snail and vimentin expression in SW480-sh-LCN2 cells.Moreover,LMB treatment remarkably abrogated LCN2-overexpression induced changes in SW620 cells.In addition,restoring snail level also effectively eliminated the increase of E-cadherin and ZO-1,and reversed the decrease of vimentin and snail expression and migration induced by LCN2 overexpression in SW620 cells,but did not appear to influence the expression of NF-κB.These results suggested a novel mechanism that LCN2 suppresses metastasis in CRC cells via inhibiting NF-KB-dependent activation of snail and EMT by targeting NF-kB promoter activity.The immunohistochemistry analysis of LCN2,E-cadherin,β-catenin,proliferation marker Ki67 and NF-κB expression in CRC tissues indicated that the positive rate of LCN2 in primary tumors was significantly higher(66.5%,266/400)than in lymph node metastases(31.2%,42/135).Interestingly,LCN2 was positively correlated with membrane E-cadherin expression and negatively associated with nuclear β-catenin and proliferation marker Ki67 expression,which are critical factors in tumor initiation and progression.Similar changes of E-cadherin,vimentin and Ki67 staining were found in mouse xenografts.According to the expression of both LCN2 and NF-κB,we divided the clinical samples into four groups:1:LCN2(+)/NF-κB(-),n=21;2:LCN2(-)/NF-κB(-),n=48;3:LCN2(+)/NF-κB(+),n=27;4:LCN2(-)/NF-κB(+),n=30,(follow-up time,1-200 months;median,52 months),and analyzed the correlation of LCN2 and NF-κB expression and prognosis of patients.The results showed that patients with LCN2(+)/NF-κB(-)expression had more favorable overall survival than those with LCN2(-)/NF-κB(+)expression(Figure 5F)and less snail accumulation in nucleus.The 5-year survival rate of group 1(86%)was also higher than other three groups(69%,63%,and 60%)。SW480 cells which are endogenously expressed LCN2 were treated with certain concentration of ATRA to assess the relationship of all-trans retinoic acid(ATRA),LCN2 and EMT in CRC.We found that ATRA inhibited phosphorylated NF-κB,snail and vimentin expression and migration but elevated LCN2 and E-cadherin expression,while neutralizing the enhanced LCN2 expression with a specific antibody blocked these actions of ATRA.In conclusion:1.In CRC cell lines and mouse model,LCN2 could upregulate the expression of epitheilial markers,downregulate mesenchymal markers expression.Meanwhile,LCN2 inhibits the ability of proliferation,migration and invasion in vitro,and tumorigenesis and metastasis in vivo,suggesting LCN2 suppresses EMT and metastasis in CRC.2.LCN2 may suppress EMT progress through inhibiting NF-κB/snail signaling pathway in CRC cells.Moreover,patients with LCN2 positive together with NF-κB negative expression had a more favorable prognosis and higher five-year survival rate.Thus,LCN2/NP-κB/snail signaling pathway may be a novel candidate for CRC therapy.3.ATRA increases LCN2 expression and it is dependent on the elevated LCN2 expression to enhance E-cadherin expression,and reduce vimentin expression and attenuated NF-icB/snail pathway and migration capacity in CRC cells.Therefore,ATRA may have the potential in CRC therapy.