| Breast cancer is a heterogeneous disease with four major subtypes according to gene expression profiling:luminal A,luminal B,HER2,basal-like.Compared with the other subtypes,basal-like breast cancer(BLBC)is associated with an aggressive clinical history,early recurrence,distant metastasis and shorter survival.BLBC usually occurs in younger and premenopausal women and has a tendency toward early metastatic spread to the brain and lungs,sites known to be associated with a poor prognosis.A lack of effective targeted therapies for BLBC often results in a rapidly fatal clinical outcome.An important prerequisite for targeted therapies is to understand the molecular mechanisms underlying the metastatic process associated with BLBC.Notably,BLBC possess the activated epithelial-mesenchymal transition(EMT)program,which is required for tumor progression and metastasisEMT is a phenotypic conversion that facilitates embryonic development,wound healing and cancer metastasis.During EMT,epithelial cells lose their epithelial properties and acquire a motile mesenchymal phenotype.Importantly,EMT causes the acquisition of cancer stem cell(CSC)-like properties.As tumor cells undergone EMT often faced a harsh environment of foreign tissue during the metastatic process,they mustrewire metabolic and oncogenic pathways to meet the demands of survival and metastasis.Characterization of the causal RelAtionship between EMT and metabolic alteration will help to develop novel interventions for targeting metastatic breast cancer.Metabolic reprograming has been accepted as a hallmark of cancer Lipid metabolism that represents an important source of bioactive molecules is implicated in tumor progression.Arachidonic acid(AA)is a polyunsaturated fatty acid that is metabolized into prostaglandins(PG),hydroperoxyeicosatetraenoic acids and epoxyeicosatrienoic acids through a series of enzyme-catalyzed reactions.AA-RelAted enzymes and their metabolites are involved in pathophysiological processes of many diseases,including a variety of cancers.Aldo-keto reductase family 1 member B1(AKR1B1),which catalyzes the reduction of PGH2 to PGF2a,is a major NADPH-dependent PGF synthase during AA metabolism.PGF2a,a major metabolite of AKR1B1,modulates cell adhesion,migration and invasion via PGF2a receptor(FP)-mediated signalingin endometrial cancer,suggesting that AKR1B1 may be involved in tumor progression by modulating lipid metabolism.Epalrestat is the only AKR1B1 inhibitor that has been approved for the targeted treatment of diabetic complications,which is easily absorbed by tissues and has minimum adverse effects.Epalrestat inhibit the activity of AKR1B1,and repress the progress of BLBC.In this study,we aimed to revealed the role of AKR1B1 in the progress of BLBC and confirm the mechanism,especially the function of epalrestat in treat BLBC.Above all,we found that AKR1B1 expression was significantly higher in BLBC than in other subtypes via bioinformatics analysis.To confirm this finding,we collected fresh frozen breast tumor tissues and cell samples,and performed Western blotting semi-quantitative RT-PCR or quantitative real-time PCR to validated the expression of AKR1B1 and EMT markers.Consistently,AKR1B1 protein level also was high in BLBC cell lines,whereas it was absent in luminal cell lines.While investigating Twist2-mediated induction of EMT in BLBC,we unexpectedly identified that AKR1B1 expression is elevated by Twist2.To investigate the correlation between AKR1B1 and Twist2,we analyzed the expression of AKR1B1 and Twist2 via bioinformatics analysis.The results indicated that AKR1B1 expression positively correlated with Twist2 expression.The positive correlation between AKR1B1 and Twist2 in breast cancer prompted us to investigate their mutually causal relationships.we established stable cell lines with high expression or knock down Twist2.As anticipated,Twist2 expression substantially downregulated E-cadherin expression and induced a morphological change indicative of EMT.These data suggest that Twist2,as a transcriptional factor,may induce AKR1B1 expression via transcriptional regulation.To determine whether AKR1B1 expression is regulated directly by Twist2.We analyzed the promoter sequence,generated several deletion mutants,introduced point mutations and construct lucifrase plasmid.We employed the luciferase assay and ChIP assay in 293T,MDA231 and SUM159 cells,We revealed that Twist2 directly bound to the AKR1B1 promoter,and E-box at-997 bp is required for Twist2-induced transcriptional activation.To study the molecular function and mechanism of AKR1B1,we established stable cell lines with high expression or knock down of AKR1B1.We employed Western blotting,transwell assay and luciferase assay.The results implied that AKR1B1 expression can significantly caused a decrease in the epithelial marker E-cadherin and an increase in mesenchymal marker,repress E-cadherin promoter luciferase activity and enhance the migration and invasion.Together,these observations indicate an important role for AKR1B1 in induction of EMT and acquisition of migratory and invasive ability in breast cancer cells.PGF2a is a major metabolite of AKR1B1.To understand the metabolic consequence of AKR1B1 expression,we first measured the production of PGF2a by a Elisa kit in stable cell lines with high expression or knock down of AKR1B1.We revealed that AKR1B1 expression can increase the PGF2a production.AKR1B1,a monomeric NADPH-dependent cytosolic enzyme,leads to a decrease in the ratio of NDAPH/NADP+.A decrease in NDAPH/NADP+ ratio could cause oxidative stress.To confirm the relationship between AKR1B1 and oxidative stress,we performed ROS assay,Our results demonstrated that AKR1B1 expression induced an obvious increase of ROS.Since NF-?B is a redox-sensitive transcription factor that has been proposed to be the sensor for ROS.AKR1B1-mediated increase of ROS may involve NF-?B signaling.We employed Western blotting to validated the exporession of RelA and luciferase.assay.The results showed that AKR1B1 expression led to a marked increase of RelA expression in both nucleus and cytoplasm and enhanced NF?B promoter luciferase activity.The NF-?B pathway has been implicated in the regulation of EMT.To investigate the role of AKR1B1-mediated NF?B signaling in EMT induction,we determined the expressions of RelA and various transcriptional factors of EMT by Western blotting in stable cell lines with high expression or knock down of AKR1B1.We observed that AKR1B1 expression significantly increase RelA and twist2 expression.It's well known that TNF-? and IL-1? contribute to NF?B activation.Indeed,RelA expression was induced by TNF-? or IL-1? in MDA-MB231 and SUM 159 cells.Importantly,Twist2 expression also was dramatically upregulated upon TNF-? or IL-1? stimulation in these cells.These data suggested that Twist2 expression was positively correlated with RelA expression.To further investigate the relationship between RelA and twist2,we established stable cell lines with high expression or knockdown of RelA.Strikingly,RelA expression resulted in an increase,in twist2 expression,indicating a critical role of RelA in regulating Twist2 expression.To demonstrate whether Twist2 expression is regulated directly by RelA,We analyzed the promoter sequence of Twist2 generated several deletion mutants,introduced point mutations and constructed lucifrase plasmid.We performed luciferase assay and ChIP assay in T47D,MDA-MB231 and 293Tcells.The results suggested that all NF?B-binding motifs are important for RelA-mediated Twist2 activation.We detected a dramatic enrichment of RelA in the Twist2 promoter.These data indicated that RelA upregulates Twist2 expression by directly binding to the Twist2 promoter.Many solid tumors contain a small population of highly tumorigenic CSCs,which contribute significantly to tumor initiation and metastasis.Emerging evidence has shown that EMT program generates cells with stem cell-like properties.As AKR1B1 is highly expressed and involved in the EMT process in BLBC,we speculated that AKR1B1 expression might confer stem cell-like properties to BLBC cells.To test this notion,we we examined tumorsphere formation and CD44high/CD24low markers using flow cytometry analysis in stable cell lines with high expression and knock down of AKR1B1.we revealed that AKR1B1 expression enhanced tumorsphere formation and induced a remarkable increase of CD44high/CD24low population.CSCs is highly tumorigenic and metastatic.We performrd soft agarose assay and in vivo tumorigenesis abilities were tested in severe combined immunodeficient(SCID)mice.We revealed that AKR1B1 expression caused a marked increase of colony-formation in vitro and dramatically enhanced tumor growth in vivo.Next,we investigated whether inhibition of AKR1B1 expression affected tumor metastasis in a xenograft metastasis model.Strikingly,knockdown of AKR1B1 expression greatly suppressed lung metastasis in vivo.Epalrestat,a specific inhibitor of AKR1B1,could inhibit the activity of AKR1B1,To investigate weather epalrestat could repress the progress of BLBC,we treated BLBC with epalrestat,and a series of experimens were employed.we revealed that epalrestat could restore E-cadherin expression,reversed the suppression of the E-cadherin promoter,and dramatically repressed the migration and invasion,significantly reduced the PGF2a production,significantly suppressed tumorsphere formation and reduced the percentage of CD44high/CD24low population,caused a dramatic decrease of colonies in vitro and significantly reduced tumor growth in vivo and greatly suppressed lung metastasis in vivo.In all,epalrestat could suppress BLBC progress.To elucidate clinical relevance of AKRlBl,we revealed that patients with high AKR1B1 expression have shorter relapse-free survival,and primary tumors with high AKR1B1 expression preferentially metastasized to the brain and lungs via bioinformatics analysis.These clinical validations strongly support the critical role of AKR1B1 in BLBC.From the above results,we could get the following conclusion:1.AKR1B1 overexpression highly correlates with BLBC and poor prognosis.2.AKR1B1 activates EMT by a Twist2-mediated positive feedback loop.3.AKR1B1 promotes tumorigenicity and metastasis by inducing cancer stem cell-like properties4.AKR1B1 inhibitor epalrestat suppresses BLBC progression... |
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