Mechanistic And Functional Study Of NF-?B/POU2F2/SLIT2/ROBO1 Pathway In Gastric Cancer Metastasis

?Background and aim?Cancer metastasis is the major cause of gastric cancer-related deaths.Our information over the regulatory networks of gastric cancer(GC)metastasis is far from clear.Mechanistic study of GC metastasis has long been the focus of interest.GC metastasis involves crosstalks among plenty of genes and signaling pathways.To uncover the key nodes regulating these pathways will definitely contribute to a better understanding of molecular mechanisms of GC metastasis as well as therapeutic treatment.Transcription factors have been implicated in regulating diverse cellular pathways.Although emerging evidences show that some transcription factors can function as either oncogenes or tumor suppressors,the role of transcription factors in mediating cancer metastasis remains unexplored.Here,we discovered that a critical transcriptional factor POU2F2 that has been reported to be required for the development of several cancer types,is also significantly important for gastric cancer(GC)metastasis.We found an aberrant upregulation of POU2F2 in metastatic GC.However,the underlying mechanisms and potential function of upregulated POU2F2 in GC have been poorly defined.?Methods?1.Westernblot was preformed to detect the expression of POU2F2 in normal gastric epithelial cells(GES-1)and gastric cancer cell lines with different metastatic abilities.POU2F2 expression was also examined in clear metastatic state of GC and cancer adjacent tissues by immunohistochemistry.Furthermore,we analyzed the correlation between the expression of POU2F2 and GC prognosis.2.Loss-of-function and gain-of-function experiments were carried out to study the role of POU2F2 in GC metastasis.Bioinformatics analysis was adopted to predict the potential downstream target of POU2F2.EMSA,CHIP-seq and luciferase assays were preformed to verify the predicted target.In vitro and in vivo functional studies were further used to validate the target gene.3.Based on reference reading,we search for candidate upstream gene of POU2F2.Bioinformatics prediction was adopted to search for the potential binding sites of the candidate gene to the promotor of POU2F2.We carried out a series of experiments ranging from EMSA and CHIP-seq to luciferase assays to validate the binding site.We also use in vitro and in vivo study to examine the role of this signaling pathway in regulating GC metastasis.4.Luciferase assays were adopted to study the regulatory role of miR-218 on multiple targets of this signaling pathway.In vitro and in vivo functional studies were also carried out to further confirm the function of miR-218 on modulating multiple targets of this pathway to affect GC metastasis.?Results?1.POU2F2 expression is extremely low in GES-1 cells.Compared with GES-1 cells,protein levels of POU2F2 increase by 2 folds in GC sublines with weak metastatic ability(MKN28-NM,SGC7901-NM).However,POU2F2 expression increased by 9 folds in GC sublines with strong metastatic ability(MKN28-M,SGC7901-M).In para-cancerous tissues,the ratio of positive POU2F2 expression is 4 percent.In GC,84.97 percent of GC metastases show positive POU2F2,while only 6 percent of GC tissues without metastasis has POU2F2 expression.GC tissues with positive POU2F2 expression has higher TNM stages and shorter life expectancy.2.SGC7901-M cells transfected with POU2F2 shRNA showed impaired ability of invasion and migration.Tail vein injection experiment with SGC7901-M cells transfected with POU2F2 shRNA revealed decreased lung metastases.Overexpression POU2F2 with lentivirus restored metastatic ability.Bioinformatics analysis indicates ROBO1 as a potential target of POU2F2.Further EMSA,CHIP-seq and luciferase assays confirmed the binding of ROBO1 to POU2F2.Gain-and loss-of function study showed that POU2F2 promoted GC metastasis via ROBO1.3.Previous studies implied that POU2F2 may serve as a downstream target of NF-?B.By bioinformatics prediction,we got candidate binding site of NF-?B and POU2F2.We further validated this specific binding via EMSA,CHIP-seq and luciferase assays.Activating NF-?B directly increased POU2F2 and ROBO1 expression and enhanced GC cell invasion and migration in vitro and in vivo.These effects were blocked by inhibiting NF-?B activity.When POU2F2 expression was impaired,the elevated ROBO1 expression and cell metastatic ability due to the activation of NF-?B was declined accordingly.ROBO1 overexpression antagonized the effects of shPOU2F2 on the metastatic ability of GC cells.4.Luciferase assay results showed that miR-218 directly binds to and simultaneously regulates POU2F2,I?K-? and ROBO1.Upregulating miR-218 leads to declined expression of POU2F2,I?K-? and ROBO1,and impaired GC cell metastatic ability in vitro and in vivo.By suppressing miR-218,we could see the opposite phenotypes.Moreover,inhibiting the levels of POU2F2,I?K-? and ROBO1 resulted in a similar metastatic phenotype observed in miR-218 overexpressing condition.However,upregulating the expression of IKK-?,POU2F2 or ROBO1 by vectors without miR-218-binding sites leads to the partial rescue of the inhibition of GC metastasis derived from miR-218 overexpression.?Conclusions?1.POU2F2 positively regulates ROBO1 to promote GC invasion and migration.POU2F2 positivity closely correlates with poor GC prognosis.2.POU2F2 serves as the link between NF-?B and SLIT2/ROBO1 signaling pathway in mediating GC metastasis.3.MiR-218 inhibits GC metastasis through modulating multiple genes in the NF-?B/POU2F2/SLIT2/ROBO1 pathway.