MESP2 Suppresses The Proliferation,Migration And Invasion Of Gastric Cancer Cells Via Regulating The TCF4/SKP2/p27 Axis

MESP2(Mesodermal Posterior 2),a transcription factor with a basic helix–loop–helix(b HLH)motif that has been illustrated to play pivotal roles during the development of somite and heart in mammalian.MESP2 is a regulator of several processes in somatic morphogenesis.In the controlled segmentation program of the Notch signalling pathway reflects the important function of MESP2.In fact,boundary formation and somite compartmentalisation seem to depend on the combined action of Wnt and Notch signalling.Interrupting either pathway restrains somite formation and the MESP2 protein levels that indicates somite compartmentalisation and boundary formation.Previous functional experiments have displayed that aberrant expression of b HLH transcription factor is related with various tumorigenesis.These findings suggest that MESP2 may also participate in human cancer progression.GC(Gastric cancer),as a deadly malignancy,is the third main cause of global human cancer death,resulting in～723 000 deaths annually.Patients have poor survival,mainly due to suboptimal treatment and late diagnosis.TCF4(Transcription Factor 7-like 2)is a key component of the Wnt signalling pathway,which mostly acts as a transcriptional factor after entering the nucleus.Furthermore,Wnt ligands trigger the typical Wnt/beta-catenin signalling pathway,which in turn activates the LRP and Frizzled receptors,leading to beta-catenin stabilization and nucleus translocation.Subsequently,nuclear beta-catenin binds to TCF4 and thereby expresses relevant genes.The ultimate and functional aim in the typical Wnt signalling to beta-catenin pathway is the formation of the TCF4/beta-catenin complex,and this interaction is regarded as a valid target for treatment of cancer.Herein,molecular biology,cell biology,molecular pathology and other techniques were used to elucidate the molecular mechanisms of MESP2 in GC cells,explaining the effect of MESP2 in suppressing GC progression.The main results are as follows:(1)The low expression of MESP2 is associated with poor prognosis in GC patients.We first examined the intracellular localization of MESP2 protein in GC cells.Next,we analyzed MESP2 protein levels in 80 samples of clinical patients by IHC(immunohistochemistry)staining.MESP2 IHC staining was notably lower in gastric tumor tissues in a grade-dependent pattern in comparison to the normal tissues.Consistently,analysis of MESP2 expression levels in GC patient tumors using the R2:Genomics Analysis and Visualization Platform validated that MESP2 upregulation predicted better patient survival.Taken together,our results indicate MESP2 may be an anti-oncogene.(2)MESP2 inhibits the proliferation,migration and invasion of GC cells.We successfully knocked down MESP2 expression in HGC-27 cells and MKN-45 cells by lentiviral sh RNA,namely,sh MESP2 nos.1 and 2.In MTT and Brd U staining assays,knockdown of MESP2 promoted the proliferation behaviors of GC cells.Conversely,overexpression of MESP2 substantially suppressed cell growth.Then we utilized flow cytometry to show MESP2 overexpression arresting cell cycle at the G0/G1 phase.In addition,Transwell and Wound-healing assays demonstrated knockdown of MESP2 enhanced the migratory and invasive behaviors.The results of mouse xenograft model and lung metastasis in vivo were also confirmed.(3)MESP2 suppresses the activation of the TCF4/beta-catenin transcriptional complex by competing with beta-catenin for binding of TCF4.Through functional domains analysis,we found that ITF2 and MESP2 proteins exhibited the relatively well-conserved b HLH domain.And we assessed that MESP2 and TCF4 had a binding relationship through online website prediction.Our results were further validated by Immunofluorescence staining and Co-IP assays.Truncated experiments confirmed that the specific binding regions were the binding domains between b HLH and beta-catenin.We analyzed the amount of TCF4 in nuclear fraction was remarkably increased after knocking down MESP2 by subcellular fractionation.In addition,we examined that MESP2 overexpression drastically decreased the direct binding between beta-catenin and TCF4,on the basis that MESP2 did not bind to beta-catenin.Luciferase reporter assay showed that TCF4 transcription factor activity decreased after overexpression of MESP2.(4)MESP2 regulates the transcriptional activity of the SKP2 promoter and the subsequent ubiquitination of p27 protein to inhibit gastric cancer progression.WB and q RT-PCR assays showed that there was no significant effect on p27 m RNA levels after downregulation of MESP2,but p27 protein levels were reduced.It seemed that a post-transcriptional mechanism was involved in changes in p27 expression levels.Our study unveiled that overexpression of MESP2 significantly lengthened the half-life of p27 protein.Consistently,knockdown of MESP2 induced p27 degradation was blocked by MG132 treatment.Additionally,the ubiquitylation assay showed that downregulation of MESP2 enhanced the poly-ubiquitylation of p27.Finally,transcriptome profiling showed MESP2 depletion suppressed the p27 pathway.We found that TCF4 could bind to the SKP2 promoter by Ch IP.Importantly,overexpression of MESP2 significantly inhibited the combination of TCF4 with SKP2 promoter.Next,Luciferase reporter assay showed that transfection of TCF4 increased the luciferase activity of SKP2 reporter in comparison with that in control group.Impressively,the luciferase activity of SKP2 reporter was notably inhibited after co-transfection of MESP2 and TCF4.We detected that knockdown of SKP2 and MESP2 suppressed the proliferation and migration of GC cells.Consistently,these related proteins expression levels were partially inhibited.These results suggest that overexpression of MESP2 leads to p27 accumulation due to the inhibition of SKP2 transcription mediated by the TCF4/beta-catenin complex.In summary,we first reported that MESP2 functions as a new tumour suppressor in GC.Mechanistically,MESP2 competitively binds with beta-catenin to TCF4,and blocks the transcriptional activation activity of TCF4/beta-catenin complex on SKP2 promoter,which in turn enhances the stability and expression levels of p27 through suppression of p27 ubiquitination.Accordingly,this study revealed that MESP2 as novel player in inhibiting proliferation,migration and invasion of GC cells,implying that MESP2 is a prospective therapeutic target for GC patients.