MSC Induce Emt In Melanoma By Paracrine Of TGF-? Mediated Activation Of ERK1/2 Signaling Pathway

Backgrounds and ObjectiveMelanoma,one kind of malignant tumor specifically derived from melanocytes,dominates the most serious and deadliest rates of skin cancer.In recent years,with the growing incidence of melanoma around the world,it still remains an extremely tough task to deal with the disease though international clinicians have explored tremendous fundamental researches and effective treatment strategies.Thus deeper and more comprehensive studies focusing on the pathogenesis of melanoma and the involved complicate signaling pathways may be important to pharmacological therapeutic improvements.It has been documented that gene mutation(including oncogene or the suppressor)majorly contributes to melanoma formation,while during the following process of tumor invasion and metastasis,epithelia-mesenchymal transforming(EMT)shows great potential to promote it.EMT was primarily mentioned in the context of embryonic development,and it is a means by which a cell transforms morphologically and functionally from an epithelial phenotype to a mesenchymal one.Most important hallmarks of EMT were reported as the totally divided expression of E-cadherin and N-cadherin.This switch leads melanoma cells to intimately contact with stromal fibroblasts and endothelia cells instead of adjacent keratinocytes,thus providing assistance for dermal and vascular melanoma invasion.It has been reported that activation of IL-6 induced STAT3 signaling pathway promotes uveal melanoma development,suggesting that EMT could be regulated by diverse molecular signaling network.Therefore,basis on comprehensive investigation of involved signaling pathways,direct attempts to explore the specific inhibitors or targeted antibodies towards EMT process will become the prevalent treatment strategy.Transforming growth factor ?(TGF-?)serves as a multifunctional cytokine,which triggers intercellular signaling pathway of melanoma cells by autocrine and paracrine in the tumor microenvironment.TGF-? has been demonstrated to elicit diverse cellular reactions including inflammation,anti-apoptosis and immune escape.What's more,TGF-? is well known to promote melanoma invasion and metastasis by inducing EMT.While in ex vivo incubation of B16 melanoma cells,silencing of TGF-? by RNAi technology significantly blocked the tumor improvement.It seemsthat TGF-? signaling play an important role to melanoma progress,thus making it more necessary to excavate the extraordinary role of TGF-? signaling in EMT.It is reported that MAPK,ERK and NF-?B were important modulators of melanoma EMT,which was accelerated by phosphorylation of MAPK and ERK,while as an important pro-inflammatory transcript factor,NF-?B phosphorylation was another major cause of EMT formation.Activation of MAPK,ERK and NF-?B was reported certainly dependent on TGF-? stimulation,although it still remains obscure when it comes to EMT regulation.Mesenchymal stem cell(MSC)is a kind of multi-potential differential stem cell,acting as a double-edged sword in clinical tumor treatment.MSC have long been considered of effective carriers delivering targeted therapeutic agents to tumor cells,while upon stimulations of inflammatory cytokines,MSC could become highly immunosuppressive by immensely unleashing nitric oxide(NO)and other chemokines.In recent years it has been reported that tight interactions between melanoma cells and MSC of the tumor environment may promote EMT progress and accelerate tumor growth and migration,suggesting that something like cytokines and chemokines secreted by MSC may be involved in inducing melanoma cells undergo EMT.However,whether MSC being able to secrete functional TGF-?to promote EMT of melanoma cells is scarcely known.Moreover,the role of MAPK/ERK and NF-?B signaling pathways in it still needs to be elucidated.Therefore,the major purpose of this study is to determine the role of MSC secreted TGF-? and the related signaling pathways on EMT progress.Consequently,on the basis of backgrounds above,the present study was designed to determine: 1)whether paracrine secretion of TGF-? by MSC is participated in EMT of melanoma cells;2)the role of MAPK/ERK and NF-?B signaling pathways in TGF-? mediated EMT process.MethodsExperimental C57BL/6 mice at 6-8 weeks age were purchased from the Shanghai Experimental Animal Center at the Chinese Academy of Sciences.MSC were obtained from the bone marrow of C57BL/6 mice by flushing the tibias and femurs with normal saline.After being centrifuged and filtered,the MSC-conditioned medium(MSC-CM)was collected and used to stimulate B16 melanoma cells undergo EMT.Wound-healing and transwell invasion assay were used to detect changes in B16 cells' ability of proliferation and invasion by MSC-CM.Q-PCR and immunofluorescence were used to measure changes in specificbiomarkers of EMT by MSC-CM.The effect of TGF-? on MSC-CM mediated EMT of B16 cells was determined by using TGF-? specific inhibitor SB431542,and was validated by in vivo experiments.The TGF-?-MAPK or TGF-?-ERK signaling was determined by detecting the phosphorylation levels of MAPK and ERK using Western Blot,and their specific inhibitor SB203580 and SCH772984 were administrated as a method to observe the blocking effect on TGF-? induced EMT process.Results1.MSC significantly enhanced the B16 cells' ability of proliferation and invasion.B16 melanoma cells were co-cultured with MSC-CM and the PRIM 1640 medium as the control,while the MSC-CM cultured B16 cells represented obvious increases in wound closure area(41.7 ± 7.62 vs 92.0 ± 7.00 %,P<0.05,n=3)and numbers of invasive cells(74.0 ± 8.08 vs 198.3 ± 20.48,P<0.05,n=3).What's more important,B16 cells cultured with MSC-CM showed higher expression of E-cad(1.0± 0.03 vs 0.5 ± 0.09 fold,P<0.05,n=3)and lower of Vimentin(1.0 ± 0.23 vs 2.6 ±0.23 fold,P<0.05,n=3)in transcription level compared with the control group.These results were then verified by immunofluorescence test.Next we conducted the injection of B16 melanoma cells co-cultured with MSC-CM or PRIM medium into the tail vein of C57BL/6 mice to establish the standard lung metastasis model.As a result,B16 cells exposed to MSC-CM caused much higher relative ratio of metastasis to the whole lung area(0.3 ± 0.05 vs 0.8 ± 0.12 %,P<0.05,n=3)compared with the control group.Together,these data above demonstrated that MSC promote B16 melanoma cells proliferation and invasion as well as the EMT process though paracrine.2.MSC significantly enhanced the B16 cells' ability of proliferation and invasion.The ELISA assay showed that MSC CM showed higher concentration of TGF-?,and then we administrated exogenous TGF-? to make it clear whether TGF-? is participated in inducing EMT in B16 cells.As shown in changes of E-cad andVimentin gene expression,TGF-? induced a notable downregulation of E-cad(1.0 ±0.03 vs 0.5 ± 0.06 fold,P<0.05,n=3)while upregulation of Vimentin(1.0 ± 0.03 vs2.2 ± 0.15 fold,P<0.05,n=3)compared with the control group.What is more,TGF-?receptor specific inhibitor SB431542 significantly raised the m RNA levels of E-cad(0.5 ± 0.06 vs 1.1 ± 0.07 fold,P<0.05,n=3)and reduced that of Vimentin(2.2 ± 0.15 vs 1.0 ± 0.10 fold,P<0.05,n=3)compared with TGF-? treatment.Consistently,MSC CM treatment significantly lowered the gene expression of E-cad in B16 cells and Vimentin exactly showed the opposite.What is more paramount,MSC CM induced changes in E-cad and Vimentin expression were suppressed by TGF-? inhibitor,which was also verified in in vivo experiment.Those data above proved that MSC induced EMT of melanoma cells was dependent on TGF-? paracrine.3.ERK1/2 inhibition significantly blocked TGF-? induced EMT in melanoma cellsWhen exogenous TGF-? was added to stimulate B16 cells,Western blot assay represented a remarkable increase in phosphorylation of p38 MAPK and ERK1/2compared with the control group.Meanwhile,the levels of phosphorylation of NF-?B and inflammatory chemokines(IL-6 and IL-8)were similarly upregulated by TGF-? treatment.In order to determine roles of MAPK and ERK in TGF-?'regulation of EMT in melanoma cells,we used SB203580 and SCH772984 to specifically blockade p38 MAPK and ERK1/2 activation.As a result,ERK1/2instead of p38 MAPK inhibition significantly reversed TGF-? induced increase in Vimentin expression and decrease in Snail expression using immunofluorescence and Western Blot methods.Together,these data above delivered us an important massage that TGF-?-ERK1/2 signaling pathway was responsible for MSC paracrine triggered EMT in melanoma cells.Moreover,TGF-? evoked NF-?B activation may underlie the pro-inflammation effect.ConclusionParacrine of TGF-? by MSC promotes EMT in melanoma cells,augmenting their ability of proliferation and invasion.TGF-? mediated ERK1/2 signaling pathway was pivotal to MSC induced EMT in melanoma cells.At the same time,we have noted that paracrine of TGF-? was responsible for NF-?B activation and inflammatory excitation which may accelerate the ETM process.In general,thisstudy have made a point that paracrine of TGF-? by MSC is intimately involved in promoting EMT in melanoma cells,thus providing a novel outlook for clinicians designing effective strategy on melanoma treatment at an early state.