The Role Of α5-nAChR/STAT3/ADAM10 Signaling Axis In The Progression Of Cutaneous Melanoma

BackgroundSkin melanoma(SKCM)is the skin malignancy with the highest mortality rate,and its morbidity and mortality are on the rise worldwide.Smoking promotes the metastasis of melanoma and is an independent marker of poor prognosis in cutaneous melanoma.Nicotine is a major pathogenic component of tobacco and promotes proliferation,migration,stemness and epithelial-mesenchymal transition(EMT)in a variety of tumors.α5-nicotinic acetylcholine receptor(α5-nAChR)promotes melanoma progression by activating pathways such as PI3K/AKT.Nicotine promotes the expression of a disintegrin and metalloprotease 10(ADAM 10),which is involved in melanoma proliferation,invasion,metastasis and immune escape.It is not clear whether α5-nAChR mediates the expression of ADAM10 in response to nicotine in the melanoma.The aim of this paper is to investigate the role and mechanism of the α5-nAChR/ADAM10 axis in the progression of cutaneous melanoma and to provide new strategies for melanoma prevention and treatment.Objective1.To clarify the expression and correlation of α5-nAChR and ADAM 10 in databases and tissue samples of SKCM.2.To verify the role of the α5-nAChR/STAT3/ADAM10 signaling axis on the migration of melanoma cells in vitro.3.To reveal the role of α5-nAChR and AD AM10 in the tumorigenesis and metastasis of melanoma in the animal study.Methods1.Bioinformatics analysis was performed using the online databases GEPIA,UALCAN,TIMER,UCSC.We analyzed the expression levels of CHRNA5 and ADAM10 in human cutaneous melanoma tissues and corresponding normal tissues and the correlation between the expression levels of CHRNA5 and AD AM10.We analyzed the correlation between the expression levels of each and the prognosis of melanoma patients,as well as the prognostic significance of the high coexpression of CHRNA5 and ADAM 10 in melanoma patients.2.Immunohistochemical staining was used to measure the expression levels of α5-nAChR and AD AM10 in melanoma tissues and paraneoplastic tissues and the correlation between the expression of α5-nAChR and AD AM10.3.Western blot was used to detect the expression of α5-nAChR,ADAM10,pSTAT3,N-cadherin and Zebl after nicotine treatment in A375 and M14 cells.4.Stable transfected cell lines with low expression of α5-nAChR(abbreviated as sh-CHRNA5)were constructed using lentiviruses.Western blot was used to investigate the expression of α5-nAChR,ADAM10,pSTAT3,N-cadherin and Zebl in the sh-NC group and the sh-CHRNA5 group.5.Stable transfected cell lines with high expression of α5-nAChR(abbreviated as oe-CHRNA5)were constructed using lentiviruses.The above cells were treated with the STAT3 inhibitor(Niclosamide)and the expression of α5-nAChR,AD AM10,pSTAT3 in the oe-NC group and the oe-CHRNA5 group was detected by Western blot.6.Chromatin immunoprecipitation assay was used to verify the binding of the transcription factor STAT3 to the ADAM 10 promoter in the oe-NC group and the oe-CHRNA5 group of A375 cells.7.The effects of α5-nAChR and AD AM 10 on melanoma cell migration were investigated using wounding healing assay and transwell assay after treatment with the ADAM10 inhibitor(GI254023X)in the sh-NC group and the sh-CHRNA5 group.8.Immunohistochemistry was used to detect the expression of α5-nAChR,AD AM10 and Zeb1 in the cutaneous melanoma homograft and the effect of nicotine on the expression of these proteins.Results1.The expression levels of CHRNA5 and AD AM10 were higher in melanoma tissues than that in normal tissues in the TCGA cutaneous melanoma database,and the expression levels of both were positively correlated.High expression level of CHRNA5 or AD AM10 was associated with poor prognosis in melanoma patients.The survival time of melanoma patients with high coexpression of CHRNA5 and ADAM 10 was significantly shorter than those with high expression of CHRNA5 or ADAM 10 alone.2.Immunohistochemical results suggested that both α5-nAChR and ADAM 10 were highly expressed in melanoma tissues and the expression levels of both were positively correlated.3.Western blot showed that the expression levels of α5-nAChR,AD AM10,pSTAT3,N-cadherin and Zebl were all increased after the treatment of nicotine in A375 and M14 cells compared to the control group,while there were no significant changes in the expressions of tSTAT3.4.Western blot demonstrated that the expression levels of α5-nAChR,ADAM10,pSTAT3,N-cadherin and Zebl were lower in the sh-CHRNA5 group than those in the sh-NC group.5.Western blot demonstrated the expression levels of pSTA3 and ADAM 10 were lower in the Niclosamide-treated group than those in the corresponding non-inhibitor-treated group.6.Chromatin immunoprecipitation assay showed that the transcription factor STAT3 could bind to the promoter of ADAM10,and the amount of STAT3 binding to the ADAM10 promoter was higher in the oe-CHRNA5 group than that in the oe-NC group.7.Wound healing assay and transwell assay demonstrated that the migration ability of cells with high expression of α5-nAChR was enhanced compared to the oe-NC group.The migration ability of cells in the GI254023X-treated group was lower than that in the corresponding non-inhibitor-treated group.8.Immunohistochemistry of homograft tumors of melanoma showed that the expression levels of α5-nAChR,ADAM10 and Zebl were lower in the KD group than those in the NC group.The expression levels of α5-nAChR,ADAM10 and Zebl were higher in the nicotine-treated group than those in the corresponding non-nicotine-treated group.Conclusion1.The expression levels of α5-nAChR and ADAM 10 are higher in cutaneous melanoma tissues than those in normal tissues,and their expression levels are positively correlated,and they are all associated with poor prognosis.2.In vitro,nicotine regulates the expression of ADAM 10,pSTAT3,N-cadherin and Zebl in melanoma cells through α5-nAChR.α5-nAChR/STAT3/ADAM10 signaling axis regulates the migration of melanoma cells.3.Immunohistochemistry of mice homograft tissues suggests that the nicotine/α5-nAChR/ADAM10 signaling axis promotes melanoma growth.