Nicotine And Neuronal Acetylcholine Receptor Subunit Alpha-3(CHRNA3) Regulate Hippo Pathway To Promote The Growth And Migration Of Esophageal Carcinoma

In this research we compared genome wide DNA methylation status of esophageal cancer cell lines with normal tissue with DNA bisulfate sequencing. We identified the promoter region of CHRNA3as one of the hypermethlyated region in the ES cancer cell lines. We used western blot anaylsis to verify CHRNA3expression level in clinical ES cancer samples, among which3of the5sample tested showed with down regulation of CHRNA3.Functional study indicated knockdown of CHRNA3in ES cancer cell lines increased the growth rate, invasion and migration of ES cancer cells. Further mechanism studies indicated nicotine treatment or CHRNA3knockdown leads to nuclear translocation of YAP1, further substantiated by decreased S127phosphorylation of YAP1. The activation of YAP1was verified the induction of YAP1downstream genes such as CTGF by realtime PCR. Co-IP, GST pull down and immunofluorescent colocalizaiton experiments indicated the physical interaction and colocalization between YAP1and CHRNA3. And YAP1activation by nicotine treatment was inhibited by PKC inhibitor Enzastaurin.The negative regulation complex of YAP1contains β-catenin,14-3-3which showed of decreased interaction with YAP1after CHRNA3knockdown or Nicotine treatment, furthermore, the interaction between YAP1and P63is decreased after nicotine treatment or CHRNA3knock down, which decreases the stability of p63and inhibit the apoptosis of ES cancer cell. Immunohistochemistry chip analysis indicated upregulation of YAP1in esophargeal clinical samples, whereas CHRNA3level is upregualted in some of the samples and downregulated in some other samples.We demonstrated that CHRNA3plays an important role in the formation and cell membrane localization of the negative regulation complex of YAP1. In general in this research we have discovered a new mechanism of nicotine carcinogenesis which is to hijack the Hippo pathways transcription factor YAP1to promote the growth and migration of ES cancer cells. Intervention of molecules invovoled this processes might shed new insight for cancer therapy and prevention.