The Role Of C-Myc In Promoting Cholangiocarcinoma Cells To Overcome Contact Inhibition

Objective:To investigate the effects and mechanisms of c-Myc on abrogating contact inhibition in human cholangiocarcinoma(CCA)cells.Methods : In this experiment,,human bile duct epithelial cells,HIBEC,cholangiocarcinoma QBC939 cells and RBE cells were incubated in low cell density(about 50% confluent cells)and high density(confluent cells).The cell cycle distributions were detected by flow cytometry.The c-Myc inhibitor 10058-F4(F4),mTOR inhibitor rapamycin(Rap)and Yap inhibitor verteporfin(VP)were used to inhibite the activity of these proteins.Western blot analysis was used to detect the expression level of related proteins.The siRNA interference assay was used to interfere the expression of c-Myc protein.Results:(1)The loss of contact inhibition in human cholangiocarcinoma cells: flow cytometry analysis showed that the normal bile duct epithelial HIBEC cells contact inhibition at high cell density,cell cycle arrest in GO/G1 phase,while in the cholangiocarcinoma cell line QBC939 and RBE high density still maintain higher proliferation ability.(2)c-Myc is involved in the regulation of human cholangiocarcinoma cell resistance: Western blot analysis showed that the protein level of c-Myc obviously decreases in contact-inhibited normal biliary epithelial cells.However,CCA cells sustain high protein levels of c-Myc and keep strong proliferation ability in confluent conditions.The inhibition of c-Myc restores contact inhibition in CCA cells.(3)The mTOR pathway is implicated in c-Myc-mediated contact inhibition loss in human CCA cells.The results of western blot showed that the expression of phosphorylation of P70S6 K and S6 K,mTOR downstream effectors,decreased significantly in confluent HIBEC cells,but not in CCA cells.Inhibition of c-Myc significantly suppressed the phosphoration of p70S6 K and S6 in confluent CCA cells.And mTOR inhibition restored contact inhibition in CCA cells.(4)Deregulated c-Myc in confluent human CCA cells is regulated by YAP signaling: Western blot analysis showed that YAP was inactivated in HIBEC cells,but sustains high levels of activity in confluent CCA cells.YAP inhibition by verteporfin lead to decreased c-Myc in confluent CCA cells.(5)Merlin is down-regulated in confluent CCA cells.Western blotting showed that phosphor-Merlin decreased in confluent HIBEC cells but increased in confluent QBC939 and RBE cells.Conclusion: We find that c-Myc sustains high protein levels in confluent human CCA cells,but not in human normal biliary epithelial cells.The inhibition of c-Myc restores contact inhibition of human CCA cells.Furthermore,we show that c-Myc promotes the loss of contact inhibition in human CCA cells through the mTOR pathway,and the deregulation of c-Myc is controlled by Yes-associated protein(YAP).Our findings indicate that c-Myc is an important promoter which mediating the loss of contact inhibition in human CCA cells.