TCP1 Promotes The Proliferation Of Tumor Cells By Regulating The Stability Of C-Myc Through AKT/GSK-3β And ERK Signaling Axes

Objective: Previous study showed that TCP1 was highly expressed in many solid tumors,and was related to the clinical classification and poor prognosis of pancreatic cancer and hepatocarcinoma.Phenotypic experiments in vivo and in vitro showed that TCP1 regulated the proliferation and metastasis of tumor cells.However,the detail mechanism remains unclear.In this thesis,the molecular mechanism of TCP1 regulating c-Myc was studied from the perspective of protein homeostasis.Methods:(1)Knock down TCP1 cell lines(sh TCP1)and negative control cell lines(scramble)were constructed by sh TCP1 lentivirus infection with PANC-1,Bx PC-3,Huh-7 and SMMC-7721 cell lines.The levels of c-Myc protein and c-Myc m RNA in all cell lines were detected by Western blot and RT-q PCR respectively;(2)Treated with the protein synthesis inhibitor cycloheximide(CHX)and 26 S proteasome inhibitor MG132 for 0 min,15 min,30 min,60 min,90 min and 120 min in the sh TCP1 and scramble cell lines,and exploring the degradation pathway of c-Myc preliminarily;(3)Exogenous c-Myc and human ubiquitin were transformed or co-transformed in sh TCP1 and scramble cell lines and treated with MG132 for 1 h,the level of exogenous c-Myc binding ubiquitin was detected by Co-IP and Western blot experiments,and the degradation pathway of c-Myc protein was further determined;(4)The effect of knock-down TCP1 on the degradation rate of c-Myc protein was compared and analyzed after CHX treating sh TCP1 and scramble cell lines for a shorter time;(5)The phosphorylation levels of p c-Myc(Ser 62)and p c-Myc(Thr 58)were detected by knock down TCP1 and overexpress TCP1 in four kinds of cancer cells.(6)Detect the phosphorylation level of the main proteins in the AKT/GSK-3β and ERK pathway by knock down and overexpress TCP1;(7)The expression level of c-Myc protein was detected by using GSK-3β inhibitor(ARA014418)in four cancer cell lines and knock down TCP1 cancer cell lines,and it was determined that TCP1 regulated AKT/GSK-3β pathway;(8)AKT specific inhibitor(AKTi-VIII)was used to detect the effect of AKT/GSK-3βpathway on the expression of TCP1 protein in four cancer cell lines,and it was determined that TCP1 played a regulatory role in the upstream of this pathway;Results:(1)In PANC-1,Bx PC-3,Huh-7 and SMMC-7721 cell lines,the protein level of c-Myc was significantly decreased after knock down TCP1,but the m RNA level had no significant change.(2)After treating CHX and MG132 in sh TCP1 and scramble cell lines,MG132 inhibited the degradation of c-Myc protein caused by knock down TCP1.In this paper,we preliminarily confirmed that the decrease of TCP1 protein level led to the degradation of c-Myc protein from the proteasomal pathway.(3)Ubiquitin experiments showed that the decrease of TCP1 protein resulted in more ubiquitin molecules bound to exogenous c-Myc protein.Therefore,it was determined that TCP1 knock-down led to the degradation of c-Myc protein by the ubiquitin proteasome pathway.(4)After CHX intervention in sh TCP1 and scramble cells for a shorter time,it was found that knocking down TCP1 accelerated the degradation of c-Myc protein.(5)In four kinds of cancer cells,the ratio of phosphorylation level of c-Myc Ser 62 to c-Myc Thr 58 decreased by knocking down TCP1,and the ratio of phosphorylation level of c-Myc Ser 62 to c-Myc Thr 58 increased by overexpressing TCP1.(6)After knocking down TCP1 or overexpressing TCP1,the phosphorylation levels of various proteins in the AKT/GSK-3β and ERK pathways changed significantly,but the total protein levels in the pathways did not change significantly.(7)The results of GSK-3β inhibitor treatment and AKT inhibitor treatment showed that the protein level of c-Myc recovered after specifically inhibiting the activity of GSK-3βin the four TCP1 knock-down cell lines,which was equivalent to that of the scramble group.Inhibition of AKT/GSK-3β pathway did not affect the protein expression of TCP1.Conclusions:(1)The knock down TCP1 could promote the degradation of c-Myc by ubiquitin proteasome pathway in liver cancer and pancreatic cancer cell lines.(2)TCP1 regulated the phosphorylation of c-Myc at Thr58 and Ser62 through the AKT/GSK-3β and ERK pathways.TCP1 knockdown significantly reduced the ratio of phosphorylation levels at c-Myc Ser62 and c-Myc Thr58,and then c-Myc was degraded by ubiquitin proteasome pathway by binding more ubiquitin proteins through E3 ubiquitin ligase.Overexpression TCP1 could significantly increase the ratio of phosphorylation levels at sites of c-Myc Ser62 and c-Myc Thr58,and c-Myc protein tended to be stable and increase its transcriptional activity.(3)In summary,TCP1 could stabilize c-Myc protein through AKT/GSK-3β and ERK pathways,and promote the transcription of c-Myc-targeted proliferation genes,and then promoting the proliferation of pancreatic and liver cancers.