The Function And Mechanism Of CHIP Protein In Pancreatic Cancer

BackgroundPancreatic cancer(PC)is hard to be diagnosed in the early stage and is one of the most malignant gastrointestinal cancers with poor prognosis,It is the fourth leading cause of cancer deaths in America and has the lowest 5-year survival rate among all malignancies.The high mortality rate is primarily due to the reason that most patients present with advanced stage disease that becomes apparent after the tumor invades surrounding tissues or metastasizes to distant organs.Furthermore,the conventional treatment approaches have little impact on the natural course of this aggressive neoplasm due to its drug resistance characteristics.Therefore,It will improve the prognosis of patients with early diagnosis,effective management of the disease by prohibiting tumor growth and metastasis,as well as increasing the drug sensitivity.CHIP is a chaperone-associated ubiquitin ligase that plays an important role in the regulation of intracellular proteins.Though binding to the carboxyl termini of Hsp70/Hsp90,CHIP mediates the ubiquitylation of chaperone-bound client proteins in conjunction with E2 enzymes and induces client degradation by the 26S proteasome.CHIP plays a key role in the regulation of misfolded proteins or other proteins that have functions in the cell signaling pathway.The dysfunctional expression of CHIP is one of the important factors that lead to tumor development or neurodegeneration.The possible function of CHIP in the development and progression of pancreatic cancer has not been elucidated.In this study,the biological function and clinical significance of CHIP in pancreatic cancer was examined in order to find a new way to an early diagnosis and effective treatment of pancreatic cancer.Objective1)To explore the influence of CHIP on the rate of tumor growth,apoptosis,invasion and migration.2)To find the protein that is the target of CHIP,to verify the role of CHIP in the cell signaling pathway.3)To explore the impact of CHIP on the tumor characteristics in xenograft mouse model.4)To evaluate the potential of CHIP in the early diagnosis and prognosis of pancreatic cancer.Methods1)DNA fragment encoding microRNA of CHIP or full-length CHIP were subcloned into plasmids and were then packaged to lentiviral vectors.Pancreatic cancer cell line Panc-1,Bxpc-3 and SW1990 were transfected/infected with plasmids/lentiviruses in order to produce CHIP knockdown or overexpression PC cells.2)Colorimetric MTT assays were used to test the viability of cancer cells that were CHIP knockdown or overexpression.Transwell chambers were used to test the invasive or metastatic potential of pancreatic cells with CHIP silence or overexpression.Flow cytometrys were performed to measure the apoptotic rate that was induced by targeted drug erlotinib in tumor cells with different CHIP expression.3)Immunoprecipitation was used to identify the protein that could be associated with CHIP,the expression of target protein was measured after plasmid transfection in pulse-chase experiments.SDS-PAGE and Western blot was used to test the expressions of proteins that play pivotal roles in the cell signaling pathway.4)Heterologous subcutaneous pancreatic cancer models in nude mice were eastablished to monitor the tumor growth.Tumor growth was measured after treated with targeted drug erlotinib.The spleen injections of pancreatic cancer cells with CHIP silence or overexpression were used to test the metastatic potential by counting the number of micrometastasis in mouse liver.Immunohistochemistrys were performed to explore the different expression of CHIP and its target protein.5)Histological examinations were performed to identify the influence of tissues CHIP expression levels on the prognosis of patients.ELISA assays were investigated to assess the expression levels of CHIP in blood sera as a biomarker for the early diagnosis of pancreatic cancer.Results1)Successful establishment of stable knockdown or overexpression of CHIP in pancreatic cancer cell lines.2)The growth rate of pancreatic cancer cells that has CHIP knockdown was higher than that of the control group,the apoptotic rate was lower than that of the control group after treated with erlotinib.CHIP silence enhanced the ability of cells to migration and invasiveness.One the country,proliferation rate was significantly lower in cells that were CHIP overexpression than that of the control group,the apoptotic rate was higher than that of the control group after treated with erlotinib.CHIP overexpression dampened the cell migration and invasiveness.The differences between control group and experimental group tested above were all statistically significant(p<0.05).3)In pancreatic cancer cell line Bxpc-3,immunoprecipitation and immunoblot analysis showed that endogenous CHP protein could combine with endogenous EGFR protein,the same as the exogenous CHIP with the exogenous EGFR.The binding region of CHIP with EGFR relied on both the TPR region and U-box region,and EGFR phosphorylation of tyrosine 845 was needed to bind to CHIP protein.CHIP decreased the steady-state levels of EGFR in a time-dependent manner.Immunofluorescent assays showed that EGFR levels were significantly decreased after transfection with Flag-CHIP vector,CHIP also promoted the degradation of the EGF-induced EGFR in the cytoplasm and nucleus.In pancreatic cancer cells,CHIP inhibited the activation of PI3K/AKT/mTOR pathway by degradation of EGFR to prohibit the tumor growth rate,CHIP can also suppress the activation of Src/FAK pathway in order to inhibitor the potential of migration and invasiveness.4)In nude mice xenograft models,tumors were significantly larger in mice injected with CHIP knockdown cells compared to control group(p<0.01),little tumor growth was observed in the mice injected with CHIP overexpression cells(p<0.01).The CHIP knockdown Bxpc-3 cells treated with erlotinib showed an increased tumor volume compared with the control group treated with erlotinib(p=0.034),while CHIP overexpression cells showed a decreased tumor volumn after treated with erlotinib compared to contro group(p<0.01).In pancreatic cancer issues,CHIP expression was eliminated in CHIP knockdown group,while the EGFR expression levels increased sharply compared to control growth,an inverse correlation between CHIP expression and EGFR positivity has been observed(p=0.045),the rate of strongly positive cells stained with Ki67 antibody was higher than that of the control group(p=0.021).Tissues were stongly stained after incubation with CHIP antibody in CHIP overexpression group,the EGFR expression levels decreased compared to control growth,an invserse correlation between CHIP and EGFR was also found(p<0.01),a lower numbers of cells stained with ki67 antibody in CHIP overexpression issues were measured compared with the control group(p=0.026).After injection CHIP knockdown or overexpression cells into the spleens of nude mice,liver metastasis was enhanced in mice given the CHIP knockdown cells(p<0.01).In contrast,CHIP overexpression cells reduced liver metastasis(p<0.01).5)In cancerous tissues from patients clinically determined to be pancreatic cancer,the level of CHIP protein expression was found to be comparable to that observed in adjacent paired normal mucosa(p=1).Protein expression of CHIP in the cancerous tissues significantly correlated with clinicopathological features such as lymph node metastasis(p=0.041)and metastasis after surgery(p=0.01).Low CHIP staining was significantly correlated with a poorer overall survival in a single log rank test and the expression of CHIP could be a potential risk factor in the evaluation of prognosis of pancreatic cancer cohort(p=0.039).The blood sera expression levels of CHIP in chronic pancreatitis(CP),pancreatic benign tumor(PBT),pancreatic endocrine tumor(PET)and pancreatic adenocarcinoma(PDAC)were significantly lower compared with those in normal people(p<0.01),increased levels were found in CP compared with PBT,PET and PDAC(p<0.05).The sensitivity and specificity was 94.4%and 74.5%for detecting CP from normal volunteers,the sensitivity and specificity was 93.6%and 85.1%for detecting PDAC from normal volunteers,the sensitivity and specificity was 68.09%and 77.78%for detecting PDAC from CP patients.The expression of CHIP in PDAC patients sera was reversely correlated with tumor metastasis(p=0.01).Conclusions1)CHIP expression level was inversely correlated with the malignant phenotype of pancreatic cancer cells,which was characterized by decreased cell growth and metastatic potential.2)The influence of CHIP on the pancreatic cancer relied on the ubiquitylation of EGFR,EGFR phosphorylation of tyrosine 845 was needed to bind to CHIP protein.CHIP inhibited the activation of PI3K/AKT/mTOR and Src/FAK pathway so as to suppress the ability of tumor growth and the potential of migration and invasiveness.3)CHIP can inhibit tumor volume and liver micrometastasis in xenograft mice models,the expression of CHIP can also sensitize the apoptotic efficacy induced by erlonitib.4)The level of CHIP protein expression was comparable to that of adjacent paired normal mucosa,CHIP expression significantly correlated with lymph node metastasis and could be a potential risk factor in evaluation of prognosis of pancreatic cancer patients.The expression of CHIP was lower in sera of pancreatic cancer patients and was inversely correlated with tumor metastasis.It could be an important biomarker for detecting PD AC or CP among outpatients.