Characterization Of CUL4B In Cholangiocarcinoma And Its Meolecular Mechanisms In Promotion Of Cancer Progression

BACKGROUNDCholangiocarcinoma(CCA),anatomically classified into intrahepatic cholangiocarcinoma(IHCC)and extrahepatic cholangiocarcinoma(EHCC),is a malignancy originating from bile duct epithelial cells.Its incidence is about 3%of malignant tumors of the digestive system,which is the second of hepatobiliary cancers.The incidence of male and female was about 1.5:1.With the unclear pathogenesis,high degree of malignant,difficult early diagnosis,low surgical resection rate,CCA has a high fatality rate.The mean survival time of untreated CCA is about 3?4 months after clinical symptom.There is an increasing trend in the incidence of CCA recent years.Complete resection is the only way to cure the disease at present,but postoperative survival rate of 5-year is still lower than 5%.It was late when diagnosed because without obvious early symptoms of the disease.Clinically,the prognostic factors of CCA have not been well established yet.So far,certain molecular biomarkers have been reported to correlate with poor survival and tumor progression,such as MUC1,MUC4,fascin,and epidermal growth factor receptor(EGFR),but most of them were not used routinely in clinical practice.Therefore,novel biomarkers for prognostic stratification and individualized therapy are urgently required.Ubiquitin ligase can connect ubiquitin molecules to lysine on a purpose of protein.According to the structure and varying lengths of ubiquitin chain on the target protein,the different proportions of substrate can result in single ubiquitin modification and poly-ubiquitination.Under normal circumstances,ubiquitin ligase can add multiple ubiquitin molecules to target protein,namely,multi-ubiquitination and the poly-ubiquitinated target protein can be degradated by the 26S proteasome.This approach is called ubiquitin-proteasome pathway,ubiquitination pathway for short.Ubiquitin-proteasome pathway in eukaryotic organisms is known as the most important protein degradation pathway.However,in some cases,ubiquitin ligase connect only one pigment molecule to the purpose protein and the uni-ubiquitinated proteins will not be degradated by protease.Single ubiquitin modifications as a signal can cause the target protein activity,location and changes in protein structure so that protein function and the position in the nucleus can be adjusted.Protein ubiquitination process does not necessarily lead to cell death.Cullin-Ring E3 ligase(CRLs)complexes are the largest known class of ubiquitin ligases.Cullin as a scaffold protein are involved in a wide variety of physiological and developmental process,such as DNA replication,cell cycle progression,DNA damage response,tumor suppression,growth and development,signal transduction and transcriptional regulation.E3 as a bridge,transfer activated ubiquitin from E2 to a target protein,which does not function with the ubiquitin itself.In human,there are eight Cullin proteins,CUL1?CUL2,CUL3?CUL4A?CUL4B?CUL5?CUL7 and CUL9.Cu14A and Cu14B are highly homologous.It is interesting that Cu14B,unlike Cu14A,carries a nuclear localization signal in its N terminus and is also localized in the nucleus,suggesting that Cul4B might be involved in the nucleus-based functions.Recently,we and others have reported that Cu14B is overexpressed in many types of solid tumors,such as cancers of the lung,colon and liver and so on.With the pathological aggravation,the expression of Cu14B is increasing,which suggested that Cu14B exerts an oncogenic effect.However,the expression of Cu14B in CCA and its role in the progression of CCA remains largely unknown.It might serve as indicators of diagnosis,treatment and prognosis of malignant solid tumor and it may be a cancer treatment target.Now it has become a hotspot.In conclusion,Cu14B is overexpressed in many types of solid tumors.but whether it is overexpressed in CCA,what is the pathogenesis,and whether it can be used as indicators for diagnosis,prognosis and treatment targets should be further explored.OBJECTUsing immunohistochemical technique in CCA clinical specimens detect the expression of Cu14B and combined with clinical pathology and follow-up data to explore the relationship between the expression of Cu14B and progression and prognosis of CCA.By building Cu14B on the overexpression and interference vectors,and using them transfect different CCA cells respectively by liposome transfection technique,so that we can build overexpression and silence the CCA cells line with Cu14B.And then through the cell experiment we will research the biological effect of Cu14B for CCA cell proliferation,invasion and metastasis,and further investigate the molecular biological mechanism of Cu14B's influence on cholangiocarcinoma development.METHODS1.In order to define the role of Cu14B in the progression of CCA,we performed IHC staining of Cul4B in a cohort of 219 CCA cases.We analyzed the relationship between Cu14B expression and clinicopathological factor,such as the pathological staging,age,gender,tumor size,the tissue differentiation degree and lymph node metastasis,and the relationship between Cul4B and EGFR expression in IHCC and EHCC.2.To assess the possible association between Cu14B expression and patient survival,Kaplan-Meier curves with a log-rank test for overall survival(OS)were undertaken3.We determined whether combining Cu14B and EGFR further improved prognostic value in EHCC patients.For this analysis,we grouped all EHCC patients according to their Cu14B and EGFR status(not overexpressed vs.overexpressed).The Kaplan-Meier analyses were therefore conducted using the group with neither Cu14B overexpression nor EGFR overexpression as the reference.4.To explore the biological role of Cu14B in CCA in vitro,we first evaluated the endogenous Cu14B expression in the CCA cells lines(QBC939,HUCCT1,RBE).Cu14B was silenced in QBC939 cells by siRNA.Cu14B was overexpressed in RBE cells by transfections with of Cu14B.MTS assay were performed to determine the proliferation of CCA cells.Transwell experiments were performed to determine the migration ability of CCA cells.5.To explore whether Cu14B is a regulator of EMT in CCA,expression of epithelial markers(E-cadherin)and mesenchymal markers(Vimentin,and N-cadherin)were analyzed after knockdown of Cu14B with siRNA transfection.6.To investigate the modulation of these tumor suppressing genes by Cu14B,we knocked down Cu14B by siRNA transfection in the QBC939 cell lines,then determined the change of the level of p16 and PTEN by Real-time PCR.RESULTS1.Cu14B protein levels were greater in cancer cells than in adjacent benign ductal epithelia cells.Overall,Cu14B was overexpressed in 28.6%(40/140)of EHCC patients and 26.6%(21/79)of IHCC patients,respectively.Among the EHCC cases,Cu14B overexpression was significantly associated with higher pathological tumor stage(P=0.024)and lymph node metastasis(P=0.013).No associations were identified between Cu14B overexpression and gender(P=0.821),age(P=0.520),tumor size(P=0.650)or histological differentiation(P=0.299).By contrast,Cu14B expression was not associated with any clinicopathologic variables in IHCC cases.Overexpression of EGFR was present in 19(25.0%)of the 76 IHCCs,and 23(16.7%)of the 138 EHCCs,respectively.There is a marginal association between Cu14B overexpression and EGFR expression(P=0.093)in EHCC cases,but not in IHCC cases.2.In EHCC,patients with Cul4B overexpression had a lower OS rate than patients who were not.The estimated mean OS time was significantly different between patients with Cul4B-overexpressed and Cul4B-nonoverexpressed tumors(55.029± 2.595 vs 86.974 ± 0.882 months,respectively;P<0.001).By contrast,no statistical significance was identified between Cul4B overexpression and overall survival in IHCC.In univariate Cox regression analysis,Cul4B overexpression was a prognostic factor for cancer mortality(hazard ratio=1.779,95%CI=1.102-2.690,P=0.028)in EHCC.Additionally,histological differentiation,tumor stage,UICC stage and EGFR expression were also significantly related to overall survival.In a multivariate analysis,UICC stage and EGFR expression remained its predictive value,whereas Cul4B expression lost.In IHCC,Cul4B expression failed to be related to overall survival of CCA patients.4 factors including tumor size,lymph node metastasis,UICC stage,and EGFR expression were identified as prognostic factors by univariate analysis.In multivariate analysis,only UICC stage and lymph node metastasis were independent prognostic factors.Collectively,these data suggested that Cul4B was an unfavorable prognostic indicator in Chinese patients with EHCC.3.Kplan-Meier analysis showed that the group that comprised those who had both EGFR and Cul4B overexpression,had the worst cancer-related survival.4.QBC939 cells showed the highest level of Cul4B,whereas HUCCT1 and RBE cells exhibited relatively lower level(QBC939>HUCCT1>RBE).MTS assay showed that the proliferation of QBC939 cells was significantly attenuated after silencing Cul4B compared to its negative control.After 48h and 72h of Cul4B siRNA treatment,the number of QBC939 cells was reduced to 20.7±3.5%and 27.9±5.6%,respectively(P<0.05).Meanwhile,we also performed overexpression of Cul4B in RBE cells.The growth of RBE cells significantly enhanced after transfections with expression plasmid of Cul4B.Notably,overexpression of Cul4B significantly increased colony formation of HUCCT1 cells compared to negative control,vice versa.Next,transwell experiments were performed to determine the migration ability of CCA cells,either transfected with siRNA or expression plasmid of Cul4B.Our data clearly showed that the migration and invasive capacity of QBC939 and HUCCT1 cells decreased after knocking down Cu14B;By contrast,overexpression of Cu14B significantly increased migration and invasive capacity of HUCCT1and RBE cells.5.Western blot showed that expression of E-cadherin was significantly down-regulated,while Vimentin and N-cadherin were up-regulated in QBC939 and HUCCT1 cells after Cu14B was knocked down by siRNA.It indicated that Cu14B might promote EMT.6.Our data showed that knocking down of Cul4b by siRNA transfection would lead to an up-regulation of both p16 and PTEN in QBC939 cells,which suggested that Cu14B could promote tumor progression partially through the repression of these tumor suppressing genes.CONCLUSION1.Cu14B is overexpressed in CCA tissue and Cu14B could promote tumor progression partially through the repression of these tumor suppressing genes.2.In the current study,we demonstrated that co-overepression of Cu14B and EGFR could define a subset of EHCC patients with poor prognosis.3.Cu14B is a poor prognostic factor in extrahepatic CCA in Chinese.4.How Cu14B contributes to CCA invasion and progression may be related to Cu14B promoting EMT.SIGNIFICANCE1.To the best of our knowledge,for the first time,we demonstrated that Cu14B can promote the epithelial mesenchymal transformation in CCA cells,which Contribute to discover the mechanism of CCA.2.We showed that the overexpression of Cu14B is a poor prognostic factor in Chinese CCA patients with EHCC.Cu14B overexpression in surgically excised CCA tissues might help predict overall survival of the patients.Notably,our findings suggested Cu14B and EGFR expression in a subset of CCA cases with poor prognosis.3.Our findings provided the basis for Cu14B as the new CCA therapeutic targets.