| Thyroid carcinoma is the most common malignancy of the endocrine organs.In recent years,the incidence of thyroid carcinoma has rapidly increased all over the word.Papillary thyroid carcinoma(PTC)is the most common type,which accounts for 80%of all thyroid carcinoma cases.The overall survival rate of PTC is excellent above 90%at 10 years,even at 20 years.However,there are still several patients would face the morbidity of recurrences.The reported overall recurrence rate is 15-35%.Recurrence may be associated with increased mortality and poor prognosis.Therefore,there is an urgent need to identify biomarkers associated with the progression and recurrence of PTC.Cullin 4B(CUL4B)belongs to the Cullin family.It is an important component of Cullin-Ring E3 ubiquitin ligases(CRLs).CRLs represent the biggest ubiquitin ligases that direct cell proteins for degradation through the ubiquitin-proteasome pathway.They play important roles in signal transduction,cell cycle regulation,and DNA damage response.Nowadays,researchers were attracted by the role of CUL4B in cancer,they confirmed that CUL4B might be a novel oncogene.Our previous studies found that CUL4B is overexpressed in cholangiocarcinoma,gastric and prostate carcinoma and promotes the invasion of tumor cells.However,the research of CUL4B in tumor is still in its infancy and the roles of CUL4B in thyroid carcinoma are unknown.Therefore,in our study,we utilized bioinformatics analysis,clinical tissues,cell and animal models to investigate the expression of CUL4B in thyroid and make an initial discussion of the role and underlying mechanism of CUL4B in PTC.This will help us to choose patients with high risk of recurrence and poor prognosis,and provide new ideas for clinical treatment of these patients.Part 1 The expression of CUL4B and its relation to clinico-pathological factors and prognostic in PTCRecently,several clinico-pathological risk factors or biological markers have been used in the stratification of PTC,such as age,tumor size,TNM staging,and distant metastasis,as well as TERT promoter mutations,BRAF V600E mutation and multiple concurrent mutations,however,the results of different analyses are distinct and the role of these risk factors remains more controversial.Previous researchers have suggested that CUL4B is overexpressed in several types of malignancies,and its expression was correlated with tumor recurrence and patient's prognosis.In this part,we utilized clinical tissues and bioinformatics analysis to investigate the expression of CUL4B in thyroid,and analyzed the relationship between CUL4B overexpression and clinic-pathological characters.Besides,we also discussed the risk factors associated with recurrent PTC.The results were as follows:1.CUL4B was significantly overexpressed in thyroid carcinoma and its expression was correlated with tumor recurrence.Firstly,immunohistochemistry was used to examine CUL4B expression in a cohort of 36 normal thyroid tissues,159 PTC tissues and 19 undifferentiated thyroid carcinoma tissues.The expression of CUL4B was also analyzed in TCGA and GEO dataset.The results showed that CUL4B expression was significantly upregulated in thyroid carcinoma tissues,especially in undifferentiated carcinoma tissues,compared with normal thyroid tissues.Kaplan-Meier survival analysis showed that CUL4B overexpression was correlated with a high recurrence rate.CUL4B expression was significantly correlated with histologic subtypes,tumor size,nodal metastasis,TNM staging and extrathyroidal extension.2.Factors associated with recurrent PTC include patient's age,tumor size,TNM staging,nodal and distant metastasis,the recurrence rates were increased in patients over 55 years old compared with those younger than 55.Compared with non-aggressive phenotypes of PTC include the classic variant and the follicular variant,the tall cell variant has statistical difference in patient's age,tumor size,multiple tumor,and TNM stage.Multivariate analysis showed that TNM staging and distant metastasis were independent factors of recurrence.Conclusion:1.CUL4B was significantly overexpressed in thyroid carcinoma and its expression was correlated with tumor recurrence.2.TNM staging and distant metastasis were independent factors of recurrence.Part ?:The expression of CUL4B in aggressive histologic subtypes of PTCRecently,aggressive histologic subtypes of PTC is gradually concerned by researchers.In this part,we utilized clinical thyroid tissue samples to clarify the expression of CUL4B in aggressive histologic subtypes of PTC.Interestingly,we found a case of ?-hCG-secreting columnar cell variant of PTC(CCV-PTC)with high expression of CUL4B in the process of data collection,which is still not reported in the literature so far.So we described this first case and studied the expression of?-hCG in a cohort of follicular cell differentiated thyroid carcinoma.The results were as follows:1.CUL4B was significantly overexpressed in aggressive histologic subtypes of PTC compared with non-aggressive histologic subtypes.In particular,we focused on the expression of CUL4B in aggressive histologic subtypes of PTC.The results showed that the overall expression rate of CUL4B in aggressive histologic subtypes of PTC was significantly increased compared with non-aggressive histologic subtypes,there was significant statistical difference between the two groups.The expression of CUL4B was highest in solid variant and columnar cell variant,among them,high expression of CUL4B was found in all the four CCV-PTC enrolled.2.Columnar cell variant of PTC with ectopic autocrine of ?-hCG is rarely seen,the expression of ?-hCG was more common in anaplastic thyroid carcinoma.In the case of ?-hCG-secreting CCV-PTC,the ?-hCG produced by the carcinoma cells was believed to be the cause of patient's hyperthyroidism.Columnar cell variant is a rare subtypes of aggressive PTC.It is characterized by columnar tumor cells and nuclear pseudostratification.The clinical behavior mainly depends on the encapsulation or infiltration.Then,immunohistochemistry was used to examine the expression of ?-hCG in thyroid tumors.Besides,we also reviewed literatures of paraneoplastic syndrome in tumors of thyroid follicular cell origination.The results showed that the expression of ?-hCG was more common in anaplastic thyroid carcinoma,but the significance of ?-hCG in thyroid malignancy is also confused.Paraneoplastic syndromes in thyroid tumors of follicular cell origination are rare,and include dermatologic,hematologic,endocrine,and neurologic abnormalities.Conclusion:1.CUL4B was significantly overexpressed in aggressive histologic subtypes of PTC compared with non-aggressive histologic subtypes.2.CCV-PTC with ectopic autocrine of ?-hCG is rarely seen,the expression of?-hCG was more common in anaplastic thyroid carcinoma.Part III:The role and molecular mechanism of CUL4B-mediated proliferation and invasion in PTCPrevious researchers have suggested that CUL4B is closely related to proliferation,invasion and metastasis in several types of malignancies,they confirmed that CUL4B might be a novel oncogene.Our group also found overexpression of CUL4B can promotes invasion and metastasis in cholangiocarcinoma,prostate cancer and gastric carcinoma.However,the role of CUL4B in thyroid cancer remains largely unknown.In this part,we utilized thyroid cell and xenografts model to clarify the biological function and mechanisms of CUL4B in PTC.The results were as follows:1.CUL4B promotes proliferation in thyroid cells.MTS,EdU and colony formation assays demonstrated that down-regulation of CUL4B could reduce cell proliferation ability in PTC cells IHH4,and overexpression of CUL4B could promote the proliferation of normal thyroid follicular epithelial cells Nthy-ori 3-1 and PTC cells BHP10-3.2.CUL4B promotes invasion in thyroid cells.Scratch and transwell assays showed that down-regulation of CUL4B could reduce cell migration and invasive abilities in PTC cells IHH4,and overexpression of CUL4B could promote the migration and invasive abilities of normal thyroid follicular epithelial cells Nthy-ori 3-1 and PTC ce ls BHP10-3.3.CUL4B promotes PTC tumor growth in vivo.Tumor xenografts assay showed that shRNA mediated knockdown of CUL4B significantly inhibited the growth of tumor xenografts of PTC cells IHH4,the average volume and weight of tumor mass decreased notably.4.CUL4B promotes EMT in PTC cells.IHH4 cells with CUL4B knock down appeared round,with the epithelial morphology,whereas BHP10-3 cells with CUL4B overexpression acquired a spindle-shaped morphology.RT-qPCR and western-blot methods showed that CUL4B silencing in IHH4 cells up-regulated the expression of epithelial marker E-cadherin,and down-regulated the expression of mesenchymal markers N-cadherin,Vimentin and transcriptional factors snail and slug.Whereas overexpression of CUL4B in BHP10-3 cells down-regulated the expression of epithelial marker E-cadherin,and up-regulated the expression of mesenchymal markers N-cadherin,Vimentin and transcriptional factors snail and slug.5.CUL4B functions as a positive regulator of Wnt/?-catenin/c-myc signaling pathway and PTEN/PI3k/AKT/mTOR signaling pathway in PTC.To further characterize the mechanism by which CUL4B regulates cell proliferation and invasion in PTC.Firstly,we conducted gene expression profiling of CUL4B-depletion(si4B)vs control(NC)in IHH4 cells,differentially expressed genes were identified enriched in Wnt signaling pathway by gene set enrichment analysis(GSEA).CUL4B expression was found to be significantly correlated with CTNNB1(the key factor of Wnt/?-catenin signaling pathway)in TCGA datasets.Western-blot showed that knockdown of CUL4B significantly reduced the total and nuclear protein level of?-catenin(the key factor of Wnt/?-catenin/c-myc signaling pathway)in PTC cells IHH4,the level of downstream factor c-myc was also reduced.Whereas overexpression of CUL4B in BHP10-3 cells up-regulated the expression level of(3-catenin and c-myc.Immunofluorescence assay also showed that knockdown of CUL4B in IHH4 cells also significantly reduced ?-catenin in the cell nucleus.Besides,Western-blot and RT-qPCR showed that knockdown of CUL4B in IHH4 cells significantly up-regulated the expression level of PTEN(the antagonism molecular of PI3k/AKT signaling pathway).CUL4B expression was also found to be significantly correlated with PI3KCA,AKT3 and mTOR(the key factor of PI3k/AKT/mTOR signaling pathway)in TCGA datasets.Western-blot showed that knockdown of CUL4B significantly reduced the phosphorylation level of AKT,phosphorylation and total level of mTOR in PTC cells,although the total AKT were unaffected.Whereas overexpression of CUL4B in BHP10-3 cells up-regulated the expression level of AKT and mTOR.Taken together,these results indicate that CUL4B functions as a positive regulator of Wnt/?-catenin/c-myc signaling pathway and PTEN/PI3k/AKT/mTOR signaling pathway in conferring its oncogenic activity in PTC.Conclusion:1.In vitro,CUL4B increases migratory and invasive capacity and induced EMT in PTC.In vivo,CUL4B promotes PTC tumor growth.2.CUL4B functions as a positive regulator of Wnt/?-catenin/c-myc signaling pathway and PTEN/PI3k/AKT/mTOR signaling pathway in PTC. |
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