| Renal cell carcinomas (RCCs), which arise from the renal epithelium, account for approximately 90% of kidney cancers. With the changes of people’s life style and living environment, the incidence of RCC is increasing. In the United States, an estimated 61,560 kidney cancer cases and 14,080 cancer-related deaths are expected in 2015. In our country, the incidence of RCC is also increasing year by year, and RCC became the second important urogenital cancer, which was just less than bladder cancer. Immunotherapy and targeted therapy are the recommended routine treatment strategies for patients with metastatic RCC (mRCC), who would not usually be cured by nephrectomy. With the application of molecular genetics to RCC, more and more important molecules and pathways were found and possessed vital roles in RCC. In addition, more and more drugs are being approved in the USA, Europe and China for its treatment. However, the efficacy of these drugs is generally limited by various resistance mechanisms with the development of targeted drugs. Therefore, it is very important to find the novel and important target and pathways in the pathogenesis and progression of RCC, to predict the prognosis of RCC, to choose an appropriate targeted drug and to overcome the resistance to targeted drugs in advanced metastatic RCC.Cullins are evolutionarily conserved and participate in constituting the largest known class of E3 ubiquitin ligases which involved in cell cycle regulation, transcription, signal transduction, development and so on. Human gene encodes eight kinds Cullin members, including CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7 and PARC. CUL4B play an important role in cell proliferation, DNA replication and damage. X-linked CUL4B mutations can cause mental retardation and CUL4B knockout mice are embryonic lethal. In addition, because CUL4B possess a nuclear localization signal, CUL4B might be involved in the nucleus-based functions. CUL4B could down-regulated miR-372 and miR-373, and promote the expression of CDK2, which induced DNA replication, according to a previous study. In addition, CUL4B could block the expression of tumour suppressor gene p16 and PTEN, promoting tumour genesis. Thus, in recent years in the development of CUL4B effect and mechanism of tumour genesis are more and more important. There are some studies, which proved the close correlation of CUL4B with liver cancer, colorectal cancer, osteosarcoma, glioma and tumour microenvironment. However, the role and mechanism of CUL4B in RCC has not been reported in the literature.c-Met is a high-affinity transmembrane receptor tyrosine kinase (RTK) with only one known ligand:hepatocyte growth factor (HGF). The binding of HGF to c-Met leads to receptor homodimerization and phosphorylation, and the further activation of multiple downstream intracellular signalling pathways, including Ras/Raf/MEK/ERK, PI3K/Akt, and focal adhesion kinase (FAK). The HGF/MET signalling pathway is a crucial regulator of cell proliferation, invasion, migration, and angiogenesis. Although c-Met is important in the control of tissue homeostasis under normal physiological conditions, it becomes aberrantly activated in human cancers via mutation, amplification or protein over-expression.Previous studies indicated that c-Met was overexpressed in clear cell renal cell carcinoma and papillary renal cell carcinoma. The expression level of c-Met was closely related to the pathological features and prognosis of RCC according to the previous studies. Therefore, c-Met inhibitors such as Cabozantinib have been used in clinical trials about treatment of RCC. Recent studies have shown that c-Met activation might lead to the resistance of targeted therapy. Therefore, the role of c-Met in the pathogenesis, progression and target drug resistance of RCC is worth studying. In addition, whether CUL4B could regulate c-Met pathway, has not yet been reported in the literature.Based on the above background, the following research was carried out. First, the expression level and the role of CUL4B in RCC were investigated. Then, to clarify the role of CUL4B in RCC, the protein chip was used. We found that c-Met might involve in the regulation of CUL4B in RCC. Finally, the mechanism of CUL4B regulating c-Met pathway in RCC was further investigated. We want to find the novel targets and molecular signalling pathways in the pathogenesis and progression of RCC via the present study. Besides, we can better predict the prognosis, guide the selection of target drugs and solve the problems of drug resistance.Part Ⅰ The expression level and role of CUL4B in RCCIt well known that CUL4B play an important role in a variety of tumours with the high expression levels.In order to further investigate whether CUL4B could promote tumour progression in RCC, the present part of study was performed. We use the human renal cell carcinoma cell lines, human renal cell carcinoma tissue samples as well as nude mice. Details are as follows:1. CUL4B was high expressed in renal cell carcinoma cell lines as well as renal cell carcinoma specimens. Renal cell carcinoma cell line 769-P,786-O and OS-RC-2 and immortalized normal renal tubular epithelial cell line HK-2 were cultured. The protein level was detected in these cells by western blot. We found that the expression level of CUL4B in three kinds of renal cell carcinoma cell lines were significantly higher than that in normal renal tubular epithelial cell line HK-2. We collected 33 cases of kidney tumours and adjacent normal tissues, which were confirmed to be RCC by pathological examination. Western blot method was used to detect the expression level of CUL4B in 33 pairs of tissues. We found that the protein levels of CUL4B were higher in the RCC than the adjacent normal tissues in 27 of the 33 (81.8%) of pairs.2. The expression level of CUL4B was positively correlated with nuclear grading, pathologic staging and prognosis in RCC. We used tissue microarrays (TMAs) to assess CUL4B expression in tumour tissues from 90 patients with RCC. Immunohistochemical staining was used to detect the CUL4B expression level. The total expression of CUL4B was calculated by multiplying the staining intensity score by the positive rate score:CUL4B was considered overexpressed when the product was greater than 6. We found that CUL4B expression level was not related to age (P= 0.278), sex (P=0.876) and tumour size (P=0.054). However, the expression level of CUL4B was positively correlated with tumour nuclear grade (P=0.006) and pathological stage (P=0.02). Most importantly, CUL4B expression was significantly correlated with disease-specific survival (P=0.031) by Kaplan-Meier survival analysis and log-rank test.3. The expression level of CUL4B had an important effect on the biological behaviour of the renal carcinoma cells, including proliferation, colony formation, invasion and migration. The CUL4B interference and overexpression of renal carcinoma cells (shControl、shCUL4B、PLOC、PLOC-CUL4B) were successfully constructed. Western blot was used to detect CUL4B expression level in the cells. Cell proliferation was detected by MTT method. The proliferation rate was significantly lower in CUL4B interference RCC cells than that in control cells (P<0.05). On the contrary, the cell proliferation rate was significantly increased in CUL4B overexpressing cells compared with the control cells (P<0.01). Colony formation ability was detected. The colony formation was significantly lower in CUL4B interference RCC cells than that in control cells (P<0.01). On the contrary, the colony formation rate was significantly increased in CUL4B overexpressing cells compared with the control cells (P<0.001). The migration capacity was detected by scratch test.The migration rate was significantly lower in CUL4B interference RCC cells than that in control cells (P<0.05). On the contrary, the migration rate was significantly increased in CUL4B overexpressing cells compared with the control cells (P<0.05). The invasiveness was detected by transwel assay. The invasive rate was significantly lower in CUL4B interference RCC cells than that in control cells (P<0.05). On the contrary, the invasive rate was significantly increased in CUL4B overexpressing cells compared with the control cells (P<0.01).4. The CUL4B expression level influences the tumourigenesis of RCC in nude mice. In order to further clarify the influence of CUL4B on the growth of renal cell carcinoma, CUL4B interference RCC cell lines and BALB-c male mice Nude Mice were applied. "We found that the tumour volume formed by CUL4B interference cell was significantly reduced compared with the control cell (P<0.001).In conclusion, CUL4B expression level increased significantly in renal cell carcinoma, and CUL4B expression level is closely related to the grading and stage of RCC. Increased expression of CUL4B is related to the poor nuclear grade and pathological stage. Most importantly, c-Met expression was significantly correlated with disease-specific survival. Further study proved that CUL4B expression level was closely related to o cell proliferation, colony formation, invasion, migration, and tumourigenesis of RCC.PartⅡ Influences of CUL4B Regulating c-Met Signaling Pathway in Renal Cell CarcinomaAccording to the first part of the study, we found that CUL4B play an important role in RCC, but its specific mechanism is still obscure. To further investigate the mechanism of CUL4B in RCC, the second part of the study was performed.1. The phosphorylation level of c-Met was decreased in CUL4B interference RCC cells. Because the activation of RTK plays an important role in RCC, the current treatment of advanced RCC mostly targeted tyrosine kinase inhibitor drugs (TKI). Given the above, the protein array about the phosphorylation levels of RTK was used in CUL4B interference RCC cells. We found that the phosphorylation level of c-Met, PDGFR-α, TNK1, Tie-1, PDGFR-β and other RTK were reduced to some extent. However, the phosphorylation level of c-Met decreased 80% and was the most obvious one.2. c-Met was high expressed and positively correlated with CUL4B in RCC.(1) we selected 10 pairs RCC samples, in which CUL4B was high expressed, from 33 pairs tissues. Western blot method was used to detect protein level of c-Met. In the 10 pairs of samples, the protein level of c-Met was significantly increased in tumours, which was consistent with CUL4B.(2) Western blot was used to detect the expression of c-Met in three kinds of renal carcinoma cell line 769-P,786-O, OS-RC-2 and immortalized normal renal tubular epithelial cell HK-2. c-Met was high expressed in three kinds of renal cell carcinoma cells HK-2 expression levels, which was consistent with CUL4B.(3) RCC tissue microarray was used to detect the expression of c-Met. c-Met expression levels were not related to age (P=0.688) and sex (P=0.398). However, c-Met expression levels were positively correlated with tumour nuclear grade (P= 0.008), pathological stage (P=0.002), and tumour size (P=0.002). Subsequently, c-Met expression was significantly correlated with disease-specific survivalby Kaplan-Meier survival analysis and log-rank test (P<0.001). CUL4B expression levels were positively correlated with c-Met expression levels (P=0.02).3. CUL4B positively regulated c-Met pathway. The CUL4B interference and overexpression of renal carcinoma cells (shControl、shCUL4B、PLOC、 PLOC-CUL4B) were used. Western blot was used to detect the key molecule in c-Met pathway. The expression of c-Met, phosphorylation level of c-Met and AKT were significantly reduced in CUL4B interference cells than that in control cells. In contrast, the expression of c-Met, phosphorylation level of c-Met and AKT were significantly increased in CUL4B overexpressing cells than that in control cells. Real time quantitative RT-PCR was used to detect the mRNA levels of c-Met in 4 types of cells. Interestingly, we found that there was no significant difference about the mRNA levels of c-Met in 4 types of cells. To further validate our results, we use CUL4B knockout mice. Western blot was used to detect c-Met expression level in kidneys of CUL4B knockout mice. The c-Met expression was significantly reduced in kidney of CUL4B knockout mice.4. CUL4B promoted renal carcinoma cell proliferation, colony formation, invasion and metastasis by up-regulating c-Met. c-Met inhibitor SU11274 was used to treat CUL4B overexpression cells and the abilities of cell proliferation, colony formation, invasion and metastasis were detected. The MTT method was used to detect cell proliferation. We found that cell proliferation rate was significantly lower in SU 11274 treated CUL4B overexpression cells than the untreated CUL4B overexpression cells (P<0.01). Colony formation ability was also detected. Colony formation ability was significantly lower in SU 11274 treated CUL4B overexpression cells than the untreated CUL4B overexpression cells (P<0.001). The transwel assay was used to detect cell invasiveness. Cell invasive ability was significantly lower in SU 11274 treated CUL4B overexpression cells than the untreated CUL4B overexpression cells (P<0.001).5. CUL4B induced drug resistance to first-line TKI in RCC by up-regulating the c-Met and the resistance could be reversed after applying c-Met inhibitors in RCC.(1) The CUL4B interference and overexpression of renal carcinoma cells (shControl、shCUL4B、PLOC、PLOC-CUL4B) were used. The first-line TKI sunitinib was used to treat these cells and MTT method was used to detect the cellular activities under different treating time and concentration of sunitinib. After treatment with 10μM and 20μM sunitinib for 48h, the cell activity was significantly decreased in CUL4B interference cells than that in control cells (P<0.01). In contrast, after treatment with 10μM and 20μM sunitinib for 48h, the cell activity was significantly increased in CUL4B overexpression cells than that in control cells (P<0.01 and P <0.001). After treatment with 20μM sunitinib for 24h,48h and 72h, the cell activity was significantly decreased in CUL4B interference cells than that in control cells (P <0.05, P<0.01 and P<0.001). On the contrary, After treatment with 20μM sunitinib for 24h,48h and 72h, the cell activity was significantly increased in CUL4B overexpression cells than that in control cells (P<0.05, P<0.01 and P<0.01)(2) Previously treating the cells with the c-Met inhibitor SU11274, then the first-line TKI sunitinib was used. MTT method was used to detect the cellular activities under different treating time and concentration of sunitinib. However, no significant difference was observed in the CUL4B interference and overexpression of renal carcinoma cells (shControl、shCUL4B、PLOC、PLOC-CUL4B).In summary, according to the study of the second part, we found that CUL4B influences the cell proliferation, invasion, metastasis and prognosis by regulating c-Met pathway in RCC. In addition, CUL4B induced drug resistance to first-line TKI in RCC by up-regulating the c-Met and the resistance could be reversed by blocking c-Met pathway. |
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