The Expression Of CUL4 And The Effects And Mechanisms Of CUL4 In The Carcinogenesis And Apoptosis Of Lung Cancer

Objective:Ubiquitination represents a very important post translation modification of protein,is the signal of protein degradation.Cullin 4-Ring E3 ligases(CRL4)is one of the key enzyme in the process of ubiquitination.The CUL4 ubiquitin ligase is a tetrameric complex in which the elongated CUL4 A scaffold utilizes the adaptor proteins DDB1 and Rbx1/Roc1 to recruit the DCAF substrate receptors,which in turn bind specific substrates,and the E2 ubiquitin conjugating enzyme with activated ubiquitin,thereby bringing substrates and activated ubiquitin into close proximity to facilitate ubiquitin transfer.CUL4 A and CUL4 B are important members of CUL4,which participate in a broad varietyof physiologically and developmentally controlled processessuch as cell cycle progression,DNA damageresponse,activation of apoptosis relative protein,and the activation or suppression of proto-oncogene and anti-oncogene.Thus,CUL4 A and CUL4 B play important role in the carcinogenesis and progression of a wide variety of cancer.We propose to investigate the effects and mechanisms of CUL4 A and CUL4 B in the carcinogenesis and progression of lung cancer.Methods:1.352 cases of lung cancer paraffin-embedded tissue and 62 cases normal lung tissue were collected in Tianjin Medical University Cancer Institute&Hospital from January,2000 to June,2011.(1)Clinical pathological data for all patients was collected;(2)Tissue array was prepared;(3)The expression of CUL4 A and CUL4 B was analyzed using immunohistochemical staining.(4)Extract DNA of 120 cases samples,real-time PCR was performed to detect copy number of CU4 A and CUL4 B.(5)Relationship between CUL4A/B expression and clinicopathological factors including prognosis was analyzed.2.shCUL4 A and shCUL4 B lentivirus particles were prepared.NCI-H520 and NCI-H446 cells were infected by the lentivirus and selected by puromycin to built clones that stably express shCUL4 A or shCUL4 B.The protein and mRNA expression of NCI-H520 and NCI-H446 cells before and after infection wasexamined by Western Blot and RT-PCR.3.The impact of degradation of CUL4 A and CUL4 B on cell progression and cell cycle were evaluated by flow cytometric analysis through BrdU/7-AAD staining.The effects on expression level of XPC and P21 was measured by Western blot.The4.correlation between expression level of CUL4A/B and XPC or P21 was measured by IHC in tumor samples.Brd U/7-AAD flow cytometric analysis was performed to evaluate the rescue effect of knockdown P21 on cell cycle.5.The impact of degradation of CUL4 A and CUL4 B on apoptosis by flow cytometric analysis through Annexin?/7-AAD staining.The correlation between CUL4 B and FOXO3 a were measured by IHC.Changes of the expression level of FOXO3 a and apoptosis related protein and signaling pathways after knockdown CUL4 A and CUL4 B were measured by western blot.siRNA interfere FOXO3 a to evaluate the role of FOXO3 a plays in lung cancer cell apoptosis.6.The impact of degradation of CUL4 A and CUL4 B on cell proliferation and apoptosis was analysied in nude mice model.Result:1.The expression of CUL4 A and CUL4 B in lung cancer tissue:(1)CUL4A predominately express in the nucleus and cytoplasm.CUL4 B predominately express in the nucleus.The expression of CUL4A/B was significantly increased in tumor tissues comparing with that in normal tissue(P<0.001).(2)DNA copy number of CUL4 A and CUL4 B high expression group was significantly higher than that of CUL4 A and CUL4 B low expression group(P<0.001).(3)Expression of CUL4 A was correlated with smoking status(P<0.001).CUL4A/B high expression was also related to clinical stage,tumor burden,lymph node metastasis,OS and PFS.2.The roles and mechanisms of CUL4 A on cell growth: After knockdown CUL4 A in NCI-H520 and NCI-H446 cells,there showed a shorten S phase,and an arrested G0/G1 phase.At the same time,the expression of P21 was elevated.Then we knockdown the elevated P21,the effects of CUL4 A on cell cycle was restored.3.The roles and mechanisms of CUL4 B on cell apoptosis:(1)Knockdown CUL4 B led to the elevated expression of FOXO3 a,which triggered apoptosis of lung cancer cells.(2)There is a negative correlation between the expression of CUL4 B and FOXO3 a in lung cancer samples.(3)Targeted degradation of CUL4 B led to attenuation of ERK signaling pathway,which results in reducing phosphorylation of FOXO3 a.4.In vivo experiment: Knockdown of CUL4 A and CUL4 B suppressed tumor growth in nude mice.The degradation of CUL4A?CUL4B have the same effect on the cell proliferation,cell apoptosis,cell cycle in vivo as that in vitro.Conclusions:1.In lung cancer,the expression of CUL4 A and CUL4 B protein and DNA wasincreased comparing with normal tissue.The elevated expression of CUL4 A and CUL4 B was correlated with prognosis of lung cancer patients.2.In lung cancer cells,elevated expression of CUL4 A influenced DNA reair and promoted cell proliferation by degradation of XPC and P21.3.Knock-down of CUL4 B resulted in the lost of activation of ERK signaling pathway,which lead to the decrease of the degradation of FOXO3 a.The accumulation of FOXO3 a affected lung cancer cell apoptosis.4.The results of transplantation Mouse Models showed that knock-down of CUL4 A and CUL4 B inhibited tumor growth.