Numb在三阴乳腺癌中抑制HDM2泛素化降解p53
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摘要
目的:探究Numb蛋白在三阴乳腺癌患者中的表达降低情况,及Numb蛋白在三阴乳腺癌中对抑癌因子p53蛋白水平的影响及调控机制,进一步研究Numb蛋白的降低与三阴乳腺癌发生发展的相关性,从而为缺乏有效治疗方法的三阴乳腺癌提供一个潜在的治疗新靶点。方法:40例三阴乳腺癌患者病理组织切片取自重庆医科大学临床病理诊断中心,采用免疫组化法检测Numb蛋白在三阴乳腺癌患者中的表达情况。MCF-10A细胞株和MDA-MB-231细胞株均为ATCC来源,采用qPCR和Western blot法检测对比Numb、HDM2、p53三者的转录水平和蛋白质水平在以上两个细胞株中差异。采用增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)质粒转染的方法在MDA-MB-231细胞中重表达Numb,采用q PCR和Western blot法验证Numb、HDM2、p53三者表达的变化。结果:转染NUMB-EGFP后MDA-MB-231细胞中Numb的mRNA和蛋白质水平均明显上调,HDM2无显著改变,p53在转录水平无明显变化,但在蛋白质水平显著升高。在231细胞中上调Numb蛋白可以在转录后水平调节p53水平,使p53蛋白随之显著升高。结论:Numb蛋白在三阴乳腺癌患者中表达降低的比列很高,为55%,且Numb蛋白在三阴乳腺癌细胞MDA-MB-231中可以调控抑癌因子p53蛋白水平,Numb蛋白水平与p53蛋白水平呈正相关。
Objective : To investigate the decrease of Numb protein expression in triple-negative breast cancer patients and the effect of Numb protein on the p53 protein levels in triple-negative breast cancer and its regulation mechanism,and to further study the correlation between the decrease of Numb protein and the occurrence and development of triple-negative breast cancer,so as to provide a potential new therapeutic target for triple-negative breast cancer without effective treatment. Methods: 40 cases of triple negative breast cancer were taken from Clinical pathology diagnosis centre of Medical University of Chongqing. The expression of Numb protein in triple negative breast cancer patients was detected by immunohistochemistry. MCF-10A cell line and MDA-MB-231 cell line were all ATCC-derived. The mRNA and protein levels of Numb,HDM2 and p53 were compared by q PCR and Western blot. Numb was re-expressed in MDA-MB-231 cells by plasmid transfection.The expression of Numb,HDM2 and p53 was verified by qPCR and Western blot. Results: After transfection with NUMB-EGFP,the mRNA and protein levels of Numb in MDA-MB-231 cells were significantly increased,HDM2 did not change evidently,and p53 did not change significantly at the mRNA levels,but increased remarkably at the protein levels. The increased of Numb protein in NUMB-EGFP-transfected cells can regulate the p53 levels at post-transcriptional level,resulting in a significant increase of p53 protein levels. Conclusion:The expression of Numb protein in triple-negative breast cancer patients decreased by 55%,and Numb protein can regulate the level of p53 protein in triple-negative breast cancer cell line MDA-MB-231. The level of Numb protein is positively correlated with the level of p53 protein.
Objective : To investigate the decrease of Numb protein expression in triple-negative breast cancer patients and the effect of Numb protein on the p53 protein levels in triple-negative breast cancer and its regulation mechanism,and to further study the correlation between the decrease of Numb protein and the occurrence and development of triple-negative breast cancer,so as to provide a potential new therapeutic target for triple-negative breast cancer without effective treatment. Methods: 40 cases of triple negative breast cancer were taken from Clinical pathology diagnosis centre of Medical University of Chongqing. The expression of Numb protein in triple negative breast cancer patients was detected by immunohistochemistry. MCF-10A cell line and MDA-MB-231 cell line were all ATCC-derived. The mRNA and protein levels of Numb,HDM2 and p53 were compared by q PCR and Western blot. Numb was re-expressed in MDA-MB-231 cells by plasmid transfection.The expression of Numb,HDM2 and p53 was verified by qPCR and Western blot. Results: After transfection with NUMB-EGFP,the mRNA and protein levels of Numb in MDA-MB-231 cells were significantly increased,HDM2 did not change evidently,and p53 did not change significantly at the mRNA levels,but increased remarkably at the protein levels. The increased of Numb protein in NUMB-EGFP-transfected cells can regulate the p53 levels at post-transcriptional level,resulting in a significant increase of p53 protein levels. Conclusion:The expression of Numb protein in triple-negative breast cancer patients decreased by 55%,and Numb protein can regulate the level of p53 protein in triple-negative breast cancer cell line MDA-MB-231. The level of Numb protein is positively correlated with the level of p53 protein.
引文
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[9]Shen Q,Zhong W,Jan Y,et al.Asymmetric numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts.Development,2002,129(20):4843-4853.
[10]Colaluca I N,Tosoni D,Nuciforo P,et al.NUMB controls p53tumour suppressor activity.Nature,2008,451(7174):76-81.
[11]Zhang Y,Li F Y,Song Y L,et al.Numb and Numbl act to determine mammary myoepithelial cell fate,maintain epithelial identity,and support lactogenesis.FASEB,2016,30(10):3474-3488.
[12]Rhyu M S,Jan L Y,Jan Y N.Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells.Cell,1994,76(3):477-491.
[13]Juven-Gershon T,Shifman O,Unger T,et al.The mdm2oncoprotein interacts with the cell fate regulator Numb.Mol Cell Biol,1998,18(7):3974-3982.
[14]Berdnik D,T9r9k T,Gonzalez-Gaitan M,et al.The endocytic proteina-adaptin is required for numb-mediated asymmetric cell division in drosophila.Dev Cell,2002,3(2):221-231.
[15]Vassilev L T,Vu B T,Graves B,et al.In vivo activation of the p53pathway by small-molecule antagonists of MDM2.Science,2004,303(5659):844-848.
[16]Tosoni1 D,Pambianco1 S,Ekalle Soppo B,et al.Re-clinical validation of a selective anti-cancer stem cell therapy for Numbdeficient human breast cancer.EMBO Mol Med,2017,7(5):656-671.
[17]Ray-Coquard I,Blay J Y,Italiano A,et al.Effect of the MDM2antagonist RG7112 on the P53 pathway in patients with MDM2-amplified,well-differentiated or dedifferentiated liposarcoma:an exploratory proof-of-mechanism study.Lancet Oncol,2012,13(11):1133-1140.
[18]Pece S,Confalonieri S,R Romano P,et al.NUMB-ing downcancer by more than just a NOTCH.Biochim Biophys Acta,2011,1815(1):26-43.
[19]Enari M,Ohmori K,Kitabayashi I,et al.Requirement of clathrin heavy chain for p53-mediated transcription.Genes Dev,2006,20(9):1087-1099.
[20]Cayouette M,Raff M,et al.Asymmetric segregation of Numb:a mechanism for neural specification from Drosophila to mammals.Nature Neurosci,2002,5(12):1265-1269.
[2]Di Marcotullio L,Ferretti E,Greco A,et al.Numb is a suppressor of hedgehog signallingand targets gli1 for itch-dependent ubiquitination.Nature,2006,8(12):1415-1423.
[3]Cahilly-Snyder L,Yang-Feng T,Francke U,et al.Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line.Somat Cell Mol Genet,1987,13(3):235-244.
[4]Vousden K H,Prives C.P53 and prognosis:new insights and further complexity.Cell,2005,120(1):7-10.
[5]Momand J,Wu H H,Dasgupta G,et al.MDM2-master regulator of the p53 tumor suppressor protein.Gene,2000,242(1/2):15-29.
[6]Hutterer A,Knoblich J A.Numb and a-adaptin regulate sanpodo endocytosis to specify cell fate in drosophilaexternal sensoryorgans.EMBO Rep,2005,6(9):836-842.
[7]Foulkes W D,Smith I E,Reis-Filho J S,et al.Triple-negative breast cancer.N Engl J Med,2010,363(20):1938-1948.
[8]Pece S,Confalonieri S,Di Fiore P P,et al.NUMB-ing down cancer by more than just a NOTCH.Biochim Biophys Acta,2011,1815(1):26-43.
[9]Shen Q,Zhong W,Jan Y,et al.Asymmetric numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts.Development,2002,129(20):4843-4853.
[10]Colaluca I N,Tosoni D,Nuciforo P,et al.NUMB controls p53tumour suppressor activity.Nature,2008,451(7174):76-81.
[11]Zhang Y,Li F Y,Song Y L,et al.Numb and Numbl act to determine mammary myoepithelial cell fate,maintain epithelial identity,and support lactogenesis.FASEB,2016,30(10):3474-3488.
[12]Rhyu M S,Jan L Y,Jan Y N.Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells.Cell,1994,76(3):477-491.
[13]Juven-Gershon T,Shifman O,Unger T,et al.The mdm2oncoprotein interacts with the cell fate regulator Numb.Mol Cell Biol,1998,18(7):3974-3982.
[14]Berdnik D,T9r9k T,Gonzalez-Gaitan M,et al.The endocytic proteina-adaptin is required for numb-mediated asymmetric cell division in drosophila.Dev Cell,2002,3(2):221-231.
[15]Vassilev L T,Vu B T,Graves B,et al.In vivo activation of the p53pathway by small-molecule antagonists of MDM2.Science,2004,303(5659):844-848.
[16]Tosoni1 D,Pambianco1 S,Ekalle Soppo B,et al.Re-clinical validation of a selective anti-cancer stem cell therapy for Numbdeficient human breast cancer.EMBO Mol Med,2017,7(5):656-671.
[17]Ray-Coquard I,Blay J Y,Italiano A,et al.Effect of the MDM2antagonist RG7112 on the P53 pathway in patients with MDM2-amplified,well-differentiated or dedifferentiated liposarcoma:an exploratory proof-of-mechanism study.Lancet Oncol,2012,13(11):1133-1140.
[18]Pece S,Confalonieri S,R Romano P,et al.NUMB-ing downcancer by more than just a NOTCH.Biochim Biophys Acta,2011,1815(1):26-43.
[19]Enari M,Ohmori K,Kitabayashi I,et al.Requirement of clathrin heavy chain for p53-mediated transcription.Genes Dev,2006,20(9):1087-1099.
[20]Cayouette M,Raff M,et al.Asymmetric segregation of Numb:a mechanism for neural specification from Drosophila to mammals.Nature Neurosci,2002,5(12):1265-1269.