CSN4基因干扰对乳腺癌MDA-MB-231细胞增殖和凋亡的影响
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摘要
乳腺癌目前是危害女性常见的恶性肿瘤,研究发现COP9复合体中的各个亚基与恶性肿瘤的发生发展密切相关,且CSN4亚基对整个复合体具有很重要的调节作用。为探讨CSN4基因在乳腺癌细胞中的生物学功能及其分子作用机制,本研究首先在乳腺癌细胞MDA-MB-231中,成功建立了慢病毒介导的CSN4基因的干扰表达体系,并通过细胞表型实验、CCK8实验和细胞克隆形成实验证实CSN4基因表达的干扰能显著抑制乳腺癌MDA-MB-231细胞的增殖速率和克隆形成能力。流式细胞检测结果表明,干扰CSN4的表达能显著增加Sub G_1期乳腺癌MDA-MB-231细胞比例;凋亡检测结果表明,干扰CSN4的表达能显著增加早期凋亡和晚期凋亡细胞的比例,这证明干扰CSN4的表达能够促进乳腺癌细胞凋亡。最后,通过Western blot实验证实CSN4能调控CDK6和Caspase3蛋白的表达,激活Caspase3切割PARP,揭示CSN4可调控CDK6和Caspase3的表达从而影响乳腺癌细胞的增殖和凋亡。本研究结果进一步加深了人们对乳腺癌细胞凋亡和细胞生长的分子机制的认识,同时也进一步揭示了CSN4在肿瘤生物学中的作用和机制。
Breast cancer is one of the most common malignant tumors endangering women. It has been found that the subunits of the COP9 complex are closely related to the occurrence and development of malignant tumors, and the CSN4 subunit plays an important role in regulating the whole complex. In the breast cancer cell line MDA-MB-231, we successfully established a lentivirus-mediated CSN4-knockdown cell line. CCK8 cell proliferation assays and colony formation experiments confirmed that CSN4 knockdown significantly decreased the cellular proliferation rate. Cell cycle analysis showed that CSN4 knockdown increased sub-G_1 population and induced apoptosis. In addition, Western blotting assays confirmed that CSN4 regulates the expression of CDK6 and Caspase3, suggesting that CSN4 modulates the proliferation and apoptosis of breast cancer cells by regulating the expression of CDK6 and Caspase3 genes and thereby tumorigenesis. This study has deepened our understanding of the molecular mechanism of apoptosis and cell growth in breast cancers, and further revealed the role and mechanism of CSN4 in cancer biology.
Breast cancer is one of the most common malignant tumors endangering women. It has been found that the subunits of the COP9 complex are closely related to the occurrence and development of malignant tumors, and the CSN4 subunit plays an important role in regulating the whole complex. In the breast cancer cell line MDA-MB-231, we successfully established a lentivirus-mediated CSN4-knockdown cell line. CCK8 cell proliferation assays and colony formation experiments confirmed that CSN4 knockdown significantly decreased the cellular proliferation rate. Cell cycle analysis showed that CSN4 knockdown increased sub-G_1 population and induced apoptosis. In addition, Western blotting assays confirmed that CSN4 regulates the expression of CDK6 and Caspase3, suggesting that CSN4 modulates the proliferation and apoptosis of breast cancer cells by regulating the expression of CDK6 and Caspase3 genes and thereby tumorigenesis. This study has deepened our understanding of the molecular mechanism of apoptosis and cell growth in breast cancers, and further revealed the role and mechanism of CSN4 in cancer biology.
引文
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[3]Corbex M,Bouzbid S,Traverse-Glehen A,Aouras H,McKay-Chopin S,Carreira C,Lankar A,Tommasino A,Gheit T.Prevalence of papillomaviruses,polyomaviruses,and herpesviruses in triple-negative and inflammatory breast tumors from algeria compared with other types of breast cancer tumors.PLoS One,2014,9(12):e114559.
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[5]Rajan KE,Rajkumar R,Liao CC,Ganesh A,Marimuthu G.Light-induced COP9 signalosome expression in the Indian false vampire bat Megaderma lyra.J Physiol Sci,2010,60(1):43-49.
[6]Dubiel D,Rockel B,Naumann M,Dubiel W.Diversity of COP9 signalosome structures and functional consequences.FEBS Lett,2015,589(19 Pt A):2507-2513.
[7]Lingaraju GM,Bunker RD,Cavadini S,Hess D,Hassiepen U,Renayus M,Fischer ES,Thom?NH.Crystal structure of the human COP9 signalosome.Nature,2014,512(7513):161-165.
[8]Emberley ED,Mosadeghi R,Deshaies RJ.Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate,and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism.J Biol Chem,2012,287(35):29679-29689.
[9]Yu YS,Tang ZH,Pan QC,Chen XH,Liu XN,Zang GQ.Inhibition of CSN3 expression induces growth arrest and apoptosis of hepatocellular carcinoma cells.Cancer Chemoth Pharm,2012,69(5):1173-1180.
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[11]Zhao R,Yeung SC,Chen J,Iwakuma T,Su CH,Chen B,Qu C,Zhang F,Chen YT,Lin YL,Lee DF,Jin F,Zhu R,Shaikenov T,Sarbassov D,Sahin A,Wang H,Wang H,Lai CC,Tsai FJ,Lozano G,Lee MH.Subunit 6 of the COP9signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers.J Clin Invest,2011,121(2):851-865.
[12]Zhang XC,Chen J,Su CH,Yang HY,Lee MH.Roles for CSN5 in control of p53/MDM2 activities.J Cell Biochem,2008,103(4):1219-1230.
[13]Tomoda K,Kubota Y,Kato J.Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1.Nature,1999,398(6723):160-165.
[14]Yang X,Menon S,Lykke-Andersen K,Tsuge T,Di Xiao,Wang X,Rodriguez-Suarez RJ,Zhang H,Wei N.The COP9 signalosome inhibits p27(kip1)degradation and impedes G1-S phase progression via deneddylation of SCFCul1.Curr Biol,2002,12(8):667-672.
[15]Bech-Otschir D,Kraft R,Huang X,Henklein P,Kapelari B,Pollmann C,Dubiel W.COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system.Embo J,2001,20(7):1630-1639.
[16]Kim BC,Lee HJ,Park SH,Lee SR,Karpova TS,McNally JG,Felici A,Lee DK,Kim SJ.Jab1/CSN5,a component of the COP9 signalosome,regulates transforming growth factor beta signaling by binding to Smad7 and promoting its degradation.Mol Cell Biol,2004,24(6):2251-2262.
[17]Rhodes DR,Kalyana-Sundaram S,Mahavisno V,Varambally R,Yu J,Briggs BB,Barrette TR,Anstet MJ,Kincead-Beal C,Kulkarni P,Varambally S,Ghosh D,Chinnaiyan AM.Oncomine 3.0:genes,pathways,and networks in a collection of 18,000 cancer gene expression profiles.Neoplasia,2007,9(2):166-180.
[18]Rhodes DR,Kalyana-Sundaram S,Mahavisno V,Barrette TR,Ghosh D,Chinnaiyan AM.Mining for regulatory programs in the cancer transcriptome.Nat Genet,2005,37(6):579-583.
[19]Rhodes DR,Yu J,Shanker K,Deshpande N,Varambally R,Ghosh D,Barrette T,Pandey A,Chinnaiyan AM.ONCOMINE:a cancer microarray database and integrated data-mining platform.Neoplasia,2004,6(1):1-6.
[20]Pollmann C,Huang X,Mall J,Bech-Otschir D,Naumann M,Dubiel W.The constitutive photomorphogenesis 9signalosome directs vascular endothelial growth factor production in tumor cells.Cancer Res,2001,61(23):8416-8421.
[21]Kob R,Kelm J,Posorski N,Baniahmad A,von Eggeling F,Melle C.Regulation of the anaphase-promoting complex by the COP9 signalosome.Cell Cycle,2009,8(13):2041-2049.
[22]Leal JF,Fominaya J,Cascón A,Guijarro MV,BlancoAparicio C,Lleonart M,Castro ME,Ramon Y Cajal S,Robledo M,Beach DH,Carnero A.Cellular senescence bypass screen identifies new putative tumor suppressor genes.Oncogene,2008,27(14):1961-1970.
[23]Boutonnet C,Tanguay PL,Julien C,Rodier G,Coulombe P,Meloche S.Phosphorylation of Ser72 does not regulate the ubiquitin ligase activity and subcellular localization of Skp2.Cell Cycle,2010,9(5):975-979.
[24]Bashir T,Pagan JK,Busino L,Pagano M.Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization.Cell Cycle,2010,9(5):971-974.
[25]Bhansali M,Shemshedini L.COP9 subunits 4 and 5 target soluble guanylyl cyclaseα1 and p53 in prostate cancer cells.Mol Endocrinol,2014,28(6):834-845.
[26]Lingaraju GM,Bunker RD,Cavadini S,Hess D,Hassiepen U,Renatus M,Fischer ES,Thom?NH.Crystal structure of the human COP9 signalosome.Nature,2014,512(7513):161-165.
[27]Kollmann K,Heller G,Schneckenleithner C,Warsch W,Scheicher R,Ott RG,Sch?fer M,Fajmann S,Schlederer M,Schiefer AI,Reichart U,Mayerhofer M,Hoeller C,Z?chbauer-Müller S,Kerjaschki D,Bock C,Kenner L,Hoefler G,Freissmuth M,Green AR,Moriggl R,Busslinger M,Malumbres M,Sexl V.A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis.Cancer Cell,2016,30(2):359-360.
[28]Thomas JW,Lee-Lin SQ,Green ED.Human-mouse comparative mapping of the genomic region containing CDK6:localization of an evolutionary breakpoint.Mamm Genome,1999,10(7):764-767.
[29]Schulze-Gahmen U,Kim SH.Structural basis for CDK6activation by a virus-encoded cyclin.Nat Struct Biol,2002,9(3):177-181.
[30]Hetfeld BK,Peth A,Sun XM,Henklein P,Cohen GM,Dubiel W.The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis.Apoptosis,2008,13(2):187-195.
[31]Sang MM,Du WQ,Zhang RY,Zhang JN,Pei DS.Suppression of CSN5 promotes the apoptosis of gastric cancer cells through regulating p53-related apoptotic pathways.Bioorg Med Chem Lett,2015,25(15):2897-2901.
[32]da Silva Correia J,Miranda Y,Leonard N,Ulevitch RJ.The subunit CSN6 of the COP9 signalosome is cleaved during apoptosis.J Biol Chem,2007,282(17):12557-12565.
[33]Lei D,Li F,Su H,Liu J,Wei N.Hepatic deficiency of COP9 signalosome subunit 8 induces ubiquitin-proteasome system impairment and Bim-mediated apoptosis in murine livers.PLoS One,2013,8(7):e67793.
[34]Dubois EL,Gerber S,Kisselev A,Harel-Bellan A,Groisman R.UV-dependent phosphorylation of COP9/signalosome in UV-induced apoptosis.Oncol Rep,2016,35(5):3101-3105.
[35]Akiyama H,Fujisawa N,Tashiro Y,Takanabe N,Sugiyama A,Tashiro F.The role of transcriptional corepressor Nif3l1in early stage of neural differentiation via cooperation with Trip15/CSN2.J Biol Chem,2003,278(12):10752-10762.
[2]Chen W,Zheng R,Baade PD,Zhang S,Zeng H,Bray F,Jemal A,Yu XQ,He J.Cancer statistics in China,2015.CA:A Cancer Journal for Clinicians,2016,66(2):115-132.
[3]Corbex M,Bouzbid S,Traverse-Glehen A,Aouras H,McKay-Chopin S,Carreira C,Lankar A,Tommasino A,Gheit T.Prevalence of papillomaviruses,polyomaviruses,and herpesviruses in triple-negative and inflammatory breast tumors from algeria compared with other types of breast cancer tumors.PLoS One,2014,9(12):e114559.
[4]Maytal-Kivity V,Pick E,Piran R,Hofmann K,Glickman MH.The COP9 signalosome-like complex in S.cerevisiae and links to other PCI complexes.Int J Biochem Cell Biol,2003,35(5):706-715.
[5]Rajan KE,Rajkumar R,Liao CC,Ganesh A,Marimuthu G.Light-induced COP9 signalosome expression in the Indian false vampire bat Megaderma lyra.J Physiol Sci,2010,60(1):43-49.
[6]Dubiel D,Rockel B,Naumann M,Dubiel W.Diversity of COP9 signalosome structures and functional consequences.FEBS Lett,2015,589(19 Pt A):2507-2513.
[7]Lingaraju GM,Bunker RD,Cavadini S,Hess D,Hassiepen U,Renayus M,Fischer ES,Thom?NH.Crystal structure of the human COP9 signalosome.Nature,2014,512(7513):161-165.
[8]Emberley ED,Mosadeghi R,Deshaies RJ.Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate,and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism.J Biol Chem,2012,287(35):29679-29689.
[9]Yu YS,Tang ZH,Pan QC,Chen XH,Liu XN,Zang GQ.Inhibition of CSN3 expression induces growth arrest and apoptosis of hepatocellular carcinoma cells.Cancer Chemoth Pharm,2012,69(5):1173-1180.
[10]Wang XM,Cui JW,Li W,Cai L,Song W,Wang GJ.Silencing of the COPS3 gene by siRNA reduces proliferation of lung cancer cells most likely via induction of cell cycle arrest and apoptosis.Asian Pac J Cancer P,2012,13(3):1043-1048.
[11]Zhao R,Yeung SC,Chen J,Iwakuma T,Su CH,Chen B,Qu C,Zhang F,Chen YT,Lin YL,Lee DF,Jin F,Zhu R,Shaikenov T,Sarbassov D,Sahin A,Wang H,Wang H,Lai CC,Tsai FJ,Lozano G,Lee MH.Subunit 6 of the COP9signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers.J Clin Invest,2011,121(2):851-865.
[12]Zhang XC,Chen J,Su CH,Yang HY,Lee MH.Roles for CSN5 in control of p53/MDM2 activities.J Cell Biochem,2008,103(4):1219-1230.
[13]Tomoda K,Kubota Y,Kato J.Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1.Nature,1999,398(6723):160-165.
[14]Yang X,Menon S,Lykke-Andersen K,Tsuge T,Di Xiao,Wang X,Rodriguez-Suarez RJ,Zhang H,Wei N.The COP9 signalosome inhibits p27(kip1)degradation and impedes G1-S phase progression via deneddylation of SCFCul1.Curr Biol,2002,12(8):667-672.
[15]Bech-Otschir D,Kraft R,Huang X,Henklein P,Kapelari B,Pollmann C,Dubiel W.COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system.Embo J,2001,20(7):1630-1639.
[16]Kim BC,Lee HJ,Park SH,Lee SR,Karpova TS,McNally JG,Felici A,Lee DK,Kim SJ.Jab1/CSN5,a component of the COP9 signalosome,regulates transforming growth factor beta signaling by binding to Smad7 and promoting its degradation.Mol Cell Biol,2004,24(6):2251-2262.
[17]Rhodes DR,Kalyana-Sundaram S,Mahavisno V,Varambally R,Yu J,Briggs BB,Barrette TR,Anstet MJ,Kincead-Beal C,Kulkarni P,Varambally S,Ghosh D,Chinnaiyan AM.Oncomine 3.0:genes,pathways,and networks in a collection of 18,000 cancer gene expression profiles.Neoplasia,2007,9(2):166-180.
[18]Rhodes DR,Kalyana-Sundaram S,Mahavisno V,Barrette TR,Ghosh D,Chinnaiyan AM.Mining for regulatory programs in the cancer transcriptome.Nat Genet,2005,37(6):579-583.
[19]Rhodes DR,Yu J,Shanker K,Deshpande N,Varambally R,Ghosh D,Barrette T,Pandey A,Chinnaiyan AM.ONCOMINE:a cancer microarray database and integrated data-mining platform.Neoplasia,2004,6(1):1-6.
[20]Pollmann C,Huang X,Mall J,Bech-Otschir D,Naumann M,Dubiel W.The constitutive photomorphogenesis 9signalosome directs vascular endothelial growth factor production in tumor cells.Cancer Res,2001,61(23):8416-8421.
[21]Kob R,Kelm J,Posorski N,Baniahmad A,von Eggeling F,Melle C.Regulation of the anaphase-promoting complex by the COP9 signalosome.Cell Cycle,2009,8(13):2041-2049.
[22]Leal JF,Fominaya J,Cascón A,Guijarro MV,BlancoAparicio C,Lleonart M,Castro ME,Ramon Y Cajal S,Robledo M,Beach DH,Carnero A.Cellular senescence bypass screen identifies new putative tumor suppressor genes.Oncogene,2008,27(14):1961-1970.
[23]Boutonnet C,Tanguay PL,Julien C,Rodier G,Coulombe P,Meloche S.Phosphorylation of Ser72 does not regulate the ubiquitin ligase activity and subcellular localization of Skp2.Cell Cycle,2010,9(5):975-979.
[24]Bashir T,Pagan JK,Busino L,Pagano M.Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization.Cell Cycle,2010,9(5):971-974.
[25]Bhansali M,Shemshedini L.COP9 subunits 4 and 5 target soluble guanylyl cyclaseα1 and p53 in prostate cancer cells.Mol Endocrinol,2014,28(6):834-845.
[26]Lingaraju GM,Bunker RD,Cavadini S,Hess D,Hassiepen U,Renatus M,Fischer ES,Thom?NH.Crystal structure of the human COP9 signalosome.Nature,2014,512(7513):161-165.
[27]Kollmann K,Heller G,Schneckenleithner C,Warsch W,Scheicher R,Ott RG,Sch?fer M,Fajmann S,Schlederer M,Schiefer AI,Reichart U,Mayerhofer M,Hoeller C,Z?chbauer-Müller S,Kerjaschki D,Bock C,Kenner L,Hoefler G,Freissmuth M,Green AR,Moriggl R,Busslinger M,Malumbres M,Sexl V.A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis.Cancer Cell,2016,30(2):359-360.
[28]Thomas JW,Lee-Lin SQ,Green ED.Human-mouse comparative mapping of the genomic region containing CDK6:localization of an evolutionary breakpoint.Mamm Genome,1999,10(7):764-767.
[29]Schulze-Gahmen U,Kim SH.Structural basis for CDK6activation by a virus-encoded cyclin.Nat Struct Biol,2002,9(3):177-181.
[30]Hetfeld BK,Peth A,Sun XM,Henklein P,Cohen GM,Dubiel W.The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis.Apoptosis,2008,13(2):187-195.
[31]Sang MM,Du WQ,Zhang RY,Zhang JN,Pei DS.Suppression of CSN5 promotes the apoptosis of gastric cancer cells through regulating p53-related apoptotic pathways.Bioorg Med Chem Lett,2015,25(15):2897-2901.
[32]da Silva Correia J,Miranda Y,Leonard N,Ulevitch RJ.The subunit CSN6 of the COP9 signalosome is cleaved during apoptosis.J Biol Chem,2007,282(17):12557-12565.
[33]Lei D,Li F,Su H,Liu J,Wei N.Hepatic deficiency of COP9 signalosome subunit 8 induces ubiquitin-proteasome system impairment and Bim-mediated apoptosis in murine livers.PLoS One,2013,8(7):e67793.
[34]Dubois EL,Gerber S,Kisselev A,Harel-Bellan A,Groisman R.UV-dependent phosphorylation of COP9/signalosome in UV-induced apoptosis.Oncol Rep,2016,35(5):3101-3105.
[35]Akiyama H,Fujisawa N,Tashiro Y,Takanabe N,Sugiyama A,Tashiro F.The role of transcriptional corepressor Nif3l1in early stage of neural differentiation via cooperation with Trip15/CSN2.J Biol Chem,2003,278(12):10752-10762.