Substantial evidence supports the oncogenic role of the E3 ubiquitin ligase S-phase kinase-associated protein 2 (Skp2) in many types of cancers through its ability to target a broad range of signaling effectors for ubiquitination. Thus, this oncogenic E3 ligase represents an important target for cancer drug discovery. In this study, we report a novel mechanism by which CG-12, a novel energy restriction-mimetic agent (ERMA), down-regulates the expression of Skp2 in prostate cancer cells. Pursuant to our previous finding that upregulation of β-transducin repeat-containing protein (β-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this β-TrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets β-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif 412SP. This Skp2 downregulation was attributable to Sirt1-dependent suppression of COP9 signalosome (Csn)5 expression in response to CG-12, leading to increased cullin 1 neddylation in the Skp1-cullin1-F-box protein complex and consequent Skp2 destabilization. Moreover, we determined that Skp2 and β-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of β-TrCP reduced the expression of Sp1, a β-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-β-TrCP-Sp1 feedback loop represents a novel crosstalk mechanism between these two important F-box proteins in cancer cells with aberrant Skp2 expression under energy restriction, which provides a proof-of-concept that the oncogenic Csn5/Skp2 signaling axis represents a “druggable” target for this novel ERMA.
References
[1]
Nakayama KI, Nakayama K (2005) Regulation of the cell cycle by SCF-type ubiquitin ligases. Semin Cell Dev Biol 16: 323–333.
[2]
Frescas D, Pagano M (2008) Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer. Nat Rev Cancer 8: 438–449.
[3]
Carrano AC, Eytan E, Hershko A, Pagano M (1999) SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1: 193–199.
[4]
Chan CH, Li CF, Yang WL, Gao Y, Lee SW, et al. (2012) The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis. Cell 149: 1098–1111.
[5]
Wu J, Zhang X, Zhang L, Wu CY, Rezaeian AH, et al. (2012) Skp2 E3 ligase integrates ATM activation and homologous recombination repair by ubiquitinating NBS1. Mol Cell 46: 351–361.
[6]
Fuchs SY, Spiegelman VS, Kumar KG (2004) The many faces of beta-TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer. Oncogene 23: 2028–2036.
[7]
Hart M, Concordet JP, Lassot I, Albert I, del los Santos R, et al. (1999) The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell. Curr Biol 9: 207–210.
[8]
Spencer E, Jiang J, Chen ZJ (1999) Signal-induced ubiquitination of IkappaBalpha by the F-box protein Slimb/beta-TrCP. Genes Dev 13: 284–294.
[9]
Lassot I, Segeral E, Berlioz-Torrent C, Durand H, Groussin L, et al. (2001) ATF4 degradation relies on a phosphorylation-dependent interaction with the SCF(betaTrCP) ubiquitin ligase. Mol Cell Biol 21: 2192–2202.
[10]
Jin J, Shirogane T, Xu L, Nalepa G, Qin J, et al. (2003) SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase. Genes Dev 17: 3062–3074.
[11]
Ding Q, He X, Hsu JM, Xia W, Chen CT, et al. (2007) Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization. Mol Cell Biol 27: 4006–4017.
[12]
Wei S, Yang HC, Chuang HC, Yang J, Kulp SK, et al. (2008) A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells. J Biol Chem 283: 26759–26770.
[13]
Wei S, Chuang HC, Tsai WC, Yang HC, Ho SR, et al. (2009) Thiazolidinediones mimic glucose starvation in facilitating Sp1 degradation through the up-regulation of beta-transducin repeat-containing protein. Mol Pharmacol 76: 47–57.
[14]
Guardavaccaro D, Kudo Y, Boulaire J, Barchi M, Busino L, et al. (2003) Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo. Dev Cell 4: 799–812.
[15]
Margottin-Goguet F, Hsu JY, Loktev A, Hsieh HM, Reimann JD, et al. (2003) Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase. Dev Cell 4: 813–826.
[16]
Bashir T, Dorrello NV, Amador V, Guardavaccaro D, Pagano M (2004) Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature 428: 190–193.
[17]
Wei W, Ayad NG, Wan Y, Zhang GJ, Kirschner MW, et al. (2004) Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex. Nature 428: 194–198.
[18]
Wei S, Kulp SK, Chen CS (2010) Energy restriction as an antitumor target of thiazolidinediones. J Biol Chem 285: 9780–9791.
[19]
Yang J, Wei S, Wang DS, Wang YC, Kulp SK, et al. (2008) Pharmacological exploitation of the peroxisome proliferator-activated receptor gamma agonist ciglitazone to develop a novel class of androgen receptor-ablative agents. J Med Chem 51: 2100–2107.
[20]
Huang YC, Hung WC (2006) 1,25-dihydroxyvitamin D3 transcriptionally represses p45Skp2 expression via the Sp1 sites in human prostate cancer cells. J Cell Physiol 209: 363–369.
[21]
Wang H, Sun D, Ji P, Mohler J, Zhu L (2008) An AR-Skp2 pathway for proliferation of androgen-dependent prostate-cancer cells. J Cell Sci 121: 2578–2587.
[22]
Signoretti S, Di Marcotullio L, Richardson A, Ramaswamy S, Isaac B, et al. (2002) Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer. J Clin Invest 110: 633–641.
[23]
Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, et al. (2004) Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science 305: 390–392.
[24]
Denti S, Fernandez-Sanchez ME, Rogge L, Bianchi E (2006) The COP9 signalosome regulates Skp2 levels and proliferation of human cells. J Biol Chem 281: 32188–32196.
[25]
Wolf DA, Zhou C, Wee S (2003) The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases? Nat Cell Biol 5: 1029–1033.
[26]
Luo J, Nikolaev AY, Imai S, Chen D, Su F, et al. (2001) Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 107: 137–148.
[27]
Adler AS, Littlepage LE, Lin M, Kawahara TL, Wong DJ, et al. (2008) CSN5 isopeptidase activity links COP9 signalosome activation to breast cancer progression. Cancer Res 68: 506–515.
[28]
Furukawa M, Zhang Y, McCarville J, Ohta T, Xiong Y (2000) The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1. Mol Cell Biol 20: 8185–8197.
[29]
Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H (1999) p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr Biol 9: 661–664.
[30]
Kamura T, Hara T, Kotoshiba S, Yada M, Ishida N, et al. (2003) Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation. Proc Natl Acad Sci U S A 100: 10231–10236.
[31]
Tedesco D, Lukas J, Reed SI (2002) The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2). Genes Dev 16: 2946–2957.
[32]
Jiang H, Chang FC, Ross AE, Lee J, Nakayama K, et al. (2005) Ubiquitylation of RAG-2 by Skp2-SCF links destruction of the V(D)J recombinase to the cell cycle. Mol Cell 18: 699–709.
[33]
Nie L, Wu H, Sun XH (2008) Ubiquitination and degradation of Tal1/SCL are induced by notch signaling and depend on Skp2 and CHIP. J Biol Chem 283: 684–692.
[34]
Lin YW, Yang JL (2006) Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling. J Biol Chem 281: 915–926.
[35]
Nie L, Xu M, Vladimirova A, Sun XH (2003) Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities. EMBO J 22: 5780–5792.
[36]
Liu Y, Hedvat CV, Mao S, Zhu XH, Yao J, et al. (2006) The ETS protein MEF is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCFSkp2. Mol Cell Biol 26: 3114–3123.
[37]
Lu KP, Liou YC, Zhou XZ (2002) Pinning down proline-directed phosphorylation signaling. Trends Cell Biol 12: 164–172.
[38]
Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11: 725–732.
[39]
Shackleford TJ, Claret FX (2010) JAB1/CSN5: a new player in cell cycle control and cancer. Cell Div 5: 26.
[40]
Wang Z, Gao D, Fukushima H, Inuzuka H, Liu P, et al. (2012) Skp2: A novel potential therapeutic target for prostate cancer. Biochim Biophys Acta 1825: 11–17.
[41]
Hershko DD (2008) Oncogenic properties and prognostic implications of the ubiquitin ligase Skp2 in cancer. Cancer 112: 1415–1424.
[42]
Gao D, Inuzuka H, Tseng A, Chin RY, Toker A, et al. (2009) Phosphorylation by Akt1 promotes cytoplasmic localization of Skp2 and impairs APCCdh1-mediated Skp2 destruction. Nat Cell Biol 11: 397–408.
