Neddylation inhibitor MLN4924 suppresses cilia formation by modulating AKT1

Abstract

The primary cilium is a microtubule-based sensory organelle. The molecular mechanism that regulates ciliary dynamics remains elusive. Here, we report an unexpected finding that MLN4924, a small molecule inhibitor of NEDD8-activating enzyme (NAE), blocks primary ciliary formation by inhibiting synthesis/assembly and promoting disassembly. This is mainly mediated by MLN4924-induced phosphorylation of AKT1 at Ser473 under serum-starved, ciliary-promoting conditions. Indeed, pharmaceutical inhibition (by MK2206) or genetic depletion (via siRNA) of AKT1 rescues MLN4924 effect, indicating its causal role. Interestingly, pAKT1-Ser473 activity regulates both ciliary synthesis/assembly and disassembly in a MLN4924 dependent manner, whereas pAKT-Thr308 determines the ciliary length in MLN4924-independent but VHL-dependent manner. Finally, MLN4924 inhibits mouse hair regrowth, a process requires ciliogenesis. Collectively, our study demonstrates an unexpected role of a neddylation inhibitor in regulation of ciliogenesis via AKT1, and provides a proof-of-concept for potential utility of MLN4924 in the treatment of human diseases associated with abnormal ciliogenesis.

Key words： AKT; Cilia; MLN4924; neddylation; siRNA; VHL

References

1 Abe Y, Tanaka N (2017) Roles of the hedgehog signaling pathway in epidermal and hair follicle development, homeostasis, and cancer. J Dev Biol 5(4):12

2 Brownell JE, Sintchak MD, Gavin JM, Liao H, Bruzzese FJ, Bump NJ, Soucy TA, Milhollen MA, Yang X, Burkhardt AL (2010) Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ. Mol Cell 37(1):102–111

3 Cao M, Li G, Pan J (2009) Regulation of cilia assembly, disassembly, and length by protein phosphorylation. Methods Cell Biol 94:333–346

4 Castaneda A, Serrano C, Hernandez-Trejo JA, Gutierrez-Martinez IZ, Montejo-Lopez W, Gomez-Suarez M, Hernandez-Ruiz M, Betanzos A, Candelario-Martinez A, Romo-Parra H (2017) pVHL suppresses Akt/beta-catenin-mediated cell proliferation by inhibiting 14-3-3zeta expression. Biochem J 474(16):2679–2689

5 Cicenas J (2008) The potential role of Akt phosphorylation in human cancers. Int J Biol Mark 23(1):1–9

6 Conduit SE, Ramaswamy V, Remke M, Watkins DN, Wainwright BJ, Taylor MD, Mitchell CA, Dyson JM (2017) A compartmentalized phosphoinositide signaling axis at cilia is regulated by INPP5E to maintain cilia and promote Sonic Hedgehog medulloblastoma. Oncogene 36(43):5969–5984

7 Dere R, Perkins AL, Bawa-Khalfe T, Jonasch D, Walker CL (2015) beta-catenin links von Hippel-Lindau to aurora kinase A and loss of primary cilia in renal cell carcinoma. J Am Soc Nephrol 26(3):553–564

8 Deshaies RJ (1999) SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev Biol 15:435–467

9 Deshaies RJ, Emberley ED, Saha A (2010) Control of cullin-RING ubiquitin ligase activity by Nedd8. In: Groettrup M (ed) Conjugation and deconjugation of ubiquitin family modifiers: subcellular biochemistry, vol 54. Springer, Berlin, pp 41–56

10 D’Angiolella V, Donato V, Vijayakumar S, Saraf A, Florens L, Washburn MP, Dynlacht B, Pagano M (2010) SCF(Cyclin F) controls centrosome homeostasis and mitotic fidelity through CP110 degradation. Nature 466(7302):138–142

11 Eggenschwiler JT, Anderson KV (2007) Cilia and developmental signaling. Annu Rev Cell Dev Biol 23:345–373

12 Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M (2004) PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 30(2):193–204

13 Frew IJ, Krek W (2007) Multitasking by pVHL in tumour suppression. Curr Opin Cell Biol 19(6):685–690

14 Frew IJ, Thoma CR, Georgiev S, Minola A, Hitz M, Montani M, Moch H, Krek W (2008) pVHL and PTEN tumour suppressor proteins cooperatively suppress kidney cyst formation. EMBO J 27(12):1747–1757

15 Goetz SC, Anderson KV (2010) The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 11(5):331–344

16 Gu Q, Tan M, Sun Y (2007) SAG/ROC2/Rbx2 is a novel activator protein-1 target that promotes c-Jun degradation and inhibits 12-O-tetradecanoylphorbol-13-acetate-induced neoplastic transformation. Cancer Res 67(8):3616–3625

17 Guo J, Chakraborty AA, Liu P, Gan W, Zheng X, Inuzuka H, Wang B, Zhang J, Zhang L, Yuan M (2016) pVHL suppresses kinase activity of Akt in a proline-hydroxylation-dependent manner. Science 353(6302):929–932

18 Han YG, Kim HJ, Dlugosz AA, Ellison DW, Gilbertson RJ, Alvarez-Buylla A (2009) Dual and opposing roles of primary cilia in medulloblastoma development. Nat Med 15(9):1062–1065

19 Hasanov E, Chen G, Chowdhury P, Weldon J, Ding Z, Jonasch E, Sen S, Walker CL, Dere R (2017) Ubiquitination and regulation of AURKA identifies a hypoxia-independent E3 ligase activity of VHL. Oncogene 36(24):3450–3463

20 Hossain D, Tsang WY (2018) The role of ubiquitination in the regulation of primary cilia assembly and disassembly. Semin Cell Dev Biol. https://doi.org/10.1016/j.semcdb.2018.09.005

21 Ishikawa H, Marshall WF (2011) Ciliogenesis: building the cell’s antenna. Nat Rev Mol cell Biol 12(4):222–234

22 Jenks AD, Vyse S, Wong JP, Kostaras E, Keller D, Burgoyne T, Shoemark A, Tsalikis A, de la Roche M, Michaelis M (2018) Primary cilia mediate diverse kinase inhibitor resistance mechanisms in cancer. Cell Rep 23(10):3042–3055

23 Kaelin WG Jr (2008) The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat Rev Cancer 8(11):865–873

24 Khan NA, Willemarck N, Talebi A, Marchand A, Binda MM, Dehairs J, Rueda-Rincon N, Daniels VW, Bagadi M, Raj DB (2016) Identification of drugs that restore primary cilium expression in cancer cells. Oncotarget 7(9):9975–9992

25 Kim S, Zaghloul NA, Bubenshchikova E, Oh EC, Rankin S, Katsanis N, Obara T, Tsiokas L (2011) Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry. Nat Cell Biol 13(4):351–360

26 Kuehn EW, Walz G, Benzing T (2007) Von hippel-lindau: a tumor suppressor links microtubules to ciliogenesis and cancer development. Cancer Res 67(10):4537–4540

27 Kwon YW, Kim IJ, Wu D, Lu J, Stock WA Jr, Liu Y, Huang Y, Kang HC, DelRosario R, Jen KY (2012) Pten regulates Aurora-A and cooperates with Fbxw7 in modulating radiation-induced tumor development. Mol Cancer Res 10(6):834–844

28 Lehman JM, Laag E, Michaud EJ, Yoder BK (2009) An essential role for dermal primary cilia in hair follicle morphogenesis. J Invest Dermatol 129(2):438–448

29 Liang Y, Meng D, Zhu B, Pan J (2016) Mechanism of ciliary disassembly. Cell Mol Life Sci 73(9):1787–1802

30 Liu H, Kiseleva AA, Golemis EA (2018) Ciliary signalling in cancer. Nat Rev Cancer 18:511–524

31 Najafov A, Sommer EM, Axten JM, Deyoung MP, Alessi DR (2011) Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem J 433(2):357–369

32 Nawrocki ST, Griffin P, Kelly KR, Carew JS (2012) MLN4924: a novel first-in-class inhibitor of NEDD8-activating enzyme for cancer therapy. Expert Opin Investig Drugs 21(10):1563–1573

33 Nigg EA, Raff JW (2009) Centrioles, centrosomes, and cilia in health and disease. Cell 139(4):663–678

34 Nikonova AS, Golemis EA (2015) The tumor suppressor FBW7 controls ciliary length. EMBO J 34(19):2388–2390

35 Persad S, Attwell S, Gray V, Mawji N, Deng JT, Leung D, Yan J, Sanghera J, Walsh MP, Dedhar S (2001) Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem 276(29):27462–27469

36 Polytarchou C, Iliopoulos D, Hatziapostolou M, Kottakis F, Maroulakou I, Struhl K, Tsichlis PN (2011) Akt2 regulates all Akt isoforms and promotes resistance to hypoxia through induction of miR-21 upon oxygen deprivation. Cancer Res 71(13):4720–4731

37 Pugacheva EN, Jablonski SA, Hartman TR, Henske EP, Golemis EA (2007) HEF1-Dependent aurora a activation induces disassembly of the primary cilium. Cell 129(7):1351–1363

38 Richards FM (2001) Molecular pathology of von HippelLindau disease and the VHL tumour suppressor gene. Expert Rev Mol Med 2001:1–27

39 Rishikaysh P, Dev K, Diaz D, Qureshi WM, Filip S, Mokry J (2014) Signaling involved in hair follicle morphogenesis and development. Int J Mol Sci 15(1):1647–1670

40 Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307(5712):1098–1101

41 Schermer B, Ghenoiu C, Bartram M, Muller RU, Kotsis F, Hohne M, Kuhn W, Rapka M, Nitschke R, Zentgraf H (2006) The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth. J Cell Biol 175(4):547–554

42 Singla V, Reiter JF (2006) The primary cilium as the cell’s antenna: signaling at a sensory organelle. Science 313(5787):629–633

43 Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, Brownell JE, Burke KE, Cardin DP, Critchley S (2009) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458(7239):732–736

44 Spektor A, Tsang WY, Khoo D, Dynlacht BD (2007) Cep97 and CP110 suppress a cilia assembly program. Cell 130(4):678–690

45 Suizu F, Hirata N, Kimura K, Edamura T, Tanaka T, Ishigaki S, Donia T, Noguchi H, Iwanaga T, Noguchi M (2016) Phosphorylationdependent Akt-Inversin interaction at the basal body of primary cilia. EMBO J 35(12):1346–1363

46 Thoma CR, Frew IJ, Hoerner CR, Montani M, Moch H, Krek W (2007) pVHL and GSK3beta are components of a primary ciliummaintenance signalling network. Nat Cell Biol 9(5):588–595

47 Valencia-Gattas M, Conner GE, Fregien NL (2016) Gefitinib, an EGFR tyrosine kinase inhibitor, prevents smoke-mediated ciliated airway epithelial cell loss and promotes their recovery. PloS ONE 11(8):e0160216

48 Walentek P, Quigley IK, Sun DI, Sajjan UK, Kintner C, Harland RM (2016) Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis. Elife 5:e17557

49 Wang Q, Chen X, Hay N (2017) Akt as a target for cancer therapy: more is not always better (lessons from studies in mice). Br J Cancer 117(2):159–163

50 Wang X, Ge J, Tredget EE, Wu Y (2013) The mouse excisional wound splinting model, including applications for stem cell transplantation. Nature protocols 8(2):302–309

51 Wei D, Li H, Yu J, Sebolt JT, Zhao L, Lawrence TS, Smith PG, Morgan MA, Sun Y (2012) Radiosensitization of human pancreatic cancer cells by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Cancer Res 72(1):282–293

52 Yadav SP, Sharma NK, Liu C, Dong L, Li T, Swaroop A (2016) Centrosomal protein CP110 controls maturation of the mother centriole during cilia biogenesis. Development 143(9):1491–1501

53 Yasar B, Linton K, Slater C, Byers R (2017) Primary cilia are increased in number and demonstrate structural abnormalities in human cancer. J Clin Pathol 70(7):571–574

54 Zhang C, Zhang W, Lu Y, Yan X, Yan X, Zhu X, Liu W, Yang Y, Zhou T (2016) NudC regulates actin dynamics and ciliogenesis by stabilizing cofilin 1. Cell Res 26(2):239–253

55 Zhang Z, Zhang G, Xu X, Su W, Yu B (2012) mTOR-rictor is the Ser473 kinase for AKT1 in mouse one-cell stage embryos. Mol Cell Biochem 361(1–2):249–257

56 Zhao Y, Morgan MA, Sun Y (2014) Targeting neddylation pathways to inactivate Cullin-RING ligases for anti-cancer therapy. Antioxid Redox Signal 21(17):2383–2400

57 Zhou X, Tan M, Nyati MK, Zhao Y, Wang G, Sun Y (2016) Blockage of neddylation modification stimulates tumor sphere formation in vitro and stem cell differentiation and wound healing in vivo. Proc Natl Acad Sci USA 113(21):E2935–2944

58 Zhou L, Zhang W, Sun Y, Jia L (2018) Protein neddylation and its alterations in human cancers for targeted therapy. Cell Signal 44:92–102