Abstract
Inhibitor of DNA-binding (Id) proteins prevent cell differentiation, promote growth and sustain tumour development. They do so by binding to E proteins and other transcription factors through the helix-loop-helix (HLH) domain, and inhibiting transcription. This makes HLH-mediated Id protein interactions an appealing therapeutic target. We have used the dominant interfering HLH dimerization mutant 13I to model the impact of Id inhibition in two human neuroblastoma cell lines: LA-N-5, similar to immature neuroblasts, and SH-EP, resembling more immature precursor cells. We have validated 13I as an Id inhibitor by showing that it selectively binds to Ids, impairs complex formation with RB, and relieves repression of E protein-activated transcription. Id inactivation by 13I enhances LA-N-5 neural features and causes SH-EP cells to acquire neuronal morphology, express neuronal proteins such as N-CAM and NF-160, proliferate more slowly, and become responsive to retinoic acid. Concomitantly, 13I augments the cell-cycle inhibitor p27Kip1 and reduces the angiogenic factor vascular endothelial growth factor. These effects are Id specific, being counteracted by Id overexpression. Furthermore, 13I strongly impairs tumorigenic properties in agar colony formation and cell invasion assays. Targeting Id dimerization may therefore be effective for triggering differentiation and restraining neuroblastoma cell tumorigenicity.
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Abbreviations
- bHLH:
-
basic helix-loop-helix
- Cdk:
-
cyclin dependent kinase
- GST:
-
glutathione S-transferase
- N-CAM:
-
neural cell adhesion molecule
- NF-160:
-
neurofilament 160 kDa
- pBP:
-
pBabePuro
- RA:
-
all-trans retinoic acid
- VEGF:
-
vascular endothelial growth factor
References
Bai G, Sheng N, Xie Z, Bian W, Yokota Y, Benezra R et al. (2007). Id sustains Hes1 expression to inhibit precocious neurogenesis by releasing negative autoregulation of Hes1. Dev Cell 13: 283–297.
Bergmann E, Wanzel M, Weber A, Shin I, Christiansen H, Eilers M . (2001). Expression of P27(KIP1) is prognostic and independent of MYCN amplification in human neuroblastoma. Int J Cancer 95: 176–183.
Brodeur GM . (2003). Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 3: 203–216.
Chassot AA, Turchi L, Virolle T, Fitsialos G, Batoz M, Deckert M et al. (2007). Id3 is a novel regulator of p27kip1 mRNA in early G1 phase and is required for cell-cycle progression. Oncogene 26: 5772–5783.
Ciarapica R, Rosati J, Cesareni G, Nasi S . (2003). Molecular recognition in helix-loop-helix and helix-loop-helix-leucine zipper domains. Design of repertoires and selection of high affinity ligands for natural proteins. J Biol Chem 278: 12182–12190.
Ciccarone V, Spengler BA, Meyers MB, Biedler JL, Ross RA . (1989). Phenotypic diversification in human neuroblastoma cells: expression of distinct neural crest lineages. Cancer Res 49: 219–225.
Cohen PS, Seeger RC, Triche TJ, Israel MA . (1988). Detection of N-myc gene expression in neuroblastoma tumors by in situ hybridization. Am J Pathol 131: 391–397.
De los Santos M, Zambrano A, Aranda A . (2007). Combined effects of retinoic acid and histone deacetylase inhibitors on human neuroblastoma SH-SY5Y cells. Mol Cancer Ther 6: 1425–1432.
Farah MH, Olson JM, Sucic HB, Hume RI, Tapscott SJ, Turner DL . (2000). Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127: 693–702.
Henke E, Perk J, Vider J, de Candia P, Chin Y, Solit DB et al. (2008). Peptide-conjugated antisense oligonucleotides for targeted inhibition of a transcriptional regulator in vivo. Nat Biotechnol 26: 91–100.
Hirata H, Yoshiura S, Ohtsuka T, Bessho Y, Harada T, Yoshikawa K et al. (2002). Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science 298: 840–843.
Iavarone A, Garg P, Lasorella A, Hsu J, Israel MA . (1994). The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein. Genes Dev 8: 1270–1284.
Iavarone A, Lasorella A . (2006). ID proteins as targets in cancer and tools in neurobiology. Trends Mol Med 12: 588–594.
Jankovic V, Ciarrocchi A, Boccuni P, DeBlasio T, Benezra R, Nimer SD . (2007). Id1 restrains myeloid commitment, maintaining the self-renewal capacity of hematopoietic stem cells. Proc Natl Acad Sci USA 104: 1260–1265.
Jogi A, Persson P, Grynfeld A, Pahlman S, Axelson H . (2002). Modulation of basic helix-loop-helix transcription complex formation by Id proteins during neuronal differentiation. J Biol Chem 277: 9118–9126.
Kondo M, Cubillo E, Tobiume K, Shirakihara T, Fukuda N, Suzuki H et al. (2004). A role for Id in the regulation of TGF-beta-induced epithelial-mesenchymal transdifferentiation. Cell Death Differ 11: 1092–1101.
Lasorella A, Noseda M, Beyna M, Yokota Y, Iavarone A . (2000). Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature 407: 592–598.
Lasorella A, Rothschild G, Yokota Y, Russell RG, Iavarone A . (2005). Id2 mediates tumor initiation, proliferation, and angiogenesis in Rb mutant mice. Mol Cell Biol 25: 3563–3574.
Li Y, Yang J, Luo JH, Dedhar S, Liu Y . (2007). Tubular epithelial cell dedifferentiation is driven by the helix-loop-helix transcriptional inhibitor Id1. J Am Soc Nephrol 18: 449–460.
Liu Y, Encinas M, Comella JX, Aldea M, Gallego C . (2004). Basic helix-loop-helix proteins bind to TrkB and p21 (Cip1) promoters linking differentiation and cell cycle arrest in neuroblastoma cells. Mol Cell Biol 24: 2662–2672.
Lofstedt T, Jogi A, Sigvardsson M, Gradin K, Poellinger L, Pahlman S et al. (2004). Induction of ID2 expression by hypoxia-inducible factor-1: a role in dedifferentiation of hypoxic neuroblastoma cells. J Biol Chem 279: 39223–39231.
Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O’Reilly R et al. (1999). Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401: 670–677.
Ohtani N, Zebedee Z, Huot TJ, Stinson JA, Sugimoto M, Ohashi Y et al. (2001). Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence. Nature 409: 1067–1070.
Penn LJ, Brooks MW, Laufer EM, Land H . (1990). Negative autoregulation of c-myc transcription. EMBO J 9: 1113–1121.
Perk J, Iavarone A, Benezra R . (2005). Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer 5: 603–614.
Reynolds CP, Matthay KK, Villablanca JG, Maurer BJ . (2003). Retinoid therapy of high-risk neuroblastoma. Cancer Lett 197: 185–192.
Roberts EC, Deed RW, Inoue T, Norton JD, Sharrocks AD . (2001). Id helix-loop-helix proteins antagonize pax transcription factor activity by inhibiting DNA binding. Mol Cell Biol 21: 524–533.
Rothschild G, Zhao X, Iavarone A, Lasorella A . (2006). E Proteins and Id2 converge on p57Kip2 to regulate cell cycle in neural cells. Mol Cell Biol 26: 4351–4361.
Ruzinova MB, Benezra R . (2003). Id proteins in development, cell cycle and cancer. Trends Cell Biol 13: 410–418.
Sakurai D, Tsuchiya N, Yamaguchi A, Okaji Y, Tsuno NH, Kobata T et al. (2004). Crucial role of inhibitor of DNA binding/differentiation in the vascular endothelial growth factor-induced activation and angiogenic processes of human endothelial cells. J Immunol 173: 5801–5809.
Schwab M . (2004). MYCN in neuronal tumours. Cancer Lett 204: 179–187.
Soucek L, Helmer-Citterich M, Sacco A, Jucker R, Cesareni G, Nasi S . (1998). Design and properties of a Myc derivative that efficiently homodimerizes. Oncogene 17: 2463–2472.
Soucek L, Whitfield J, Martins CP, Finch AJ, Murphy DJ, Sodir NM et al. (2008). Modeling MYC inhibition as a cancer therapy. Nature 455: 679–683.
Tanaka T, Williams RL, Rabbitts TH . (2007). Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J 26: 3250–3259.
Toschi E, Rota R, Antonini A, Melillo G, Caporossi MC . (2000). Wild-type p53 gene transfer inhibits invasion and reduces matrix metalloproteinase-2 levels in p53-mutated human melanoma cells. J Invest Dermatol 114: 1188–1194.
Vandeputte DA, Troost D, Leenstra S, Ijlst-Keizers H, Ramkema M, Bosch DA et al. (2002). Expression and distribution of id helix-loop-helix proteins in human astrocytic tumors. Glia 38: 329–338.
Vandesompele J, Edsjo A, De Preter K, Axelson H, Speleman F, Pahlman S . (2003). ID2 expression in neuroblastoma does not correlate to MYCN levels and lacks prognostic value. Oncogene 22: 456–460.
Voigt A, Hartmann P, Zintl F . (2000). Differentiation, proliferation and adhesion of human neuroblastoma cells after treatment with retinoic acid. Cell Adhes Commun 7: 423–440.
Wainwright LJ, Lasorella A, Iavarone A . (2001). Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells. Proc Natl Acad Sci USA 98: 9396–9400.
Wiercinska E, Wickert L, Denecke B, Said HM, Hamzavi J, Gressner AM et al. (2006). Id1 is a critical mediator in TGF-beta-induced transdifferentiation of rat hepatic stellate cells. Hepatology 43: 1032–1041.
Ying QL, Nichols J, Chambers I, Smith A . (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115: 281–292.
Acknowledgements
We thank GF Bottazzo for support and discussions, A Levi and A Musacchio for paper reading, E Giorda and N Rizzo for technical assistance, C Gaetano and B Illi for luciferase assay support, and many colleagues for plasmids. RR was supported by Italian Ministry of Health, AIRC grant 1586/06 and Il Tempo, SN by AIRC, ASI and MIUR-FIRB grants. RC and DA were recipients of OPBG postdoctoral and CNR predoctoral fellowships, respectively.
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Ciarapica, R., Annibali, D., Raimondi, L. et al. Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation. Oncogene 28, 1881–1891 (2009). https://doi.org/10.1038/onc.2009.56
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DOI: https://doi.org/10.1038/onc.2009.56
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