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Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation

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

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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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Correspondence to S Nasi or R Rota.

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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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