Calreticulin regulates MYCN expression to control neuronal differentiation and stemness of neuroblastoma
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Oncogenic N-MYC (MYCN) is widely used as a biomarker in clinics for neuroblastoma (NB) patients; nevertheless, mechanism that underlines MYCN regulation remains elusive. In the present study, we identified calreticulin (CRT) as a novel MYCN suppressor that downregulated MYCN promoter activity and protein expression to modulate neuronal differentiation and stemness. Our data showed that CRT-mediated MYCN suppression led to increased neurite length and commensurate elevation in differentiation marker GAP-43. We examined effect of radiotherapy and discovered that ionizing radiation (IR) was able to augment CRT expression dose-dependently in NB. Interestingly, neuronal differentiation and neurosphere formation (NSF) of NB were not only co-modulated by IR and CRT but were also dependent on Ca2+-buffering domain (C-domain) of CRT. Mutagenesis analysis showed that C-domain was indispensable for CRT-mediated MYCN regulation in NB differentiation and NSF. Of note, IR-induced formation of neural stem-like neurospheres (NS) was significantly impaired in CRT-overexpressed NB cells. The occupancy of CRT on MYCN 5′ proximal promoter was confirmed by chromatin immunoprecipitation assays, revealing potential CRT binding sites that coincided with transcription factor E2F1 binding elements. In addition, we identified a physical interaction between CRT and E2F1, and demonstrated that CRT occupancy on MYCN promoter prevented E2F1-mediated MYCN upregulation. In line with in vitro findings, hampered tumor latency and retarded tumor growth in xenograft model corroborated IR and CRT co-mediated neuronal differentiation of NB. Together, our data delineated a novel mechanism of CRT-mediated MYCN regulation and warranted further preclinical investigation towards new therapeutic strategy for NB. CRT suppresses MYCN expression and promotes neuronal differentiation in NB. CRT regulates MYCN via interaction with E2F1 and direct binding to MYCN promoter. Ca2+-buffering domain of CRT is critical in MYCN regulation and NB differentiation. CRT-MYCN axis impacts on NB stemness by modulating neurosphere formation. Xenograft model corroborates in vitro NB differentiation mediated by CRT and IR.
KeywordsCalreticulin Differentiation MYCN Neuroblastoma Neurosphere Radiation
Cancer stem cells
Event-free survival rate
Neural stem cell
Nerve growth factor
Pluripotent stem cells
Transcription start site
We wish to acknowledge Prof. Shin-Ru Shih at Chang Gung University for her kind assistance in general laboratory support. We also appreciate technical support from Microscopy core facility of Chang Gung Memorial Hospital at Linkou.
Conception and design: A.C. Lee, Y.Y. Shih, F. Zhou, Y.F. Liao, W.M. Hsu, J.H. Hong.
Clinical specimen acquisition: Y.Y. Shih, Y.F. Liao, W.M. Hsu.
Data acquisition, analysis, and interpretation: A.C. Lee, Y.Y. Shih, Y.F. Liao, W.M. Hsu.
Development of methodology: A.C. Lee, Y.Y. Shih, F. Zhou, Y.F. Liao, W.M. Hsu.
Technical and conceptual supervision: A.C. Lee, Y.Y. Shih, F. Zhou, H. Lee, Y.F. Liao, W.M. Hsu, J.H. Hong.
Writing and revision of manuscript: A.C. Lee, Y.Y. Shih, F. Zhou, T.C. Chao, H. Lee, Y.F. Liao, W.M. Hsu, J.H. Hong.
This work was supported by medical research grants from Chang Gung Memorial Hospital [CMRPG3F2232, CMRP3G1491, CMRPG3E1301, and CIRPG3D0141] and Ministry of Science and Technology, Taiwan [MOST106-2314-B-182A-023-MY2].
Compliance with ethical standards
Conflict of interest
The authors declare that there is no conflict of interest.
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