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Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Hallberg)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Hallberg)
College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing, China.
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2012 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 31, no 50, 5193-5200 p.Article in journal (Refereed) Published
Abstract [en]

Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and a disease with several different chromosomal gains and losses, which include MYCN-amplified neuroblastoma on chromosome 2, deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. Recently, activating mutations of the ALK (Anaplastic Lymphoma Kinase) RTK (Receptor Tyrosine Kinase) gene have been described in neuroblastoma. A meta-analysis of neuroblastoma cases revealed that ALK mutations (49 of 709 cases) in relation to genomic subtype were most frequently observed in MYCN amplified tumours (8.9%), correlating with a poor clinical outcome. MYCN proteins target proliferation and apoptotic pathways, and have an important role in the progression of neuroblastoma. Here, we show that both wild-type and gain-of-function mutants in ALK are able to stimulate transcription at the MYCN promoter and initiate mRNA transcription of the MYCN gene in both neuronal and neuroblastoma cell lines. Further, this stimulation of MYCN gene transcription and de novo MYCN protein expression is abrogated by specific ALK inhibitors, such as crizotinib (PF-2341066), NVP-TAE684, and by small interfering RNA to ALK resulting in a decrease in proliferation rate. Finally, co-transfection of ALK gain-of-function mutations together with MYCN leads to an increase in transformation potential. Taken together, our results indicate that ALK signalling regulates initiation of transcription of the MYCN gene providing a possible explanation for the poor clinical outcome observed when MYCN is amplified together with activated ALK.Oncogene advance online publication, 30 January 2012; doi:10.1038/onc.2012.12.

Place, publisher, year, edition, pages
2012. Vol. 31, no 50, 5193-5200 p.
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-53961DOI: 10.1038/onc.2012.12PubMedID: 22286764OAI: oai:DiVA.org:umu-53961DiVA: diva2:514618
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Anaplastic Lymphoma Kinase mutations and downstream signalling
Open this publication in new window or tab >>Anaplastic Lymphoma Kinase mutations and downstream signalling
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system.

By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells.

Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2012. 85 p.
Keyword
Anaplastic Lymphoma Kinase, oncogene, RTK, neuroblastoma, crizotinib, NVP-TAE684, gain-of-function, MYCN, transcription factor, small GTPase, Rap1, C3G, PC12 cells, neurite outgrowth
National Category
Cell Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:umu:diva-54562 (URN)978-91-7459-387-7 (ISBN)
Public defence
2012-08-24, NUS - Norrlands universitetssjukhus, Byggnad 6M, Betula, Umeå Universitet, Umeå, 09:00 (English)
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Available from: 2012-05-16 Created: 2012-04-30 Last updated: 2012-05-09Bibliographically approved

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Schönherr, ChristinaRuuth, KristinaKamaraj, SattuPalmer, Ruth HHallberg, Bengt

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Schönherr, ChristinaRuuth, KristinaKamaraj, SattuPalmer, Ruth HHallberg, Bengt
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Oncogene
Cell and Molecular BiologyMedical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

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