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Anaplastic Lymphoma Kinase mutations and downstream signalling
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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 [en]
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: urn:nbn:se:umu:diva-54562ISBN: 978-91-7459-387-7 (print)OAI: oai:DiVA.org:umu-54562DiVA: diva2:524239
Public defence
2012-08-24, NUS - Norrlands universitetssjukhus, Byggnad 6M, Betula, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-05-16 Created: 2012-04-30 Last updated: 2012-05-09Bibliographically approved
List of papers
1. Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells
Open this publication in new window or tab >>Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells
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2010 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 29, no 19, 2817-2830 p.Article in journal (Refereed) Published
Abstract [en]

Many different types of cancer originate from aberrant signaling from the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), arising through different translocation events and overexpression. Further, activating point mutations in the ALK domain have been recently reported in neuroblastoma. To characterize signaling in the context of the full-length receptor, we have examined whether ALK is able to activate Rap1 and contribute to differentiation/proliferation processes. We show that ALK activates Rap1 via the Rap1-specific guanine-nucleotide exchange factor C3G, which binds in a constitutive complex with CrkL to activated ALK. The activation of the C3G/Rap1 pathway results in neurite outgrowth of PC12 cells, which is inhibited by either overexpression of Rap1GAP or siRNA-mediated knockdown of Rap1 itself or the guanine nucleotide exchange factor C3G. Significantly, this pathway also appears to function in the regulation of proliferation of neuroblastoma cells such as SK-N-SH and SH-SY5Y, because abrogation of Rap1 activity by Rap1-specific siRNA or overexpression of Rap1GAP reduces cellular growth. These results suggest that ALK activation of Rap1 may contribute to cell proliferation and oncogenesis of neuroblastoma driven by gain-of-function mutant ALK receptors.

Place, publisher, year, edition, pages
Nature Publishing Group, 2010
Keyword
ALK, PC12 cells, neurite outgrowth, small
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-35795 (URN)10.1038/onc.2010.27 (DOI)000277591900007 ()20190816 (PubMedID)
Available from: 2010-09-06 Created: 2010-09-03 Last updated: 2017-12-12Bibliographically approved
2.
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3. Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684
Open this publication in new window or tab >>Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684
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2011 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 440, 405-413 p.Article in journal (Refereed) Published
Abstract [en]

Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.

Keyword
anaplastic lymphoma kinase (ALK), cancer, Crizotinib, gain-of-function mutation, neuroblastoma, NVP-TAE684
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:nbn:se:umu:diva-51470 (URN)10.1042/BJ20101796 (DOI)000298440300012 ()
Available from: 2012-01-24 Created: 2012-01-23 Last updated: 2017-12-08Bibliographically approved
4.
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5. Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells
Open this publication in new window or tab >>Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells
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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.

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:nbn:se:umu:diva-53961 (URN)10.1038/onc.2012.12 (DOI)22286764 (PubMedID)
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved

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