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Molecular and functional characterizations of the association and interactions between nucleophosmin-anaplastic lymphoma kinase and type I insulin-like growth factor receptor.

ABSTRACT: Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is aberrantly expressed in a subset of T cell lymphoma that commonly affects children and young adults. NPM-ALK possesses significant oncogenic potential that was previously documented using in vitro and in vivo experimental models. The exact mechanisms by which NPM-ALK induces its effects are poorly understood. We have recently demonstrated that NPM-ALK is physically associated with type I insulin-like growth factor receptor (IGF-IR). A positive feedback loop appears to exist between NPM-ALK and IGF-IR through which these two kinases interact to potentiate their effects. We have also found that a single mutation of the Tyr(644) or Tyr(664) residue of the C terminus of NPM-ALK to phenylalanine decreases significantly, but does not completely abolish, the association between NPM-ALK and IGF-IR. The purpose of this study was to determine whether the dual mutation of Tyr(644) and Tyr(664) abrogates the association and interactions between NPM-ALK and IGF-IR. We also examined the impact of this dual mutation on the oncogenic potential of NPM-ALK. Our results show that NPM-ALK(Y644,664F) completely lacks association with IGF-IR. Importantly, we found that the dual mutation of Tyr(644) and Tyr(664) diminishes the oncogenic effects of NPM-ALK, including its ability to induce anchorage-independent colony formation and to sustain cellular transformation, proliferation, and migration. Furthermore, the association between NPM-ALK and IGF-IR through Tyr(644) and Tyr(664) appears to contribute to maintaining the stability of NPM-ALK protein. Our results provide novel insights into the mechanisms by which NPM-ALK induces its oncogenic effects through interactions with IGF-IR in this aggressive lymphoma.

SUBMITTER: Shi B

PROVIDER: S-EPMC3664999 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found exclusively in a subset of ALK-positive anaplastic large cell lymphoma, promotes tumorigenesis by exerting its constitutively active tyrosine kinase activity. Thus, characterization of the NPM-ALK-induced changes in the phosphoproteome will likely provide insights into the biology of this oncoprotein. To achieve this goal, we used a strategy of combining sequential affinity purification of phosphopeptides and LC/MS. GP293 cells transfected with either NPM-ALK or an NPM-ALK mutant with decreased tyrosine kinase activity (negative control) were used. We identified 506 phosphoproteins detectable in NPM-ALK-expressing cells but not in the negative control. Bioinformatics analysis revealed that these phosphoproteins carry a wide diversity of biological functions, some of which have not been described in association with NPM-ALK, such as the tumor necrosis factor (TNF)/Fas/tumor necrosis factor-related apoptosis-induced ligand (TRAIL) signaling pathway and the ubiquitin proteasome degradation pathway. In particular, modulations of the TNF/Fas/TRAIL pathway by NPM-ALK were supported by our antibody microarray data. Further validation of the TNF/Fas/TRAIL pathway was performed in ALK(+) anaplastic large cell lymphoma (ALCL) cell lines with knockdown of NPM-ALK using short interference RNA, resulting in the loss of the tyrosine phosphorylation of tumor necrosis factor receptor-associated protein 1 (TRAP1) and receptor-interacting protein 1, two crucial TNF signaling molecules. Functional analyses revealed that knockdown of TRAP1 facilitated cell death induced by TRAIL or doxorubicin in ALK(+) ALCL cells. This suggests that down-regulation of TRAP1 in combination with TRAIL or doxorubicin might be a potential novel therapeutic strategy for ALK(+) ALCL. These findings demonstrated that our strategy allowed the identification of novel proteins downstream of NPM-ALK that contribute to the maintenance of neoplastic phenotype and holds great potential for future studies of cellular tyrosine kinases in normal states and diseases.

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Molecular and functional characterizations of the association and interactions between nucleophosmin-anaplastic lymphoma kinase and type I insulin-like growth factor receptor.

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