The Tyrosine Phosphatase Shp2 Interacts with NPM-ALK and Regulates Anaplastic Lymphoma Cell Growth and Migration

Abstract

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK K210R , formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration. [Cancer Res 2007;67(9):4278-86]

ALK fusion genes in children with atypical myeloproliferative leukemia

“…Importantly, these mutant proteins were as efficient as wt IKAROS at rescuing abnormal phenotypes from homozygous mutant mice, suggesting that the IK L allele is mainly, if not exclusively, a loss of function allele. 7,8 Two cohorts of BCR-ABL þ compound mice were generated that carried either two alleles of wt IKZF1 (BCR-ABL þ /0 ; IK þ / þ ), or one wt and one mutant IKZF1 allele (BCR-ABL þ /0 ; IK L/ þ ), which were compared for leukemia development. Nontransgenic IK þ / þ and IK L/ þ littermates were included as controls.…”

“…Of note, a recent massspectrometry approach identified PTPN11 as a substrate of NPM/ALK. 7 (c) G-banded chromosomes 2 and FISH analysis of patient 6 using the VYSIS LSI ALK Dual Color, Break Apart Probe and chromosome arm specific (CAP) for 2p (green) and 2q (red) probes, respectively. In interphases, one fusion and a red and a green signal were found; metaphases showed that the 3 0 ALK signal (red) was translocated on the q-arm of the other chromosome 2 and CAP probes also confirm the translocation t(2;2).…”

Reactive oxygen species and lipoxygenases regulate the oncogenicity of NPM-ALK-positive anaplastic large cell lymphomas

“…However, ROS production has never been investigated downstream of ALK. As signaling molecules, ROS were shown to oxidize critical targets for cell invasion, such as PTPs, MAPK, phosphate and tensin homolog (PTEN) or the Rac effector p21-activated kinase (PAK, proteins that are also affected by NPM-ALK (Honorat et al, 2006;Wu, 2006;Voena et al, 2007). We therefore sought to analyse the levels of ROS in NPM-ALK( þ ) cells.…”

Critical role of SHP2 (PTPN11) signaling in germinal center-derived lymphoma

“…34,35 Overexpression of SHP2 is also detected in samples from patients with leukemia and diffuse large B-cell lymphoma. [36][37][38] However, the relative expression and functional impact of SHP2 in B-cell lymphomagenesis remains unknown.…”

“…Immunoblotting and immunoprecipitation analysis were performed as described previously. 38 The electrophoretic mobility shift assay (EMSA) was conducted with 32 P-dATP-labeled oligonucleotide probes (Online Supplementary Table S2) and the products were separated on a native polyacrylamide gel.…”