Differential effects of X-ALK fusion proteins on proliferation, transformation, and invasion properties of NIH3T3 cells

Abstract

Majority of anaplastic large-cell lymphomas (ALCLs) are associated with the t(2;5)(p23;q35) translocation, fusing the NPM (nucleophosmin) and ALK (anaplastic lymphoma kinase) genes (NPM-ALK). Recent studies demonstrated that ALK may also be involved in variant translocations, namely, t(1;2)(q25;p23), t(2;3)(p23;q21), t(2;17)(p23;q23) and inv(2)(p23q35), which create the TPM3-ALK, TFG-ALK5, CLTC-ALK, and ATIC-ALK fusion genes, respectively. Although overexpression of NPM-ALK has previously been shown to transform fibroblasts, the transforming potential of variant X-ALK proteins has not been precisely investigated. We stably transfected the cDNAs coding for NPM-ALK, TPM3-ALK, TFG-ALK, CLTC-ALK or ATIC-ALK into nonmalignant NIH3T3 cells. All X-ALK variants are tyrosine phosphorylated and their subcellular distribution was in agreement with that observed in tumors. Moreover, our results show that the in vitro transforming capacity of NIH3T3-transfected cells are in relation to the level of X-ALK fusion proteins excepted for TPM3-ALK for which there is an inverse correlation. The differences between the five X-ALK variants with regard to proliferation rate, colony formation in soft agar, invasion, migration through the endothelial barrier and tumorigenicity seem to be due to differential activation of various signaling pathways such as PI3-kinase/AKT. These findings may have clinical implications in the pathogenesis and prognosis of ALKpositive ALCLs.

Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK

“…Other less frequent ALK fusion partners, e.g., tropomyosin-3 and clathrin heavy chain, have also been identified in ALCL as well as in CD30-negative diffuse large-cell lymphoma (1,2,7,8). Despite subtle differences in signaling and some biological functions, all fusions appear to be transforming to fibroblasts and hematopoietic cells (9). ALK fusion proteins have also been detected in a rare form of malignancy called inflammatory myofibroblastic tumor (10).…”

Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas

“…Inactivation of NPM-ALK resulted in a marked decrease in Rac1 activation (Figure 1a). Second, we examined Rac1 in cell lines of independent origin (NIH-3T3 fibroblasts and Ba/F3 lymphoid cells) that were demonstrated to become transformed by stable expression of ALK oncogenic fusions (Armstrong et al, 2004). NPM-ALK-dependent Rac1 activation was again observed (Figures 1b and c).…”

HuR-Mediated Control of C/EBPβ mRNA Stability and Translation in ALK-Positive Anaplastic Large Cell Lymphomas

“…1B, bottom). Similarly, in a murine model of ALKtransformed cells where NIH-3T3 cells stably expressed the NPM-ALK fusion protein (16), C/EPBb mRNA was found to be more stable in NPM-ALK-expressing cells (ALK þ ) than in the parental (ALK À ) NIH-3T3 cells (t 1/2 of %90 min vs. 55 minutes; Supplementary Fig. S1).…”

“…These cell lines were grown in Iscove's modified Dulbecco's medium (IMDM) supplemented with 15% fetal calf serum (FCS). Stable empty vector or NPM-ALK-transfected NIH-3T3 fibroblast clones were obtained and cultivated in Dulbecco's modified Eagle's medium (DMEM) as stated (16). Actinomycin D (Sigma-Aldrich) was added to a final concentration of 3 mg/mL for the indicated times.…”