Forced expression of the DEK-NUP214 fusion protein promotes proliferation dependent on upregulation of mTOR - PubMed (original) (raw)

Forced expression of the DEK-NUP214 fusion protein promotes proliferation dependent on upregulation of mTOR

Carl Sandén et al. BMC Cancer. 2013.

Abstract

Background: The t(6;9)(p23;q34) chromosomal translocation is found in 1% of acute myeloid leukemia and encodes the fusion protein DEK-NUP214 (formerly DEK-CAN) with largely uncharacterized functions.

Methods: We expressed DEK-NUP214 in the myeloid cell lines U937 and PL-21 and studied the effects on cellular functions.

Results: In this study, we demonstrate that expression of DEK-NUP214 increases cellular proliferation. Western blot analysis revealed elevated levels of one of the key proteins regulating proliferation, the mechanistic target of rapamycin, mTOR. This conferred increased mTORC1 but not mTORC2 activity, as determined by the phosphorylation of their substrates, p70 S6 kinase and Akt. The functional importance of the mTOR upregulation was determined by assaying the downstream cellular processes; protein synthesis and glucose metabolism. A global translation assay revealed a substantial increase in the translation rate and a metabolic assay detected a shift from glycolysis to oxidative phosphorylation, as determined by a reduction in lactate production without a concomitant decrease in glucose consumption. Both these effects are in concordance with increased mTORC1 activity. Treatment with the mTORC1 inhibitor everolimus (RAD001) selectively reversed the DEK-NUP214-induced proliferation, demonstrating that the effect is mTOR-dependent.

Conclusions: Our study shows that the DEK-NUP214 fusion gene increases proliferation by upregulation of mTOR, suggesting that patients with leukemias carrying DEK-NUP214 may benefit from treatment with mTOR inhibitors.

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Figures

Figure 1

Figure 1

Expression of the DEK-NUP214 fusion gene. Expression of DEK-NUP214 mRNA by quantitative real-time PCR in the generated stable clones of (A) U937 and (B) PL-21, as well as (C) a patient sample expressing the fusion gene. Values were calculated relative to the expression of the endogenous control, which did not differ between the clones. N.d. denotes not detected.

Figure 2

Figure 2

DEK-NUP214 promotes proliferation. Proliferation of (A) U937 and (B) PL-21 cells. Viability of the (C) U937 and (D) PL-21 cell cultures, as determined by trypan blue dye exclusion. (E) Proliferation of U937 cells expressing deletion mutants of DEK-NUP214. Mean values were calculated from three independent experiments, containing all clones. Error bars represent S.E.M.

Figure 3

Figure 3

DEK-NUP214 increases the expression of mTOR. A. Phosphorylated and total protein levels for components of the mTOR signaling pathway in U937 cells one day after seeding, showing increased levels of mTOR and activation of the mTORC1 substrate p70S6K without activation of the mTORC2 substrate Akt. α-tubulin is used as an equal loading control. Images show one representative experiment out of three. B. Protein abundance levels of cells expressing DEK-NUP214, relative to control cells. The mean of three experiments, error bars represent S.E.M. C. Expression of the mTOR gene in DEK-NUP214 cells relative to control cells. The mean of three experiments, error bars represent S.E.M.

Figure 4

Figure 4

DEK-NUP214 promotes translation. Global protein synthesis as assayed by the incorporation of radioactively labeled amino acids into newly synthesized protein in U937 cells. All values are normalized to the level of the control cells one day after seeding. Bars represent the mean values of three experiments. Error bars represent S.E.M.

Figure 5

Figure 5

DEK-NUP214 alters the balance between glycolysis and oxidative phosphorylation. Relative concentrations of accumulated lactate and remaining glucose in cell supernatant three days after seeding, showing decreased glycolysis but sustained glucose consumption, indicating increased oxidative phosphorylation in U937 cells expressing DEK-NUP214. Bars represent mean values from three experiments, normalized to control cells. Error bars represent S.E.M.

Figure 6

Figure 6

The proliferative effect of DEK-NUP214 depends on mTOR. A. Proliferation as determined by U937 cell density three days after seeding and with daily additions of the mTORC1 inhibitor everolimus. Viability was unaffected by the treatment. Bars represent mean values from four experiments. Error bars represent S.E.M. B. Potency of the mTORC1 inhibitor everolimus, as determined by reduced phosphorylation of the direct mTORC1 target p70 S6 kinase.

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