NDRG2 Sensitizes Myeloid Leukemia to Arsenic Trioxide via GSK3β-NDRG2-PP2A Complex Formation

Abstract

N-Myc downstream-regulated gene 2 (NDRG2) was characterized as a tumor suppressor, inducing anti-metastatic and anti-proliferative effects in several tumor cells. However, NDRG2 functions on anticancer drug sensitivity, and its molecular mechanisms are yet to be fully investigated. In this study, we investigated the mechanism of NDRG2-induced sensitization to As₂O₃ in the U937 cell line, which is one of the most frequently used cells in the field of resistance to As₂O₃. NDRG2-overexpressing U937 cells (U937-NDRG2) showed a higher sensitivity to As₂O₃ than mock control U937 cell (U937-Mock). The higher sensitivity to As₂O₃ in U937-NDRG2 was associated with Mcl-1 degradation through glycogen synthase kinase 3β (GSK3β) activation. Inhibitory phosphorylation of GSK3β was significantly reduced in U937-NDRG2, and the reduction was diminished by okadaic acid, a protein phosphatase inhibitor. NDRG2 mediated the interaction between GSK3β and protein phosphatase 2A (PP2A), inducing dephosphorylation of GSK3β at S9 by PP2A. Although the C-terminal deletion mutant of NDRG2 (ΔC NDRG2), which could not interact with PP2A, interacted with GSK3β, the mutant failed to dephosphorylate GSK3β at S9 and increased sensitivity to As₂O₃. Our findings suggest that NDRG2 is a kind of adaptor protein mediating the interaction between GSK3β and PP2A, inducing GSK3β activation through dephosphorylation at S9 by PP2A, which increases sensitivity to As₂O₃ in U937 cells.

Keywords: Mcl-1; PP2A–NDRG2–GSK3β complex; U937; apoptosis; arsenic trioxide; myeloid leukemia.

Figure 1

N-Myc downstream-regulated gene 2 (NDRG2) overexpression sensitized U937 cells to As₂O₃ in a caspase-dependent manner. (A) U937-Mock and three U937-NDRG2 lines were incubated with As₂O₃ (2 μM, 24 h). The cells were stained with Annexin V/propidium iodide (PI) and analyzed by flow cytometry. (B) The cells were incubated with As₂O₃ at the indicated time and concentration, and apoptotic cell population was validated with Annexin V/PI staining. (C) Here, 2 μM As₂O₃ was treated in the presence or absence of zVad-fmk (Pan-caspase inhibitor, 50 μM). The apoptotic population was validated with Annexin V/PI staining. (D) U937-Mock and U937-NDRG2 cells treated with As₂O₃ at the indicated dose. The cleavages (Cf) of Caspase3 and PARP were analyzed by immunoblotting. * is non-specific band. ** p < 0.01, *** p < 0.005 determined from t-tests. Data are presented as means ± standard error of the mean (SEM).

Figure 2

The sensitivity of U937-NDRG2 to As₂O₃ is mediated through Mcl-1 degradation. (A) Expression levels of pro-apoptotic Bcl-2 family protein (Bax) and pro-survival Bcl-2 family protein were analyzed by immunoblotting. (B) U937-Mock was infected by a lentivirus expressing scr or short hairpin Mcl-1 (shMcl-1) (clone #4 and #5). Successful knockdown of Mcl-1 was confirmed by immunoblotting, and apoptotic rate was analyzed using flow cytometry. (C) Mcl-1 messenger RNA (mRNA) expression in U937-Mock and U937-NDRG2 in the presence or absence of As₂O₃ was validated by qRT-PCR. (D) Cells were treated with As₂O₃ in the presence or absence of MG132, and then protein level of Mcl-1 was confirmed by immunoblotting. N.S.: Non-significant. * p < 0.05, *** p < 0.005 determined from t-test. Data are presented as means ± SEM.

Figure 3

As₂O₃-induced glycogen synthase kinase 3β (GSK3β) activation regulates Mcl-1 degradation and apoptosis in U937-NDRG2 cells. (A) Kinetics of phosphorylated GSK3β (Serine 9) in U937-Mock and U937-NDRG2 was checked after treatment with 2 μM As₂O₃ at the indicated time. Band intensity of phosphorylated GSK3β to total GSK3β was quantified using Image J program and presented graphically. U937-NDRG2 was treated with As₂O₃ in the presence or absence of a GSK3β inhibitor, SB216763 (10 μM). Cell death was determined using PI staining (B), and mitochondrial depolarization rate was done through Mitotracker CMXRos staining (C). The level of the indicated proteins was analyzed using immunoblotting (D). ** p < 0.01, *** p < 0.005 determined from t-test. Data are presented as means ± SEM.

Figure 4

GSK3β activation observed in U937-NDRG2 is mediated by phosphatase, PP2A, not by its upstream kinase AKT. (A) The phosphorylation of AKT on Thr308 was determined by immunoblotting using cell lysates of As₂O₃-treated U937-Mock and U937-NDRG2 cells at the indicated time points. (B) U937-NDRG2 cells were treated with As₂O₃ in the presence or absence of a phosphatase inhibitor, okadaic acid (20 nM). Protein levels of phosphorylated GSK3β and Mcl-1 were analyzed by immunoblotting. (C) PP2A activity acquired from U937-Mock or U937-NDRG2 treated with As₂O₃ was analyzed with Human/Mouse/Rat Total PP2A DuoSet IC ELISA. For the quantification of PP2Ac protein level, total lysate was subjected to immunoblotting. N.S. no significance determined from t-test. Data are presented as means ± SEM. (D) GST or GST–NDRG2 with HA–GSK3β was co-transfected in HEK293. Protein lysates acquired from the cells were subjected to GST pull-down assay. Whole-cell lysate (WCL) was used for loading control. (E) Mock or HA–GSK3β with GST–NDRG2 was co-expressed in HEK293. Protein lysates acquired from the cells were subjected to immunoprecipitation with HA antibody and then interaction was confirmed by immunoblotting. (F) Protein lysates from HEK293 expressing HA–GSK3β with GST or GST–NDRG2 was immunoprecipitated by HA antibody. Interactions among the indicated proteins were analyzed by immunoblotting.

Figure 5

NDRG2 interacts with PP2Ac through its C-terminal, and NDRG2 ΔC is incapable of sensitizing U937-NDRG2 to As₂O₃. (A) NDRG2 wild type (WT) or NDRG2 ΔC was co-expressed with HA–GSK3β in HEK293. Prepared protein lysates were subjected to GST pull-down assay and the interaction was confirmed by immunoblotting. (B) U937-NDRG2 WT and U937-NDRG2 ΔC cells were treated with As₂O₃. Expressions of the indicated proteins were analyzed by immunoblotting. (C) U937-Mock, U937-NDRG2, and U937-NDRG2 ΔC were incubated with 2 μM As₂O₃ for 24 h. The apoptotic rate was analyzed by Annexin V/PI staining. ** p < 0.01 determined using t-test. Data are presented as means ± SEM.

Figure 6

Diagrammatic representation of the proposed apoptotic pathway that mediates the higher sensitivity to As₂O₃ in NDRG2-expressing U937 cells.