Oncogenic STAT5 signaling promotes oxidative stress in chronic myeloid leukemia cells by repressing antioxidant defenses

Abstract

STAT5 transcription factors are frequently activated in hematopoietic neoplasms and are targets of various tyrosine kinase oncogenes. Evidences for a crosstalk between STAT5 and reactive oxygen species (ROS) metabolism have recently emerged but mechanisms involved in STAT5-mediated regulation of ROS still remain elusive. We demonstrate that sustained activation of STAT5 induced by Bcr-Abl in chronic myeloid leukemia (CML) cells promotes ROS production by repressing expression of two antioxidant enzymes, catalase and glutaredoxin-1(Glrx1). Downregulation of catalase and Glrx1 expression was also observed in primary cells from CML patients. Catalase was shown not only to reduce ROS levels but also, to induce quiescence in Bcr-Abl-positive leukemia cells. Furthermore, reduction of STAT5 phosphorylation and upregulation of catalase and Glrx1 were also evidenced in leukemia cells co-cultured with bone marrow stromal cells to mimic a leukemic niche. This caused downregulation of ROS levels and enhancement of leukemic cell quiescence. These data support a role of persistent STAT5 signaling in the regulation of ROS production in myeloid leukemias and highlight the repression of antioxidant defenses as an important regulatory mechanism.

Keywords: Bcr-Abl; STAT5; antioxidants; chronic myeloid leukemia; oxidative stress.

Figure 1. Bcr-Abl induces ROS production in leukemia cells

A. Lysates from KU812 and K562 cells treated (+) or not (−) with 1 μM of IM for 15 h were analyzed by Western blotting with indicated antibodies. Results are the mean of 3 independent experiments. B. Statistical analysis showing relative ROS levels (% of control) detected in KU812 and K562 cells treated or not (Co) with 1 μM IM for 15 h. Cells were incubated with the ROS sensitive fluorescent dye H2DCFDA (5 μM) and intracellular ROS levels were monitored by flow cytometry (n=11 data are mean ± SEM,*** p <0.001). C. Statistical analysis showing relative ROS levels (% of control) detected in KU812 and K562 cells treated or not (Co) with 1 μM IM for 15 h and stained with the ROS sensitive probe CellROX (n=3, data are mean ± SEM. *p<0.05; **p<0.01).

Figure 2. STAT5 promotes ROS production in Bcr-Abl+ leukemia cells

A. KU812, K562 cell lines were transfected with a dominant negative Flag-STAT5AΔ749-ΔCD4 (Δ5A) bicistronic construct or empty vector (Co). Cells were next incubated with H2DCFDA (5μM) and APC-conjugated anti-CD4 antibody to determine ROS levels in CD4⁺ transfected cells. (n=3, data are mean ± SEM, * p <0.05). B. Extracts from transfected KU812 and K562 cells were prepared and analyzed by Western blotting with an anti-Flag antibody to verify expression of the dominant negative STAT5AΔ749 mutant (Δ5A). Actin served as a loading control (α-actin). C. KU812 and K562 cells were transfected with shST5/GFP or control shLuc/GFP vectors. Cell lysates were prepared 3 days after transfection and analyzed by Western blotting with indicated antibodies (n=3).D. Quantification of Western blot (ImageJ software) was performed to determine the relative expression of STAT5 (ratio STAT5/actin) in cells transfected with shST5 or shLuc expression vectors (n=3). E. KU812 and K562 cells transfected with shST5/GFP or control shLuc/GFP vectors were stained with CellROX at 3 days post-transfection to quantify ROS levels in GFP⁺ cells (n=5, data are mean ± SEM, *p<0.05; **p<0.01).

Figure 3. STAT5-dependent repression of Catalase and GLRX1 expression in CML cells

A. qRT-PCR analysis of CAT, GLRX1, CISH and PIM1 transcripts in KU812 (left) and K562 (right) cells treated or not with IM (1μM) for 15 h. Results are presented as the fold changes in gene expression in IM-treated cells normalized to internal control genes (GAPDH and ACTB) and relative to untreated cells (normalized to 1) (n=3 in triplicates, data are mean +/− SEM. **p<0.01; *p<0.05). B. Protein extracts from KU812 and K562 cells treated or not with IM (1μM) were analyzed by Western blotting to detect catalase and Glrx1 protein expression. Actin served as a loading control. (n=3). C. qRT-PCR analysis of CAT and GLRX1 expression in leukemia cells from CML patients (n=35) and peripheral mononuclear (PMN) cells from healthy donors (n=10). D. Levels of catalase and Glrx1 proteins in KU812 and K562 cells transfected with shST5/GFP or shLuc/GFP vectors were also determined by Western blot analysis (n=3).

Figure 4. Tyrosine-phosphorylated STAT5 induces ROS production and inhibits catalase and Glrx1 expression in Ba/F3 cells

A. Extracts prepared from Ba/F3 cells stimulated or not with IL-3 and from Ba/F3 cells stably expressing STAT5A1*6 were analyzed by Immunoblotting with indicated antibodies. Results are the mean of 3 independent experiments. B. Representative flow cytometry histogram of Ba/F3 cells stimulated (+IL-3) or not (−IL-3) with IL-3 and Ba/F3 cells expressing STAT5A1*6 mutant. Cells were incubated with the ROS sensitive fluorescent probe H2DCFDA (5 μM) and intracellular ROS levels were determined by flow cytometry. C. Statistical analysis showing relative ROS levels (% of control) detected in Ba/F3 cells stimulated or not with IL-3 and Ba/F3 cells expressing STAT5A1*6 (n=7, data are mean ± SEM. *p<0.05). D. qRT-PCR analysis of cat, glrx1, cish and pim1 transcriptsin Ba/F3 cells transformed by constitutively active STAT5A1*6 mutant and control Ba/F3 cells grown in presence or absence of IL-3 (4h starvation). Results are presented as fold changes in gene expression in Ba/F35A1*6 and control Ba/F3 cells (+IL-3) normalized to internal control genes (gapdh) and relative to control Ba/F3 cells (−IL-3) normalized to 1 (n=3 in triplicates, data are mean +/− SEM. ***p<0.001; **p<0.01; *p<0.05). E. Expression of catalase and Glrx1 in Ba/F35A1*6 and control Ba/F3 cells grown in the presence or not of IL-3 (4hr starvation) as determined by Western blot analysis. Actin served as a loading control (n=3).

Figure 5. Effects of catalase and Glrx1 on ROS production and proliferation of Bcr-Abl+ leukemia cells

A. KU812 and K562 cells were transfected with Glrx1/GFP (Glrx1) or empty/GFP (Co) expression vectors. At 2 days post transfection, cells were stained with CellROX to detect ROS levels in GFP⁺ cells (n=3, data are mean ± SEM. * p <0.05). B. Extracts from transfected cells were subjected to Western blot analysis to evaluate expression of the Flag-Glrx1 protein with an anti-Flag antibody. Actin served as loading control. C. KU812 and K562 cells were cultured for 48h in the presence or not of catalase (0.5 mg/ml). Cells were then stained with H2DCFDA to measure intracellular ROS levels (n=3, data are mean ± SEM. ** p <0.01). D. Growth kinetics of KU812 and K562 cells cultured in absence (Co) or presence of catalase (0.5 mg/ml) were determined by MTT assays (n=3 in triplicates, data are mean ± SEM *p <0.05; **p <0.01;***pp<0.001). E. KU812 cells exposed or not (Co) to catalase were stained with 7-amino-actinomycin D (7-AAD) and an Alexa Fluor H488-conjugated anti-Ki67 antibody. Cell cycle phase distributions were then estimated by flow cytometry. One representative experiment is shown. F. The histogram presents the percentage of KU812 cells exposed or not (Co) to catalase in sub-G1 (apoptotic fraction) and in each phase of the cell cycle. (n=3, data are mean ± SEM. ** p <0.01;* p<0.05)

Figure 6. Contact with stromal cells promotes quiescence and reduction of STAT5-mediated oxidative stress in Bcr-Abl+ leukemia cells

A. KU812 (10⁵ cells/ml) cells were cultured alone in medium (Co) or in HS-27A conditioned medium (+CM HS-27A) or on HS-27A cell monolayers for 72 h (+HS-27A). Cells were stained with 7-AAD and an Alexa Fluor H488-conjugated anti-Ki67 antibody. Concomitant staining with an APC-conjugated anti-CD45 antibody was performed to distinguish leukemic and stromal cells. Cell cycle phase distributions were estimated by flow cytometry. The histogram presents the percentage of leukemic cells (CD45⁺ cells) in the sub-G1 (apoptotic fraction) and cell cycle phases (n=3, data are mean ± SEM. *p<0.05). B. Cells were stained with the ROS sensitive dye H2DCFDA (5 μM) and an APC-conjugated anti CD45 antibody. ROS levels were then determined in leukemic cells (CD45⁺ cells) by FACS analysis (n=3 data are mean ± SEM. * p <0.05). C. KU812 cells cultured alone without (Co) or with HS-27A conditioned medium (+CM HS-27A) or on HS-27A cell monolayers (+HS-27A) were isolated using an immunomagnetic CD45 selection kit. KU812 cell extracts were then prepared and subjected to Western blot analysis with indicated antibodies. (n=3). D. Hypothetical model for the role of STAT5 as an inducer of ROS production in CML cells. The pro-oxidant activity of STAT5 is regulated by tyrosine phosphorylation. Constitutive activation of STAT5 (P-Y-STAT5) promotes oxidative stress by repressing expression of catalase and Glrx1. Dephosphorylation of STAT5 might allow the re-expression of catalase and Glrx1 and the decrease of ROS levels in leukemic cells.