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Biology of Human Tumors

Jab1/Csn5–Thioredoxin Signaling in Relapsed Acute Monocytic Leukemia under Oxidative Stress

Fuling Zhou, Yunbao Pan, Yongchang Wei, Ronghua Zhang, Gaigai Bai, Qiuju Shen, Shan Meng, Xiao-Feng Le, Michael Andreeff and Francois X. Claret
DOI: 10.1158/1078-0432.CCR-16-2426 Published August 2017

Abstract

Purpose: High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway.

Experimental Design: We studied ROS levels and conducted c-Jun activation domain–binding protein-1 (JAB1/COPS5) and thioredoxin (TRX) gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1′s regulation of Trx in leukemia cell lines.

Results: Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of TRX and JAB1/COPS5 were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivo.

Conclusions: We identified a novel Jab1–Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. Clin Cancer Res; 23(15); 4450–61. ©2017 AACR.

Translational Relevance

Increased production of reactive oxygen species (ROS) or an inefficient antioxidant system leads to oxidative stress, which influences hematopoietic cell function. However, the precise mechanisms of this oxidative stress remain poorly understood. Insight into how ROS signaling leads to the development of acute monocytic leukemia (AML) will offer novel therapeutic opportunities. We investigated gene expression under oxidative stress in leukemia cell lines and blood samples obtained from AML patients at primary diagnosis and compared those with AML cells from the same patients obtained at relapse in 60 matched patients. Moreover, high expression levels of Jab1 and thioredoxin (Trx) were associated with disease progression and poor prognosis in relapsed AML-M5. In addition, depletion of Jab1 inhibited cellular proliferation and invasion, which was accompanied by decreased Trx expression. Mechanistically, Jab1 interacted with and positively regulated Trx expression. Increased ROS levels stimulate aberrant gene expression and promote the proliferation of leukemic blasts. In this study, we identified Jab1 as a new target of the ROS pathway that is implicated to have a role in the relapse and progression of malignant AML-M5. Our findings provide significant insight into ROS signaling. Specifically, we demonstrated that Jab1 interacts with and positively regulates Trx expression under oxidative stress and plays an important role in the pathogenesis of AML-M5.

Introduction

Malignant acute monocytic leukemia (AML-M5, classified on the basis of the French–American–British classification system) is a subtype of AML that is associated with hyperleukocytosis, extramedullary disease, and abnormal coagulation. We previously reported that oxidative stress is involved in myeloid leukemogenesis, but the specific mechanism of this process remains unknown (1). The results of several studies have suggested that excessive reactive oxygen species (ROS) production or defects in antioxidant defense lead to oxidative stress and contribute to leukemogenesis and therapy failure (2, 3). ROS, such as hydrogen peroxide (H2O2) and superoxide anion radicals, are produced by the redox microenvironment and cellular metabolic processes (4). Many types of genetic alteration with ROS production have been identified in myeloid diseases (2, 5), and intracellular ROS regulation is essential for maintaining cellular homeostasis (6). Tumor-derived ROS have been implicated in cell survival, proliferation, and motility, drug resistance, genetic mutation and instability, and growth factor signaling in various cell types (7).

The thioredoxin (Trx) system is one of the major cellular antioxidant systems that is integral to maintaining intracellular redox homeostasis, reducing oxidized protein levels and protein-refolding activity to limit damage caused by oxidative stress, and controlling cellular proliferation, viability, and apoptosis (8). Trx is localized in the cytoplasm and is mainly released from blood monocytes and transformed leukocytes (9). Increased levels of Trx increase the proliferation of cancer cells, inhibit apoptosis, and promote the growth of aggressive tumors. Increases in the protein expression of the antioxidant Trx (1, 10) have been shown in acute leukemia. Elevations in antioxidants may contribute to an imbalance in the redox status of leukemia cells, leading to oxidative stress. Furthermore, elevated antioxidant levels allow leukemia cells to survive otherwise lethal alterations in ROS. Inhibiting Trx can induce antileukemic effects (11). Identifying new Trx-interacting proteins will improve our current understanding of redox-dependent cellular events.

c-Jun activation domain-binding protein-1 (Jab1) was originally identified by our research group as a potent AP-1 coactivator (12) and then identified by other groups as a member of the COP9 signalosome complex fifth sub-unit (Csn5; ref. 13). Jab1 functions as a critical regulator of intracellular signaling and affects cellular proliferation and apoptosis by functionally inactivating several key negative regulatory proteins and tumor suppressors (14). Jab1 also stabilizes certain proteins, such as hypoxia-inducible factor 1α and c-Jun, and potentiates c-Jun and MYC transcription (12, 15), which is responsible for the transcriptional activation of genes involved in cell proliferation, angiogenesis, and invasion (14). Our previous studies and those of others have demonstrated that Jab1 plays integral roles in DNA damage response, DNA repair, and carcinogenesis (16, 17). However, to date, no study has demonstrated the contribution of ROS signaling, through Jab1, to malignant transformation in relapsed AML-M5. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway.

Materials and Methods

Patients

This study was approved by the Xi'an Jiaotong University Ethics and Scientific Committee and meets international standards for informed consent. From February 2006 to January 2013, we recruited patients from the Department of Clinical Hematology at the Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, Shaanxi, China). Patients had mainly been treated with standard dose cytarabine (100–200 mg/m2/day; days 1–7) and continuous infusions of mitoxantrone (4–8 mg/m2/day) or with daunorubicin (45 mg/m2/day; days 1–7) for two cycles. Patients who experienced a response then received high or intermediate doses of cytarabine as postinduction therapy for four cycles and other treatments, including allogeneic hematopoietic stem cell transplantation if available. Consolidation and maintenance therapies usually included standard-dose cytarabine and daunorubicin. All patients who experienced relapse (≥5% leukemic blasts in the bone marrow or recurrent extramedullary infiltration after experiencing a complete remission) were included in our study.

The exclusion criteria were uncontrolled infections, other malignancies (including lung cancer), diabetes, and thyroid dysfunctions. All patients with relapsed AML-M5 were initially identified as newly diagnosed disease and were followed up in this study. We collected heparinized blood samples from all AML-M5 patients before treatment and at relapse. For controls, we recruited sex- and age-matched healthy donors from the same hospital. Biological samples were blinded for research analysis.

Cell culture

Human AML cells (U937, THP-1, MOLM-13, HL-60, and KG-1) and 293T cells were obtained from ATCC and have been inspected for phenotypic variation and mycoplasma contamination. Cells were grown in RPMI1640 medium (U937, THP-1, and MOLM-13), IMDM (KG-1 and HL-60), or DMEM (293T), supplemented with 10% FBS and l-glutamine (2 mmol/L). Normal HS-5 bone marrow cells (adherent cells, a generous gift from Professor Xiang Li, Jiangnan University, Jiangsu, China) were grown in RPMI1640 medium. Cell lines were maintained at 2–10 × 105 cells/mL, and cell viability was analyzed before each assay using a Trypan blue dye exclusion assay (viability > 96%).

ROS level quantification

Peripheral blood mononuclear cell samples obtained from AML-M5 patients (blasts > 60%) at diagnosis or at relapse were isolated from heparinized venous blood using Ficoll sodium diatrizoate gradient centrifugation (Sigma-Aldrich). CD34+ cells originating from healthy donors served as the controls. Samples were sublimated using the immunomagnetic cell sorting system (Miltenyi Biotec). Intracellular ROS levels were measured using 2, 7-dichlorodihydrofluorescein diacetate (DCF-DA; Sigma-Aldrich). In brief, cells were washed with serum-free RPMI1640 and incubated in DCF-DA (40 μmol/L) for 20 minutes at 37°C to assess ROS-mediated oxidation of the fluorescence compound DCF-DA. The fluorescence intensity of oxidized DCF was measured using a flow cytometer (BD Biosciences), and the results were analyzed using FlowJo software (FlowJo).

ROS-related indices analysis

Pretreatment blood samples were collected in the early morning to avoid diurnal variations in levels of blood components and then centrifuged to extract the plasma. The total antioxidant capacity (T-AOC) was measured via the Fe3+ disoxidation (Fe3+ → Fe2+) method (18); superoxide dismutase (SOD) activity was determined according to the xanthine oxidase method (18); and catalase activity was evaluated by the ammonium molybdate method (19). Glutathione peroxidase (GSH-Px), succinate dehydrogenase, adenosine deaminase (ADA), and xanthine oxidase (XOD) levels and lactate dehydrogenase (LDH) leakage levels were determined by chemical colorimetry. Malondialdehyde (MDA) levels were estimated using the thiobarbituric acid reactivity assay. These plasma parameters were assayed according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing City, China).

RNA extraction and quantitative real-time PCR

Total RNA was extracted using the guanidinium thiocyanate method and purified with an mRNA Isolation Kit (Qiagen). Cellular RNA was primed using oligo (dT) primer and reverse transcribed with SuperScript II (Promega). Jab1 and Trx primers were purchased from ThermoFisher Scientific/Applied Biosystems. A real-time reverse transcription PCR (RT-PCR) was performed in triplicate using an ABI GeneAmp 7000 device.

Fluorescence-activated cell-sorting assays

For the immunofluorescence assays, cell lines or isolated leukemic cells were fixed in fixation buffer (BD Biosciences), incubated with monoclonal anti-Jab1 or monoclonal anti-Trx (1 μg/mL), and then analyzed immediately after being washed with PBS using a FACScan flow cytometer (BD Biosciences) and FlowJo software.

siRNA and DNA transfection

Cells were transfected using the Nucleofector Kit V from Amaxa Biosystems (Lonza). Gene expression was silenced with siRNAs from Santa Cruz Biotechnology that were provided as pools of three target-specific siRNAs designed to knock down expression of specific gene of interest, including Jab1 siRNA (catalog no. sc-35717), Trx siRNA (catalog no. sc-106984), and control siRNA oligonucleotides (catalog no. sc-37007). The HA/Flag-Trx plasmids and His-Jab1 were also transfected into U937 and THP-1 cells. Cell proliferation was determined using the CyQUANT NF cell proliferation assay (Invitrogen).

Establishment of short hairpin RNA–stable cells and the tumorigenicity assay in NOD-SCID mice

Short hairpin RNA (shRNA)-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer's instructions (Santa Cruz Biotechnology). Stable clones were selected after treating cells with puromycin (0.5 μg/mL) for 2 weeks. Positive clones were further confirmed via an immunoblot analysis and maintained in 0.2 μg/mL puromycin. Six-week-old NOD-SCID mice were obtained from Charles River Laboratories and given subcutaneous injections of 1 × 106 U937 cells with stable knockdown of Jab1 (sh-Jab1) or stable control vector (sh-Cont) in the left flank. Mice (5 mice/group) were observed every 3 days for palpable tumor development. Tumor volume (in cubic millimeters) was calculated as tumor length × tumor width2/2. At the end of the experiments, the mice were killed under anesthesia, the tumors were excised and weighed, and necropsies were performed. All animal experiments were carried out under protocols approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Xi'an Jiaotong University.

ELISA

Plasma cytochrome-c (Cyt-c) concentrations were measured using an ELISA kit (EIAab Science). Plasma 8-hydroxydeoxyguanosine (8-OHdG) levels were also assayed quantitatively using a StressXpress DNA-damage kit (Stressgen Bioreagents). Advanced oxidation protein products (AOPP) were detected as described previously (1). Secreted thioredoxins were detected using a Trx ELISA Kit (Novatein Biosciences).

Western blot analysis

We used cell lysates derived from U937 and THP-1 cells in the log phase of growth, leukemic cells from AML-M5 patients, and CD34+ cells from healthy donors. The membrane was probed with anti-Jab1 (Santa Cruz Biotechnology), anti-Trx (Abcam), Flag-Tag (Sigma-Aldrich), Nrf2, His-Tag, and HA-Tag (Cell Signaling Technology) antibodies and β-actin (Sigma-Aldrich) and visualized with a secondary antibody and Western Lightning Chemiluminescence Plus reagent (Thermo Scientific Pierce).

Coimmunoprecipitation assays

U937 and THP-1 cells were used to perform endogenous coimmunoprecipitation assays, as described previously (20). In brief, cell lysates were incubated in RIPA buffer. Supernatants were incubated overnight at 4°C with anti-Jab1 antibodies and protein G-sepharose or nonimmune mouse serum as a control. Proteins were separated by 12% SDS-PAGE, and immunoblotting was performed. The His-Jab1 and HA/Flag-Trx plasmids were transfected into 293T cells with the Lipofectamine Plus reagent (Invitrogen) at the indicated doses, and cells were lysed for Western blot analysis and coimmunoprecipitation assays.

Cell invasion, cell viability, and colony formation assays

Cell invasion assays were performed using Transwells coated with Matrigel (BD BioCoat Matrigel Invasion Chambers; BD Biosciences) inserted into a 24-well plate. The bottom chambers were filled with 600 μL of RPMI1640 medium containing 20% FBS as chemoattractant, and the top chamber of each Transwell was filled with 200 μL of serum-free medium supplementing treated or control cells (1 × 106 cells/mL). The number of migrated cells was counted after 24 hours using a fluorogenic DNA-binding dye CyQUANT NF cell proliferation assay kit (Invitrogen). Cell viability was determined using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) assay (1 × 104 cells/well) over a 72-hour period. Cells were labeled with MTT labeling reagent (0.5 mg/mL), and absorbance was determined on a spectrophotometric microplate reader (Bio-Rad). For the colony formation assay with AML cells, each well was seeded with 5,000 live cells in RPMI1640 medium containing soft agar, and colonies were allowed to form for 2 weeks until colony formation was visible. For the colony formation assay with HS-5 cells, cells were seed in 6-well plates (200 cells per well) with only RPMI1640 medium to form colonies. Colonies were counted after being stained with crystal violet (Sigma-Aldrich).

Luciferase reporter assay

293T cells were seeded in triplicate into 24-well plates at 5 × 104 cells/well 24 hours before transfection. In brief, the indicated plasmids were cotransfected with reporter plasmid pGL3-Trx-Luc containing the Trx promoter region (−975/+1) Trx-Luc (provided by Dr. Kathryn Tonissen, Griffith University, Nathan, Australia) and 10 ng of Renilla luciferase. Luciferase assays were performed 48 hours after transfection using a dual-luciferase reporter assay system (Promega). Firefly and Renilla luciferase activity was read on a Monolight 3010 luminometer (BD Biosciences). Firefly luciferase activity was normalized to Renilla luciferase readings in each well. Each experiment was conducted at least twice in triplicate.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays and a subsequent qPCR analysis were performed using the SimpleChIP Plus enzymatic chromatin IP kit (Cell Signaling Technology, catalog no. #9005). The comparative Ct method was used to determine relative expression compared with input antibodies against the proteins Jab1, c-Jun, and control IgG. The primer that included the AP-1 site was as follows: forward: 5′-GGACAACGGAGTCAAATTGCAG-3′, reverse: 5′-CCACAGCACCAATGCAGAGT-3′. The control primer was 2311 bp downstream of the AP-1 site: forward: 5′-CGGGGGTAGTGATCTCCCTT-3′; reverse: 5′-CCCCTTAGTGCGGATACAGC-3′.

Analysis of clinical mRNA microarrays for the detection of correlations between Jab1 and Trx

Transcriptome data from patient samples of AML were analyzed using the free online database R2: microarray analysis and visualization platform (http://r2.amc.nl) to determine whether the expression levels of Jab1 and Trx were correlated. We specifically used the tumor AML dataset (Delwel - 460 - MAS5.0 - u133p2; ref. 21) and selected the analysis with “2D gene overview (View 2 genes in many datasets)”.

Statistical analysis

Data are expressed as means ± SDs; the χ2 test was used to determine whether the distributions of categorical variables differed. Multivariate survival analyses were performed to identify independent factors for overall survival. A stratified overall survival analysis was performed using the Kaplan–Meier method, followed by the log-rank test. P < 0.05 was regarded as statistically significant. Calculations were performed using IBM SPSS statistics software 22.0.

Results

Clinical characteristics of AML-M5 patients

The study included matched samples from 60 patients with AML-M5 who experienced a relapse after treatment. These patients' clinical characteristics are shown in Supplementary Tables S1 and S2.

Comparison of ROS levels, antioxidant capacity, LDH levels, and oxidative damage in AML-M5 patients

We found that AML-M5 patients had significantly higher intracellular ROS levels at diagnosis and relapse than did 24 healthy donors (Fig. 1A). AML-M5 patients seemed to have the highest ROS levels among all the subtypes of leukemia patients (Supplementary Fig. S1A). Furthermore, ADA and XOD activities, which reflect high ROS production, were higher in patients with newly diagnosed and relapsed AML-M5 than in healthy individuals (Fig. 1A).

Figure 1.
Figure 1.

Comparison of ROS levels, antioxidant capacity, LDH levels, and accumulated oxidative damage in AML-M5 patients. A, Left, intracellular ROS levels were measured by flow cytometry after the cells had been incubated with the redox-sensitive fluorescent dye DCF-DA. The AML-M5 subtype is the most susceptible to oxidative stress, and these cells commonly express high intracellular ROS levels. Relative ROS levels in AML-M5 patients are indicated by the fluorescence intensities of DCF, expressed as a mean ± SD. The levels of oxidative stress were significantly higher at diagnosis and relapse (10.7- and 21.5-fold, respectively) than in healthy donors. ADA and XOD levels were measured using chemical colorimetric assays and used to measure ROS production. ADA and XOD activity in patients with newly diagnosed and relapsed AML-M5 was higher than that in healthy controls: 5.8-fold and 6.4-fold for ADA and 1.6-fold and 2.1-fold for XOD, respectively. Right, the antioxidant capacity levels of T-AOC, SOD, and GSH-Px were lower at diagnosis and relapse than in healthy donors. B, Left, the antioxidant capacity levels of CAT were lower at diagnosis and relapse than in healthy donors. Disease progression led to decreased regulation of antioxidant enzyme capacity in response to changing levels of oxidation. Elevated LDH levels were found in AML-M5 patients. Patients had higher LDH levels at relapse than did healthy donors. However, there were no significant difference in Cyt-c levels between healthy individuals and AML-M5 patients. Right, the accumulation of ROS in cells damages the resident proteins, lipids, and DNA. It is evident that the accumulation of AOPP, MDA, and 8-OhdG levels was characteristic of AML and reflected the AML-M5 stage. Data are means ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

In contrast, we observed a decrease in antioxidant enzyme capacity regulation in response to changes in oxidative stress levels. In particular, the levels of T-AOC, SOD, GSH-Px (Fig. 1A), and catalase (CAT; Fig. 1B) were significantly lower in AML-M5 patients than in 24 healthy donors. Most importantly, patients had higher LDH levels at relapse than at diagnosis and had higher LDH levels than did healthy individuals (Fig. 1B). However, no significant difference was observed in Cyt-c levels among these three measurements (Fig. 1B). There was also a significant increase in oxidative damage at relapse compared with that in healthy donors, as demonstrated by the presence of AOPPs, MDA, and 8-OHdG (Fig. 1B).

High expression levels of Jab1 and Trx in AML-M5 patients

Our data revealed that JAB1/COPS5 and TRX gene expression levels were significantly higher in AML-M5 patients at diagnosis and relapse than in healthy donors (Fig. 2A). In agreement with our clinical data, a scatter plot analysis, which was used to visually determine the expression levels of genes in AML-M5 patients, revealed a positive association between Jab1 and Trx expression or ROS levels (Fig. 2B, left and right). Furthermore, analyzing the two genes across online datasets indicated that Jab1 was correlated with Trx in AML patients (Fig. 2C). Jab1 and Trx protein levels were higher in leukemic cells derived from AML-M5 patients than in CD34+ cells from healthy donors (Fig. 2D; Supplementary Fig. S1B). Similar findings were observed when we compared the leukemic monocyte cell lines MOLM-13, U937, and THP-1 with cells from healthy individuals (Supplementary Fig. S3A). This finding was further confirmed in all leukemia: patients with AML-M1 to AML-M5 subtype, acute lymphoblastic leukemia, and chronic myeloid leukemia all had higher Jab1 levels than did healthy donors, as determined by qRT-PCR (Supplementary Fig. S1C).

Figure 2.
Figure 2.

High levels of Jab1 and Trx in AML-M5. A, Left, three groups were compared: patients at diagnosis, patients at relapse, and healthy donors (controls). As determined by quantitative RT-PCR, Jab1 and Trx were overexpressed at diagnosis and relapse compared with their levels in CD34+ cells from healthy donors. GAPDH was used as a control. Values here are means ± SE. B, Left, the expression levels of Jab1 and Trx were correlated in AML-M5 patients. Right, the expression levels of ROS and Jab1 were correlated in AML-M5 patients. The R and P values based on the Pearson Correlation. C, Left, overview of the two genes across online datasets (http://r2.amc.nl). Right, Jab1 and Trx were also overexpressed in AML-M5 cell lines compared with their levels in CD34+ cells from healthy donors, as determined by quantitative RT-PCR. Data are means ± SD. D, Jab1 and Trx protein expression levels in AML-M5 patients, and healthy donors were analyzed by Western blot analysis. The protein levels were quantified by ImageJ software.

Correlation of ROS/Jab1/Trx levels with patient survival

To determine whether ROS/Jab1/Trx levels were correlated with cancer patients' survival, we performed a survival analysis using the Kaplan–Meier method. High levels of ROS or high levels of Jab1, high levels of Trx, or high levels of XOD or low levels of T-AOC (median cutoff) and abnormal cytogenetics findings were correlated with a poor prognosis (Fig. 3; Supplementary Fig. S2). The mean survival duration of patients with low Jab1 mRNA expression (below the median Jab1 expression level) was 39 months compared with 8 months for patients with high Jab1 mRNA expression. The mean survival duration of patients with low Trx mRNA expression (below the median Trx expression level) was 53 months compared with 9 months for patients with high Trx mRNA expression. Supplementary Table S2 shows the abnormal cytogenetic results of 35 AML patients.

Figure 3.
Figure 3.

Relationship between ROS/Jab1/Trx levels and overall patient survival. AML-M5 patients with high ROS (above the median; A), Jab1 (above the median; B), Trx (above the median; C), and XOD (above the median; D) and low T-AOC (below the median; E) at diagnosis had a poor prognosis. Abnormal cytogenetic (cyto; F) findings were associated with a poor prognosis.

To determine whether Jab1 and Trx in AML patients were independent predictors of overall survival, we performed a multivariate analysis with the 16 variables (details in Table 1). XOD (P = 0.003), Jab1 (P = 0.008), and T-AOC (P = 0.014) were significant prognostic factors for AML (Table 1). The multivariate analysis identified XOD, Jab1, and T-AOC as significant independent factors for poor overall survival in AML.

View this table:
Table 1.

Multivariate Cox regression on overall survival

Jab1 regulates Trx expression in AML

To assess the effect of oxidative stress on Jab1 and Trx, we exposed AML cells to H2O2. We found that THP-1 cells had higher intracellular ROS levels but lower sensitivity to oxidative stress in response to H2O2 stimulus than did U937 cells (Supplementary Fig. S3B). When the H2O2 concentration increased progressively (0–50 μmol/L), the expression levels of Jab1 and Trx were significantly upregulated in U937 and THP-1 cells (Fig. 4A, left), whereas the antioxidant β-ME led to decreased Jab1 and Trx expression (Fig. 4A, right). We next studied the relationship between Jab1 and Trx and found that Jab1 overexpression induced higher endogenous Trx protein (Fig. 4B, top) and RNA levels (Fig. 4C). Inversely, depletion of Jab1 resulted in decreased Trx levels (Fig. 4B, bottom). We examined the subcellular distribution of the Trx protein after H2O2 exposure. As shown in Fig. 4D, the levels of both Trx and Jab1 in the cytoplasmic fraction were significantly higher after H2O2 exposure in U937 and THP-1 cells (Fig. 4D). However, the level of Jab1 in the nucleus was unchanged after H2O2 exposure (Fig. 4D). Furthermore, low concentrations of H2O2 stimulated the release of Trx, and knockdown of endogenous Jab1 reduced the release of extracellular Trx in U937 and THP-1 cells (Supplementary Fig. S3C). However, neither Trx silencing nor Trx overexpression affected Jab1 levels (Supplementary Fig. S4A and S4B). The data thus demonstrated that ROS regulated Jab1 and Trx levels in AML-M5.

Figure 4.
Figure 4.

Jab1 regulates Trx expression in AML. A, Regulation of Jab1 and Trx expression in AML cells treated with H2O2 (10, 25, and 50 μmol/L; left) and β-ME (right). B, Ectopic Jab1 increased Trx protein expression (top), whereas Jab1 knockdown remarkably reduced Trx levels (bottom). C, Ectopic Jab1 increased Trx RNA levels in U937 and THP-1 cells. D, The levels of Trx and Jab1 in the cytoplasmic (Cyto) fraction were increased significantly in U937 cells and THP-1 cells after 24 hours of H2O2 exposure compared with their levels in the control cells, as indicated. In contrast, only a slight increase in Trx levels was found in the nucleus, and the amount of nuclear Jab1 was unchanged. PARP and tubulin were used as nuclear and cytoplasmic protein markers, respectively. E, H2O2 did not increase Trx expression in Jab1-deficient U937 cells. Cont-si, control siRNA; Jab1-si, Jab1 siRNA. Data are means ± SD; *, P < 0.05; **, P < 0.01. The protein levels were quantified by ImageJ software.

Furthermore, we determined whether ROS-upregulated Trx is regulated by Jab1; we found that H2O2 did not stimulate Trx in Jab1-deficient U937 cells (Fig. 4E). In addition, we examined Nrf2, another regulator of Trx (22, 23), and found that H2O2 did not change the Nrf2 expression level in either the control or Jab1-deficient cells (Fig. 4E). Thus, our findings indicate that Jab1 is an upstream regulator and positively controls Trx expression in AML-M5 cells.

Jab1 specifically interacts with Trx and regulates Trx stability in AML-M5 cells

Next, to determine whether Jab1 and Trx interact, we performed coimmunoprecipitation assays and demonstrated that Jab1 and Trx proteins interacted specifically with each other, using either endogenous proteins in U937 and THP-1 cells (Fig. 5A) or ectopically expressed tagged proteins in 293T cells (Fig. 5B).

Figure 5.
Figure 5.

Jab1 specifically interacts with Trx and regulates Trx stability. A, U937 and THP-1 cell lysates were immunoprecipitated (IP) with nonimmune mouse serum immunoglobulin or Jab1 and immunoblotted with anti-Trx antibodies. Immunoglobulin G (IgG) pull-down assays and total (Tot.) cell lysates were analyzed by Western blotting. Cell lysates were immunoprecipitated with anti-Jab1 antibodies and immunoblotted with anti-Trx. Jab1 specifically interacted with Trx and vice versa. Immunoglobulin G heavy chains (HC) and light chains (LC) are indicated. B, 293T cells were transfected with His-Jab1, HA/Flag-Trx, or both for 48 hours. Cell lysates were immunoprecipitated with anti-His or anti-Flag antibodies and immunoblotted with anti-His, anti-HA, and anti-Flag antibodies and other antibodies as indicated. C, U937 cells were transfected with or without His-Jab1 and treated with 100 ng/mL cycloheximide (CHX) for the indicated times. Cell lysates were collected for Western blot analysis using antibodies targeting Trx, with β-actin used as a control. Bottom, quantification of Trx intensity, as measured by Image J software.

The above result strongly suggested that Jab1 is essential for maintaining Trx levels in AML cells. To test this hypothesis, we pretreated U937 cells expressing Jab1 cDNA or pcDNA with CHX to inhibit protein synthesis for measuring Trx protein stability. An immunoblotting analysis showed that turnover of Trx protein was significantly decreased in U937 cells that expressed His-Jab1 compared with that in pcDNA-expressing cells (Fig. 5C). Collectively, our data indicated that Jab1 is important for stabilizing Trx protein to maintain its levels in AML cells.

To further delineate whether Jab1 transcriptionally regulates Trx, we performed luciferase reporter assays with the human Trx promoter. The 5′-regulatory region of the human Trx gene (−975/+1) contains an AP-1–binding site (−527) that can recruit Jab1, as Jab1 is an AP-1 coactivator (12). Transient knockdown of Jab1 was associated with a notable decrease in Trx promoter activity (Fig. 6A, left). In contrast, Trx promoter activity was increased in a dose-dependent manner by transfection with His-Jab1 plasmid DNA (Fig. 6A, right). Furthermore, we compared Jab1′s effect on the Trx reporter constructs that contained a deletion or truncation of the AP-1 site, the wild-type promoter (containing the −975 bp of the Trx promoter), or the short Trx promoter (−527/+1) that contains only the AP-1 site (Up AP-1/ARE; Fig. 6B). Our data demonstrated that transactivation of Trx expression is driven by Jab1 through the AP-1–binding site (Fig. 6B), which was also confirmed by Jab1′s interactions with Trx promoter via ChIP experiments (Fig. 6C).

Figure 6.
Figure 6.

Jab1 positively regulated Trx promoter activity. A, 293T cells were transfected with either Jab1 siRNA or Jab1 plasmid DNA, along with Trx-luciferase reporter constructs and pRL as an internal control. Trx promoter luciferase activity was measured and normalized to Renilla luciferase activity. Trx promoter activity decreased following Jab1 silencing and increased in a dose-dependent manner with the level of Jab1 plasmid DNA. Rel., relative. B, Deletion analysis of the Trx gene AP-1. Trx-Luc reporter constructs showing the wild-type (WT) Trx (−975/+1) construct, a deletion (Del.) of AP-1 Trx-Luc, a shorter promoter (−527/+1) that contains only the AP-1 sites (Up AP-1) of Trx, and deletion of AP-1 (Down AP-1). Jab1-induced activation of the Trx gene requires AP-1 binding sites. 293T cells were cotransfected with the indicated Trx-Luc reporter constructs, with or without an increased concentration of Jab1 (μg). Luciferase assays revealed that Trx promoter transactivation by Jab1 occurred, in a dose-dependent manner, only when the AP-1–binding sites were present. Deletion or mutation of the AP-1 site in the Trx promoter abrogated Jab1 transactivation. The result is representative of three independent experiments performed in triplicate. C, Nuclear extracts of U937 cells were immunoprecipitated by IgG or anti-Jab1. Coprecipitated DNAs were detected by quantitative reverse transcriptase PCR using primers for Trx (including the AP-1 site) or negative control primer (2311 bp downstream of the AP-1 site). The results are expressed relative to the corresponding values for input DNAs. Data are means ± SD, *, P < 0.05; **, P < 0.01.

Depletion of Jab1 inhibits AML-M5 cell viability both in vitro and in vivo

To assess the effect of silencing Jab1 on AML, we transfected AML-M5 cells with Jab1 siRNA. Our data indicated that Jab1 knockdown inhibited cell growth (Fig. 7A, left; Supplementary Fig. S4C) and cell colony formation (Fig. 7A, right). In addition, transiently transfecting Trx plasmid DNA into Jab1-deficient cells altered and reversed their survival, indicating that adding back Trx can rescue Jab1′s function (Fig. 7A, right). Western blotting confirmed the ectopic expression of Trx in Jab1-deficient cells (Supplementary Fig. S4D). In contrast, overexpression of Jab1 in normal HS-5 bone marrow cells contributed to colonies formation (Supplementary Fig. S5A). We then measured the effect of Jab1 depletion on AML cells' invasive activity. The invasiveness of AML cells transfected with Jab1-siRNA was significantly decreased, especially in the presence of Ara-c (0.5 μmol/L). However, 50 μmol/L H2O2 slightly increased the numbers of invading cells (Fig. 7B; Supplementary Fig. S5B).

Figure 7.
Figure 7.

Depletion of Jab1 inhibits AML-M5 cell viability both in vitro and in vivo. A, Left, Jab1 knockdown inhibited cell proliferation. U937 cells were transfected with control or Jab1 siRNA, and cell viability was determined by an MTT assay. Right, loss of Jab1 inhibited U937 cell colony formation but transfection of ectopic Trx into Jab1-deficient cells rescued the colony formation. Each well was seeded with 5,000 live cells in medium containing soft agar, and colonies were allowed to form for 2 weeks. B, The invasiveness of U937 cells was enhanced by H2O2 treatment, inhibited by Ara-c treatment, and further decreased by Jab1 knockdown. Equal numbers of cells were added to the top wells of Matrigel-coated chambers in serum-free RPMI1640 medium and treated, as indicated, with H2O2 (50 μmol/L) or Ara-c (0.5 μmol/L). After 24 hours of culture, invasive cells were counted using the CyQUANT cell proliferation assay. C, Left, the tumor-suppressive effects of Jab1 depletion in NOD-SCID mice, as evidenced by tumor growth curves; representative photographs of harvested tumors (inset in panel); and tumor weights measured at the indicated time points (middle). Right, the expression levels of Jab1 and Trx proteins in tissues resected from the same mice were examined via Western blot analysis. Cont-si, control siRNA; Jab1-si, Jab1 siRNA. Data are means ± SD; *, P < 0.05; **, P < 0.01.

The aforementioned in vitro data showed the critical role of Jab1 in AML-M5 and led us to evaluate its activity in vivo. Tumor growth of human U937 cells was used to measure the tumor-suppressive effects of Jab1 depletion in NOD-SCID mice as described in the literature (24). We established U937 cells, with or without stable knockdown of Jab1, by transducing them with a lentivirus carrying sh-Jab1 or sh-Control (Scramble). Jab1 depletion suppressed tumor growth in the U937 xenograft mouse model. The sh-Jab1 xenografts grew significantly more slowly than did the sh-control xenografts (Fig. 7C). Tumor weight was consistently significantly lower in the knockdown Jab1 groups than in the control group (P = 0.02; Fig. 7C). A Western blot analysis showed that Jab1 and Trx were abundantly expressed in the sh-control tumors, whereas the levels of both were reduced in the sh-Jab1 tumors (Fig. 7C).

Discussion

Cancer cells generate ROS as a result of stimulation of oncogenes, mitochondrial dysfunction, abnormal metabolism, and the microenvironment. Oncogenic mutations promote aberrant metabolism and protein translation, resulting in increased ROS production. For example, AML patients with FMS-like tyrosine kinase 3 (FLT3) mutations have a very high relapse rate (25), and FLT3 induced elevated levels of ROS (26). In addition to the genetic changes that alter tumor cell metabolism, the abnormal tumor microenvironment has a major role in determining the metabolic phenotype of tumor cells. Intrinsically altered tumor cell metabolism creates spatial and temporal heterogeneity in oxygenation, pH, and the concentrations of glucose and many other metabolites. These extreme conditions induce a collection of cellular stress responses that further contribute to ROS production in AML patients with relapse. In an in vitro study, the bone marrow stromal cell environment induced a mitochondrial calcium influx, leading to increased ROS levels in ALL cells (27). The chemoprotective tumor microenvironment may also contribute to disease recurrence. Most current chemotherapeutic agents and radiotherapy increase oxidative stress and could therefore help drive tumor recurrence (28). These mechanisms might explain our finding that ROS production was high in relapsed AML patients.

Recent studies have begun to shed light on the ROS involved in modulating biological cell functions, cell signaling, and homeostasis in tumor cells (29). AML-M5, which is associated with older age, is an aggressive form of a malignant disorder of the hematopoietic system that is characterized by highly proliferative blast cells (30) and high WBC counts and serum LDH levels (31). Indeed, high WBC counts and serum LDH levels were found in AML-M5 patients in our study. In addition, ROS levels were significantly higher in patients than in healthy donors and were highest at relapse. ADA and XOD levels were both increased in relapsed AML-M5 patients that also reflect high ROS production.

The main antioxidant defense enzymes that are responsible for detoxifying ROS play a crucial role in maintaining cellular homeostasis and provide sensitive early warning signals of incipient oxidative stress conditions (32). In this study, significant differences were observed in the levels of antioxidant capacity, including those of T-AOC, SOD, catalase, and GSH-Px, between AML-M5 patients and healthy controls. Moreover, there was a significant increase in oxidative damage in AML-M5 patients at relapse, as evidenced by the presence of 8-OHdG, MDA, and AOPP. However, the Cyt-c levels did not differ significantly. Secreted Trx exhibits potential oncogenic effects and immunomodulatory activity, in addition to its antioxidative functions (33). Interestingly, secreted Trx levels were higher in the plasma of AML-M5 patients than in that of healthy donors. Thus, our clinical research demonstrates that increased ROS levels are involved in the physiologic development of AML-M5.

Increased ROS manifest their effects in myeloid disease via ROS-regulated pathways (2, 34). In our studies characterizing models of AML-M5, we further showed that Jab1 and Trx were aberrantly overexpressed in AML-M5 patients compared with in healthy donors and that this overexpression was related to poor survival outcomes. Moreover, abnormal cytogenetic results were correlated with poor survival outcomes. The results of a correlation analysis also indicated that Jab1 expression was associated with Trx. Thus, ROS may trigger leukemia by stimulating oncogenes, and an endogenous imbalance in antioxidant enzymes may be relevant in the cellular process.

Jab1′s multiple functions affect several proteins and signaling pathways, and the results of these interactions are generally oncogenic (12, 15). Trx expression is significantly induced under oxidative conditions (35). Our clinical data showed that Jab1′s association with Trx was predictive of poor prognosis in AML-M5 patients. Moreover, we showed that exposure of AML-M5 cells to H2O2 led to increased levels of endogenous ROS, which act as a signal transduction messenger by increasing Jab1/Trx-1 expression. A coimmunoprecipitation assay revealed that Jab1 specifically interacts with and modulates Trx's function in AML-M5 cells. Furthermore, knockdown of endogenous Jab1 expression with siRNA in cell lines led to reduced Trx levels, both extracellularly and intracellularly. We also identified Jab1 as a potential transcriptional activator of Trx, as the introduction of ectopic Jab1 dramatically increased Trx promoter activity and the suppression of endogenous Jab1 significantly reduced Trx promoter activity. However, we did not observe Jab1 expression changes after reducing or overexpressing Trx. In addition, we found that the amounts of Trx and Jab1 in the cytoplasmic fraction were significantly higher after H2O2 exposure in U937 and THP-1 cells. In agreement with our data, the results of a study presented by Hwang and colleagues indicated that Trx interacts with Jab1 but that overexpression of Trx does not affect Jab1 levels (36). In contrast, Trx may negatively regulate Jab1′s function when it interacts with Jab1; for example, Trx could compete with p27Kip1 for Jab1 binding (36).

ROS act as signaling molecules that affect redox-sensitive transcription factors, enzymes, oncogenes, and other effectors (37). Redox dysregulation that originates from ROS represents a specific leukemia cell vulnerability (38). An invasion assay revealed that a low concentration of H2O2 dramatically increased the number of invading cells. In contrast, U937 and THP-1 cell invasiveness was significantly decreased after transfection with Jab1 siRNA, especially in the presence of Ara-c. These results indicate that ROS enhances AML-M5 cell line invasion and that Jab1 plays an important role in managing invasion.

In conclusion, the cells of patients with leukemia are susceptible to damage by peroxidation owing to their decreased antioxidation ability. In our study, Jab1 overexpression was associated with a poor prognosis and specifically interacted with and positively regulated Trx levels, both in vivo and in vitro. Mechanistically, transactivation of Trx expression is driven by Jab1 through the AP-1–binding site. Our data reveal that the ROS-mediated interaction between Jab1 and Trx plays a critical role in the pathobiologic characteristics and relapse of AML-M5. Thus, targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: F. Zhou, X.-F. Le, F.X. Claret

Development of methodology: F. Zhou, Y. Pan, Y. Wei, G. Bai, Q. Shen, F.X. Claret

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): F. Zhou, Y. Pan, Y. Wei, R. Zhang, Q. Shen, S. Meng, F.X. Claret

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Zhou, Y. Pan, Y. Wei, F.X. Claret

Writing, review, and/or revision of the manuscript: F. Zhou, Y. Pan, Y. Wei, S. Meng, X.-F. Le, M. Andreeff, F.X. Claret

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): F. Zhou, Y. Pan, Y. Wei, F.X. Claret

Study supervision: F. Zhou, F.X. Claret

Grant Support

This project was supported in part by the National Natural Science Foundation of China (nos. 81270597, 81372816, 81070441); and grants from the NIH [CA-90853, including a Core grant (CA-016672)].

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

We thank Shin-ichi Oka at New Jersey Medical School for sharing the pDC316-Flag-Trx-HA plasmid. We thank Kathryn Tonissen at Griffith University for sharing the pGL3-hTrx-Luc reporter plasmids, and Xiang Li at Jiangnan University for sharing the HS-5 cells. We also thank Dawn Chalaire, Ann Sutton, and Tracy Stafford in the Department of Scientific Publications at MD Anderson Cancer Center for editing the manuscript.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • Received October 4, 2016.
  • Revision received November 1, 2016.
  • Accepted March 1, 2017.

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Jab1/Csn5–Thioredoxin Signaling in Relapsed Acute Monocytic Leukemia under Oxidative Stress
Fuling Zhou, Yunbao Pan, Yongchang Wei, Ronghua Zhang, Gaigai Bai, Qiuju Shen, Shan Meng, Xiao-Feng Le, Michael Andreeff and Francois X. Claret
Clin Cancer Res August 1 2017 (23) (15) 4450-4461; DOI: 10.1158/1078-0432.CCR-16-2426
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