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.2011 Dec 1;118(23):6097-106.
doi: 10.1182/blood-2011-03-341867. Epub 2011 Oct 13.

Acquired STAT4 deficiency as a consequence of cancer chemotherapy

Affiliations

Acquired STAT4 deficiency as a consequence of cancer chemotherapy

Ivan P Lupov et al. Blood..

Abstract

Signal Transducer and Activator of Transcription 4 (STAT4) is a transcription factor that is activated by IL-12 signaling and promotes Th1-cell differentiation and IFN-γ production. Defective IFN-γ production because of STAT4 mRNA and protein deficiency occurs after autologous stem cell transplantation for lymphoma. In the present study, we investigated the mechanisms of STAT4 deficiency in lymphoma patients. The tumor-bearing state is not responsible, because STAT4 levels were not significantly different in PBMCs obtained from healthy control subjects compared with those from lymphoma patients before treatment. STAT4 protein levels were significantly decreased in PBMCs and T cells obtained from lymphoma patients after standard-dose chemotherapy. Furthermore, treatment of control PBMC cultures or a natural killer cell line with chemotherapy drugs in vitro also resulted in reduced STAT4 protein and diminished, IL-12-induced IFN-γ production. Translation of STAT4 protein was not impaired in chemotherapy-treated cells, whereas the STAT4 protein half-life was significantly reduced. Chemotherapy drugs promoted the ubiquitination and proteasomal degradation of STAT4. Treatment with the proteasome inhibitor bortezomib reversed chemotherapy-induced STAT4 deficiency and defective IFN-γ production. We conclude that acquired STAT4 deficiency in lymphoma patients is a consequence of treatment with chemotherapy, results that have important implications for the design of optimal immunotherapy for lymphoma.

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Figures

Figure 1
Figure 1
Expression of STAT4 in cells obtained before and after chemotherapy. (A) STAT4 protein expression was analyzed by immunoblotting of PBMCs from 4 healthy controls (C1-C4) and 6 untreated lymphoma patients (P1-P6). Samples from controls and patients were run in separate gels and exposure was done at the same time. The indicated upper STAT4 band detected with anti-STAT4 mAb was confirmed with anti-STAT4 polyclonal Ab. (B) Immunoblot analysis of STAT4 protein levels in control (C1-C3 and C5-C15) and lymphoma patient (P4, P7-P10, P12, P19-P23) PBMCs. Lymphoma patient PBMCs were obtained before (lanes labeled “U” for untreated) and 3 weeks after (lanes labeled “S” for standard dose) their first cycle of standard-dose chemotherapy plus rituximab. The chemotherapy regimen was R-CVP for patients P1, P6, P10, and P20 and R-CHOP for all other patients. A vertical line has been inserted to indicate the repositioned gel lane. (C) The levels of STAT4 protein in panel B were quantified by densitometry of the corresponding bands using the National Institutes of Health ImageJ program, and each sample was normalized to endogenous control β-actin as the ratio. Results are presented as means ± SD from 14 control and 11 patient samples. Blots in panel B were run in different gels but exposed to the same extent. *P < .05 relative to controls (C) or untreated lymphoma patients (U); **P > .05 relative to controls (C). (D) Changes in STAT4 and STAT3 protein levels in PBMCs obtained from each patient after standard-dose chemotherapy (S) are presented as the percentage of reduction compared with PBMCs obtained before chemotherapy treatment (U). The average percentage of reduction was obtained from the same 11 patients as in panel B and shown as the horizontal line in the graph. (E) Analysis of STAT4 protein in mice treated without or with etoposide. Tumor-bearing mice were treated with vehicle or etoposide as described in “Methods.” Spleens were harvested from 2 mice killed on day 29 of study. CD4+ T cells were isolated from each spleen using positive selection with CD4 magnetic beads (Miltenyi Biotec). Total protein extracts from isolated cells were subjected to immunoblotting analysis. The level of STAT4 protein in each mouse was normalized to internal control β-actin and is presented as the ratio indicated below. Nondetectable STAT4 protein is presented as (-). Results shown are representative from 2 independent studies. A vertical line has been inserted to indicate the repositioned gel lane.
Figure 2
Figure 2
Expression of STAT4 in different hematopoietic-cell subsets. (A) Immunoblot analysis of STAT4 protein levels. Various subsets of cells as indicated were isolated from control PBMCs using positive selection with magnetic beads (Miltenyi Biotec). The level of STAT4 protein in each subset was determined as described in Figure 1E. Nondetectable STAT4 protein is presented as (-). Results shown are representative of 3 different control samples that were tested. (B) Flow cytometric analysis of STAT4 protein levels. Control PBMCs were stained with mAbs specific for lineage-associated antigens, fixed, permeabilized, and stained with anti-STAT4 Abs as described in “Methods.” Histograms represent data obtained by electronic gating on CD3−CD56+ cells (NK-cell subset; top panel), CD3+CD56− cells (T-cell subset; middle panel), and CD3−CD20+ cells (B-cell subset; bottom panel). Logarithm of red fluorescence is displayed on the abscissa and relative cell number on the ordinate. STAT4 Ab staining is indicated by shaded histograms and control staining by open histograms. Results shown are representative of 10 different control samples that were tested. Summary data and statistics are presented in the text.
Figure 3
Figure 3
Expression of STAT4 in cells treated in vitro with chemotherapy drugs. PBMCs obtained from controls were activated with PHA and IL-2 for 3 days and then cultured for 3 more days with the indicated concentrations of etoposide (Eto) and carmustine (Car). RNA was extracted and the first-strand cDNA was synthesized from the cells. STAT4 expression was analyzed using immunoblot (A) and real-time PCR (B). (C) STAT4 protein levels in CD4+ and CD8+ cells from control activated PBMCs treated without (-D) or with carmustine (Car) or etoposide (Eto) were analyzed using flow cytometry as described in “Methods.” Histograms represent the STAT4 expression gated on 5000 events of live CD4+ or CD8+ cells using WinMDI software. (D) NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) for 2-3 days. STAT4 protein levels were analyzed using Western blotting. (E) NKL cells treated as described in panel D were incubated with medium alone (unfilled bars) or medium containing 2 ng/mL of IL-12 (filled bars) for 1 day. The cell-free supernatants were analyzed for IFN-γ production using ELISA. The data are presented as means ± SD from 3 independent experiments.
Figure 4
Figure 4
STAT4 mRNA levels and half-life ofSTAT4 mRNA in PBMCs. PBMCs obtained from 6 healthy control subjects and 3 patients after high-dose chemotherapy and PBSCT were treated with actinomycin D at 1 μg/mL for 0, 2, 4, and 6 hours in a 5% CO2 incubator at 37°C. RNA was extracted and first-strand cDNA was synthesized, followed by the real-time PCR. The half-life ofSTAT4 mRNA from each sample was calculated accordingly. Results shown are means ± SD.
Figure 5
Figure 5
Analysis of translational regulation of STAT4 protein. (A) NKL cells treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as described in Figure 3D were used for [35S] methionine and cysteine labeling (10 μCi/μL; Perkin Elmer). Dead cells were removed from the culture using Ficoll centrifugation, and the collected viable cells at 10 × 106 cells/2 mL were subsequently pulsed for 15, 30, and 60 minutes with [35S] methionine/cysteine. The cell extract (1%) was separated by SDS-PAGE followed by exposure to X-ray film after drying on a Bio-Rad gel dryer. The molecular weight (MW) is accompanied on the side of the gel. (B) NKL cells treated as described in panel A were pulsed with [35S] methionine/cysteine for 24 hours. STAT4 and STAT3 proteins were immunoprecipitated from the total protein extracts using polyclonal anti-STAT4 and anti-STAT3 Abs, which were subjected to SDS-PAGE and then exposure to X-ray film after drying. A preimmunized rabbit serum (IgG) was used in immunoprecipitation as a negative background control. One percent of the total protein extracts from each sample was separated on the same gel as loading control (1% loading). The molecular weight (MW) at 75 kD is labeled.
Figure 6
Figure 6
Chemotherapy drugs reduce STAT4 protein levels via ubiquitin-mediated proteasomal degradation. NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as described in Figure 3D. Dead cells were removed by Ficoll centrifugation from the culture of each treatment, and the collected viable cells were subsequently incubated with cycloheximide (CHX) for 0, 2, 4, 6, 8, 10, 12, and 24 hours. STAT4 protein expression was analyzed by immunoblotting, and a vertical line has been inserted to indicate the repositioned gel lane (A). The levels of STAT4 protein were determined by the densitometry of the corresponding bands normalized to endogenous control β-actin using the National Institutes of Health ImageJ program. The half-life of STAT4 protein was calculated accordingly, and the results shown are means ± SD from a total of 3 independent experiments (B). *P < .05 relative to treatment without chemotherapy drug (-D). (C) NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as in Figure 3D. Dead cells were removed as described in panel A, followed by total protein extraction. Ubiquitin-conjugated protein was first enriched using the Ubiquitin Enrichment Kit (Thermo Scientific) with a specialized affinity resin binding to polyubiquitinylated proteins from cell lysates (Ub-beads). The negative control is the resin slurry lacking affinity to polyubiquitinylated proteins (beads). The enriched proteins were subjected to immunoblotting. Ubiquitin-conjugated STAT4 protein levels were analyzed using an anti-STAT4 mAb (left panel). Total STAT4 protein levels were analyzed using immunoblotting from 10 μg of whole-cell lysates (right panel). The ratio of Ub-STAT4 to total STAT4 is indicated below. The molecular weight (MW) at 75 and 118 kD is labeled. The panel is representative of 3 independent experiments, and a vertical line has been inserted to indicate the repositioned gel lane. (D) NKL cells were incubated with the proteasome inhibitor bortezomib at 5.2nM simultaneously with 50μM carmustine (Car) or 2μM etoposide (Eto) for 2 days. STAT4 protein levels were determined by immunoblotting. The results are representative of 3 independent experiments. Ratio of total STAT4 to β-actin is indicated below. (E) Densitometric analysis of STAT4 protein levels in NKL cells treated with carmustine or etoposide in the presence or absence of bortezomib. The results are presented as the averaged ratio of STAT4 to β-actin from 2 independent experiments as means ± SD (F) NKL cell treated as described in panel D were stimulated with IL-12 at 2 ng/mL for 1 day. The IFN-γ levels in the cell supernatants were evaluated using ELISA. Results are averaged from 2 independent experiments as means ± SD.
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