Noncanonical NF-κB activation mediates STAT3-stimulated IDO upregulation in myeloid-derived suppressor cells in breast cancer

Abstract

Immunotherapy for cancer treatment is achieved through the activation of competent immune effector cells and the inhibition of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). Although MDSCs have been shown to contribute to breast cancer development, the mechanism underlying MDSC-mediated immunosuppression is unclear. We have identified a poorly differentiated MDSC subset in breast cancer-suppressing T cell function through STAT3-dependent IDO upregulation. In this study we investigated the mechanisms underlying aberrant expression of IDO in MDSCs. MDSCs were induced by coculturing human CD33(+) myeloid progenitors with MDA-MB-231 breast cancer cells. Increased STAT3 activation in MDSCs was correlated with activation of the noncanonical NF-κB pathway, including increased NF-κB-inducing kinase (NIK) protein level, phosphorylation of cytoplasmic inhibitor of NF-κB kinase α and p100, and RelB-p52 nuclear translocation. Blocking STAT3 activation with the small molecule inhibitor JSI-124 significantly inhibited the accumulation of NIK and IDO expression in MDSCs. Knockdown of NIK in MDSCs suppressed IDO expression but not STAT3 activation. RelB-p52 dimers were found to directly bind to the IDO promoter, leading to IDO expression in MDSCs. IL-6 was found to stimulate STAT3-dependent, NF-κB-mediated IDO upregulation in MDSCs. Furthermore, significant positive correlation between the numbers of pSTAT3(+) MDSCs, IDO(+) MDSCs, and NIK(+) MDSCs was observed in human breast cancers. These results demonstrate a STAT3/NF-κB/IDO pathway in breast cancer-derived MDSCs, which provides insight into understanding immunosuppressive mechanisms of MDSCs in breast cancer.

Conflict of interest statement

Conflict of interest statement

The authors declare that they have no competing interests.

Copyright © 2014 by The American Association of Immunologists, Inc.

Figures

Figure 1

STAT3-induced IDO expression is not through a direct binding of STAT3 to the IDO promoter region and does not require C/EBPβ in MDSCs (A) Binding of STAT3 to three different regions in the IDO promoter containing 5 candidate STAT3-binding sites was measured by ChIP. (A1) Regions amplified are highlighted with bracketing the binding sites. (A2) ChIP-PCR assay was conducted to detect STAT3 binding to putative binding sequences. AKAP12 and HIC2 gene served as the positive. Quantitative values are calculated using the ratio of ChIP-PCR product to Input PCR product (relative ChIP value, enrichment ratio). * P+ controls, MDSCs and JSI-124-treated MDSCs (J-MDSCs). STAT3 or β-actin blots were used as protein loading controls. (C) The mRNA of C/EBPβ and IDO was examined by RT-PCR in MDSCs transfected with C/EBPβ-specific siRNA or negative control. (D) Western blot was performed to examine phosphorylation of STAT3, C/EBPβ and IDO expression in MDSCs and C/EBPβ-specific siRNA transfected MDSCs.

Figure 2

The noncanonical NF-κB pathway is activated in MDSCs. (A1) The phosphorylation levels of IKKβ (pIKKβ) and IKKα (pIKKα) in CD33+ controls, MDSCs and JSI-124-treated MDSCs (J-MDSCs). (A2) The levels of pIKKβ and pIKKα were compared using the density ratio of phosphorylated protein to total protein. * P<0.05. (B) The protein level of NIK and p100 phosphorylation in CD33+ controls, MDSCs and J-MDSCs. STAT3 or β-actin blots were used as protein loading controls. (C) Enzyme-linked immunosorbent assay was conducted to compare the levels of p50, p52, RelA and RelB subunits in nuclear extracts of CD33+ controls, MDSCs and J-MDSCs. The transcriptional activity was measured using absorbance at 450 nm (A450). * P<0.05.

Figure 3

The noncanonical NF-κB pathway mediates STAT3-induced IDO expression in MDSCs. (A1) STAT3 phosphorylation and expression levels of NIK and IDO proteins in MDSCs at indicated time points. (A2) Quantification of density on immunoblots was performed by normalizing the density of each band to STAT3 or β-actin. (B) The mRNA of NIK and IDO in MDSCs transfected with NIK-specific siRNA or negative control was examined by RT-PCR. (C1) Western blot examined the expression of NIK and IDO and STAT3 phosphorylation in MDSCs and siRNA transfected MDSCs. (C2) Expression levels NIK and IDO and STAT3 phosphorylation were compared using the density ratio of indicated protein to β-actin respectively. * P

Figure 4

Noncanonical NF-κB subunits RelB and p52 regulate IDO expression via direct binding to the IDO promoter region in MDSCs. (A) Four detective biotinylated probes covering 11 putative sequences were synthesized. (B) Detective probes were incubated with MDSCs nuclear extracts with or without competitive probes to test the specificity of NF-κB-IDO binding. Forty fold dose of competitive probes was used to avoid NF-κB binding to the biotinylated detective probes. * P

Figure 5

The correlation of STAT3 and NF-κB activation and IDO expression in MDSCs in human breast cancer. In this study, paraffin-embedded breast cancer tissues were collected from 30 patients who received radical mastectomy at the Department of Breast Oncology of Tianjin Medical University Cancer Institute and Hospital. (A) Breast cancer tissue sections were immunostained with phospho-STAT3 (pSTAT3) and CD33 for pSTAT3+ MDSCs, IDO and CD33 for IDO+ MDSCs, as well as NIK and CD33 for NIK+ MDSCs. (B) The percentages of pSTAT3+MDSCs, IDO+MDSCs and NIK+MDSCs in MDSCs are shown. (C) The correlations between the distribution of pSTAT3+ MDSCs, IDO+ MDSCs and NIK+ MDSCs are shown.

Figure 6

Blocking STAT3 activation inhibits MDSCs and tumor metastasis in a mouse mammary cancer model. 4T1 mouse mammary carcinoma cells (3 × 106/mouse) were injected into the mammary fat pads of BALB/c mice. Mice were then treated intraperitoneally with JSI-124 at dose of 1 mg/kg/d or vehicle control (PBS+DMSO) for 12 days. (A) Tumor size in mice was monitored during JSI-124 treatment and measured using the following equation: Tumor volume= (longer diameter) × (shorter diameter) 2/2. (B) On day 16, mice were sacrificed and metastatic nodules in lungs were counted based on HE-stained slides. * P < 0.05. (C) Single cell suspensions of whole spleen and cancer tissues were prepared from tumor-bearing mice and the population of MDSCs were calculated by detecting CD11b+ subpopulation in CD45+ myeloid cells using flowcytometry. * P < 0.05. (D1) CD11b+ MDSCs were enriched using human CD11b MicroBeads and the expression of NIK and IDO, as well as STAT3 phosphorylation in MDSCs were detected by Western blot analysis. Four representative samples were displayed. (D2) Expression levels NIK and IDO and STAT3 phosphorylation were compared using the density ratio of indicated protein to β-actin or STAT3. * P<0.05.

Figure 7

IL-6 stimulates STAT3-dependent IDO expression in MDSCs. (A) Image of Human cytokine antibody array. The spot density represents the level of cytokine in the supernatants. UC represents CD33+ cells isolated from primary cord blood. (B1) The levels of 42 cytokines in the supernatant of MDSCs, J-MDSCs and the base line for the combination of MDA-MB-231 cell and CD33+ cells were displayed. (B2) Nine cytokines were increased at least 2 folds in MDSCs, compared to the base line, and 8 cytokines decreased at least 2 folds in J-MDSCs, compared to MDSCs. (C) The concentration of IL-1β, GM-CSF, IL-6 and IL-10 were detected by ELISA method. * P<0.05. (D) The expression level of IDO in the co-culture system for MDSCs development with or without co-treatment with IL-1β, GM-CSF, IL-6 and IL-10. Quantification of protein levels was evaluated using the density ratio of IDO protein to β-actin. * P<0.05. (E) Specific IL-6 neutralizing antibody was used in the co-culturing system of CD33+ progenitors and MDA-MB-231 cells to block IL-6 signal in the introduction of MDSCs. The IL-6 neutralizing antibody significantly decreased STAT3 phosphorylation and expression of NIK and IDO. * P<0.05.