NF-κB regulates GDF-15 to suppress macrophage surveillance during early tumor development
Nivedita M. Ratnam, … , David J. Wang, Denis C. Guttridge
Nivedita M. Ratnam, … , David J. Wang, Denis C. Guttridge
Published September 11, 2017
Citation Information: J Clin Invest. 2017;127(10):3796-3809. https://doi.org/10.1172/JCI91561.
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Research Article Cell biology Immunology

NF-κB regulates GDF-15 to suppress macrophage surveillance during early tumor development

Abstract

Macrophages are attracted to developing tumors and can participate in immune surveillance to eliminate neoplastic cells. In response, neoplastic cells utilize NF-κB to suppress this killing activity, but the mechanisms underlying their self-protection remain unclear. Here, we report that this dynamic interaction between tumor cells and macrophages is integrally linked by a soluble factor identified as growth and differentiation factor 15 (GDF-15). In vitro, tumor-derived GDF-15 signals in macrophages to suppress their proapoptotic activity by inhibiting TNF and nitric oxide (NO) production. In vivo, depletion of GDF-15 in Ras-driven tumor xenografts and in an orthotopic model of pancreatic cancer delayed tumor development. This delay correlated with increased infiltrating antitumor macrophages. Further, production of GDF-15 is directly regulated by NF-κB, and the colocalization of activated NF-κB and GDF-15 in epithelial ducts of human pancreatic adenocarcinoma supports the importance of this observation. Mechanistically, we found that GDF-15 suppresses macrophage activity by inhibiting TGF-β–activated kinase (TAK1) signaling to NF-κB, thereby blocking synthesis of TNF and NO. Based on these results, we propose that the NF-κB/GDF-15 regulatory axis is important for tumor cells in evading macrophage immune surveillance during the early stages of tumorigenesis.

Authors

Nivedita M. Ratnam, Jennifer M. Peterson, Erin E. Talbert, Katherine J. Ladner, Priyani V. Rajasekera, Carl R. Schmidt, Mary E. Dillhoff, Benjamin J. Swanson, Ericka Haverick, Raleigh D. Kladney, Terence M. Williams, Gustavo W. Leone, David J. Wang, Denis C. Guttridge

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Figure 4

NF-κB is a direct regulator of Gdf-15.

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NF-κB is a direct regulator of Gdf-15. (A) p65–/– MEFs were infected wit...
(A) p65–/– MEFs were infected with retrovirus for full-length (p65WT) or truncated p65 (p65ΔTAD). Gdf-15 expression by qRT-PCR was assayed following TNF treatment (5 ng/ml) for 2 hours. n = 3. Data are shown as mean ± SEM. *P ≤ 0.005, 1-way ANOVA. (B) ELISA from conditioned media from transfected cells in A. n = 3. Data are shown as mean ± SEM. *P ≤ 0.05, 1-way ANOVA. (C) Schematic of Gdf-15 gene with NF-κB consensus site in exon 2, compared with consensus site in mouse and human. (D) EMSA from TNF-treated Ras MEFs using NF-κB consensus site probe shown in C. Supershift assay from Ras MEFs incubated with antisera specific for p65 and p50. Asterisks indicate supershifted complexes. Specificity of the complexes was tested by adding ×1000 molar excess of labeled WT probe or nonlabeled mutant probe. (E) ChIP assays for p65 binding from Ras MEFs. DNA was amplified with oligonucleotides spanning the NF-κB site on exon 2 of Gdf-15. Fold enrichment over IgG controls (normalized to input) are indicated. n = 3. *P ≤ 0.05, Student’s t test. (F) ChIP for pPol II (serine 2 version), as described in Figure 4E. DNA was amplified with the same oligonucleotides as shown in E. Fold enrichment over IgM controls (normalized to input) are indicated. n = 3. *P ≤ 0.05. Student’s t test. (G) MEFs were transfected with a luciferase reporter with WT or mutated NF-κB consensus sites, as shown in C. Cells were cotransfected with H-RasG12V, and after 48 hours, luciferase activity was measured. n = 3. *P ≤ 0.05 compared with p65+/+ MEFs with WT construct+ H-Ras, 2-way ANOVA. (H) MEFs were transfected with WT and mutant luciferase reporters and treated with 1 μl/ml of TNF for 2 hours. Luciferase activity was measured. n = 3. *P ≤ 0.05 when compared with p65+/+ MEFs with WT construct with TNF, 2-way ANOVA.