Fig. 5. MNA increases binding of Sp1 to the promoter of TNFα, increasing TNFα transcription and cytokine production.
(A) Fold change in expression of TNFα in T cells treated with MNA over T cells cultured without MNA. Means of expression (n = 5 healthy donors) with SEM shown. Data representative of at least n = 3 independent experiments. (B) NFAT and Sp1 binding to the TNFα promoter of T cells treated with or without 8 mM MNA before (Ctrl) and after 4-hour stimulation by PMA/ionomycin. Immunoglobulin G (IgG) and H3 were used as negative and positive controls, respectively, for the immunoprecipitation. Quantification of ChIP shows the fold increase in Sp1 and NFAT binding to the TNFα promoter in MNA-treated cells compared with control. Data representative of at least n = 3 independent experiments. P value determined by multiple t tests (***P < 0.01). (C) T cells (noncytotoxic) show increased expression of TNF in the tumor relative to the ascites of HGSC. Colors represent different patients. Displayed cells have been randomly subsampled to 300 and jittered to limit overplotting (**Padj = 0.0076). (D) Proposed model of MNA in ovarian cancer. MNA is produced in tumor cells and fibroblasts in the TME and taken up by the T cells. MNA increases binding of Sp1 to the promoter of TNFα, leading to increased transcription of TNFα and cytokine production of TNFα. MNA also leads to a decrease in IFN-γ. The resulting inhibition of T cell function leads to decreased killing capacity and increased tumor growth.