The Mechanism For Activation Of STAT3Promoting Immunosuppressive Impact On T Cells In MDSCs Of Breast Cancer

Objective:This study aimed to elucidate the molecular mechanisms regulated STAT3mediated IDO upregulation in breast cancer derived MDSCs.Methods:30cases of peripheral blood samples from healthy donors were collected; using magnetic beads, CD33+cells were isolated and cocultured with breast cancer cell line MDA-MB-231in vitro for3days to induce MDSCs (iMDSCs). iMDSCs were co-cultured with T cell to oberve the immunosuppression effect of iMDSCs on the proliferation and secretion of T cell. The expression and activation of downstream signalings, C/EBPβ, NF-κB and non-canonical NF-κB, were detected by Western blotting. Special siRNA against these downstream signal proteins were transformed to evaluated the regulation for IDO expression. Biotinylated probe detected the potential binding sites in IDO promoter region. ChIP assay we used to identify the direct interaction between these signal proteins and promoter region of IDO. Cytocine microarray and ELISA was used to exam the levels of factors in the supernatant of iMDSCs. In order to distinguish the key upstream cytocine of STAT3/IDO pathway, corresponding cytokines or blocking antibodies were added to detect IDO expression in iMDSCs.Finally mouse4T1mammary cancer model was established toinvestigate the the therapeutic effect of the STAT3-NF-κB-IDO pathway in vivo,Results:When iMDSCs were co-cultured with T cell, the proliferation of T cells were inhibited (P<0.05),; the IDO inhibitor1-MT or STAT3inhibitor JSI-124, significantly reverse the inhibition of T cells proliferation. MDSC significantly inhibit T cells to secrete IFN-y, promote TGF-β, IL-10release (P<0.05). And offer1-MT or JSI-124treatment, the level of IFN-y was elevated, while TGF-βand IL-10decreaseed statistically significant (P<0.05). To further explore the mechanism regulated the STAT3mediated IDO expression, bioinformatics softwares were used to find STAT3potential binding sites in IDO promoter region, however through ChIP experiments no direct STAT3binding to the promoter region were identified. Western Blotting detected STAT3downstream signaling C/EBPP, NF-κB and noncanonical NF-κB, and they were activated in iMDSCs. But STAT3inhibitor JSI-124significantly reduced C/EBPP, NF-κB and noncanonical NF-κB signaling proteins. When iMDSCs tranfected with the siRNA against C/EBPP, no significant decrease in the expression of IDO was found; while tranfected with siRNA against noncanonical NF-κB, IDO expression was significantly reduced. Nuclear ELISA detected noncanonical NF-κB pathway downstream signaling proteins p52and RelB and found them was significantly increased in nuclear of iMDSCs, suggesting that noncanonical NF-κB pathway is involved in the expression of STAT3-mediated IDO expression. Bioinformatics software shown p52-RelB potential binding sites in IDO promoter.The biotin probe detected the promoter region of IDO and found high affinity and specificity sites of p52-RelB in10000bp～2000bp upstream of IDO promoter. ChIP experiments show that p52-RelB direct binding promoter region. These demonstrated STAT3activated noncanonical NF-κB pathway, causing downstream signaling p52-RelB translocation to the nucleus, direct binding promoter region, causing IDO expression. The levels of42cytokines in the supernatant of iMDSCs were examed. GRO-α,IL-6, GM-CSF,GRO,IL-1β,IL-10and MCP-3were increased in iMDSCs, ELISA kits validated the increase levels of IL-6, GM-CSF, IL-10and IL-1β in iMDSCs (P<0.05):IL-6(296.90±47.72ng/ml increased to3684.34±381.08ng/ml), GM-CSF (412.50±51.70ng/ml increased to6001.16±502.89ng/ml), IL-10(271.170±97.45ng/ml increased to2495.55±324.78ng/ml), IL-1β(97.27±19.65ng/ml increased to2736.15±495.31ng/ml). With the JSI-124, IL-6is reduced to952.38±446.19ng/ml, GM-CSF to1129.61±145.18ng/ml, IL-10to215.96±141.85ng/ml.\Adding IL-6factor in iMDSCs culture system significantly increased IDO expression, whereas IL-6neutralizing antibody significantly reduced IDO expression, suggesting that IL-6involved in regulation of STAT3-dependant IDO expression. Tumor growth was inhibited after the JSI-124treatment. In CD11b+cells of JSI-124-treated mice the expression of pSTAT3, NIK and IDO protein significantly decreased.Conclusion:The iMDSCs displayed the same suppressive function from MDSCs in situ. Increased STAT3activation in MDSCs correlated with the activation of the noncanonical NF-κB pathway, then it in turn induce IDO expression through directly binding to the IDO promoter, which was termed as STAT3-NF-κB-IDO pathway. IL-6might be the key trigger for this pathway.