Constitutive and clinical stage-dependent down-regulation of STAT 1, 3, 4 and 5 expression and induction by Th1 cytokines in breast cancer patients

A31 Interaction of cytokines with their cognate receptors leads to the activation of latent transcription factors, STAT proteins whose biological activities ultimately regulate many critical aspects of cell growth, survival and differentiation. Dysregulation of the JAK-STAT pathway is frequently observed in both peripheral blood lymphocytes (PBL) and primary tumors, reflecting the importance of this pathway in homeostasis and immunoregulation by cytokines. In this study, we investigated the level and cytokine-mediated activation of STAT1, 3, 4, and 5 in breast cancer patients and healthy controls. PBL (5x10 6 /ml) of breast cancer patients in clinical stage I, II and IV, as well as healthy controls, were treated in vitro with culture medium (CM) alone and with IFNα (250 IU/ml CM), IL-2 (500 IU/ml), IL-12 (20 ng/ml), IL-18 (100 ng/ml) and IL-4 (20 ng/ml) for 24h at 37 C. Induction of different STAT phosphorylation was assessed in cellular lysates by Western blotting using monoclonal anti-STAT1, 3, 4, and 5 antibodies. In this study we show that in PBL of breast cancer patients, compared to healthy controls, there is a significantly lower and clinical stage-dependent decreased expression of baseline level of phosphorylated STAT 1 and undetectable baseline levels of STAT 3, 4 and 5 in clinical stage IV of disease. STAT1 expression in PBL was induced by all applied cytokines (IFNα, IL-12, IL-18, IL-12 and IL-18 combination) in a significantly lower and stage-dependent manner in breast cancer patients compared to controls. The pattern of expression of STAT 3 and 5 was the same in breast cancer patients in clinical stages I and II as in healthy controls, while there was no induction in clinical stage IV. Unlike in breast cancer patients in clinical stage II and IV, STAT4 was inducible only in clinical stage I with IL-12, IL-18 and, especially, with their combination. We also show that the induction of STAT4 by IL-12 and IL-18 is abrogated when IL-4 is added.In this study we give preliminary results that show constitutively decreased levels of investigated STATs in PBL, as well as, negative correlation of phosphorylated STAT1, 3, 4 and 5 induction with advancing clinical stage of patients with breast cancer compared to healthy controls. We show that IL-18 enhances STAT 1 and 4 induction with IL-12, whereas, IL-4, as a Th2 cytokine, significantly down-regulates these effects.These results provide evidence of disrupted STAT expression in PBL of breast cancer patients indicating that their baseline level and in vitro evaluation after treatment with different Th1 cytokines prior to immunotherapy may indicate potential benefit to cytokine-based immunotherapy in breast cancer patients.