GOTTARDO MarÍa Florencia
congresos y reuniones científicas
THERAPEUTIC BLOCKADE OF FOXP3 IN EXPERIMENTAL BREAST CANCER: IMMUNESTIMULATION AND DIRECT ANTITUMOR EFFECTS
ALEJANDRO J. NICOLA CANDIA; MARIELA A. MORENO AYALA; MARIA FLORENCIA GOTTARDO; ANTONELA S. ASAD; CAMILA ZUCCATO; ELISA BAL DE KIER JOFFE; ADRIANA SEILICOVICH; FLAVIA ZANETTI; MARIANELA CANDOLFI
Congreso; AACR Annual Meeting 2018; 2018
Our previous results indicate that systemic administration of a cell penetrating peptide (P60) that inhibits Foxp3, a transcription factor required for Treg function, improves the efficacy of antitumor vaccines in experimental breast cancer. We also found that P60 exerts direct antitumor effects, inhibiting tumor progression in Foxp3+ breast tumors. Although there is controversy over the role of Foxp3 in tumor cells, we found that P60 inhibits survival, proliferation and release of IL-10 in Foxp3+ breast tumor cells. Here we aimed to evaluate the pathways that control Foxp3 expression in murine LM3 breast tumor cells and to develop gene therapy vectors encoding P60 in order to improve the availability of this peptide in vivo. We assessed the role of TGF-β, mTOR and prostaglandins on the expression of Foxp3 in breast tumor cells, as these pathways have been shown to modulate Foxp3 expression in Tregs. Stimulation of LM3 cells with TGF-β and mTOR inhibitor rapamicyn upregulated Foxp3 expression, as assessed by flow cytometry, whereas indomethacin, an inhibitor of prostaglandin synthase cyclooxygenase, inhibited Foxp3 expression. These observations suggest that the regulatory mechanisms of Foxp3 expression are similar in breast tumor cells and Tregs. We next developed a plasmid that encodes P60 linked through an IRES sequence to Td.Tomato as a reporter gene (pCMV.P60.dTomato), as well a control plasmid. Transduction efficiency of these plasmids was evaluated in murine 4T1 breast tumor cells, which exhibit low expression of Foxp3 and thus are not affected by P60. Expression of Td.Tomato was readily detected by fluorescence microscopy. Conditioned media from pCMV.P60.dTomato-transfected 4T1 cells reduced the proliferation and the secretion of IL-10 in Foxp3+ LM3 cells when compared to control plasmid-transfected cells, which suggests that functional P60 is released from pCMV.P60.dTomato-transfected cells. In order to develop a gene therapeutic strategy to deliver P60 in vivo, we developed an adenoviral vector (Ad.P60.dTomato) encoding the therapeutic cassette, as well as a control vector (Ad.dTomato). These vectors successfully transduced breast tumor cells in vitro and in vivo, as evaluated by TdTomato expression. Our findings indicate that P60 could be delivered using gene therapy vectors, which could be useful to improve the survival of experimental breast cancer.