THERAPEUTIC BLOCKADE OF FOXP3 USING GENE THERAPY VECTORS

NICOLA CANDIA, ALEJANDRO J.; MORENO AYALA, MARIELA A.; GOTTARDO, M. FLORENCIA; ASAD, ANTONELA S.; ZUCCATO, CAMILA; BAL DE KIER JOFFé E; SEILICOVICH, ADRIANA; ZANETTI, FLAVIA A.; CANDOLFI, M

Congreso; Reunión conjunta de sociedades de biociencias; LXII Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2017

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. Although there is controversy over the role of Foxp3 in tumor cells, we found that P60 inhibits survival and release of IL-10 in Foxp3+ breast tumor cells. Here we aimed to evaluate the regulatory pathways that control Foxp3 expression in LM3 breast tumor cells and to develop gene therapy vectors encoding P60. We assessed the effect of recombinant TGF-â, mTOR inhibitor rapamycin and COX-2 inhibitor indomethacin, all of which modulate Foxp3 expression in Tregs. Stimulation of LM3 cells with TGF-â and rapamicyn upregulated Foxp3 expression (p<0.05) as assessed by flow cytometry, whereas indomethacin inhibited Foxp3 expression (p<0.05), suggesting that the regulatory mechanisms of Foxp3 expression are similar in tumor cells and Tregs. We next developed a plasmid that encodes P60 linked to dTomato as a reporter gene (pCMV.P60.dTomato) and a control plasmid. Transduction efficiency of these plasmids was evaluated in 4T1 cells, which exhibit low expression of Foxp3 and thus are not affected by P60. Expression of d.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 (p<0.05). In order to develop a gene therapeutic strategy to deliver P60 in vivo, we developed an adenoviral vector encoding the therapeutic cassette (Ad.P60.dTomato), as well as a control vector (Ad.dTomato). These vectors successfully transduced 4T1 cells, as evaluated by dTomato 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 models.