Immunotherapy against Breast Cancer Based on Cellular Senescence Induced by Targeting Stat3

TKACH, M; DE MARTINO M; MERCOGLIANO MF; VENTURUTTI, L; CORTESE E; SANTOMINGO G; BRANCATTO C; ELIZALDE PV; SCHILLACI R

New York

Simposio; 21th Annual Cancer Reseach Institute International Cancer Immunotherapy Symposium; 2013

Our laboratory has recently described an effective pre-clinical immunotherapy (IT) that inhibits breast cancer growth and metastasis dissemination based on immunization with tumor cells with blocked signal transducer and activator of transcription 3 (Stat3). This immunization protocol inhibited wild-type in vivo tumor growth of C4HD and 4T1 murine breast cancer cells in BALB/c mice, through the activation of a cellular immune response involving CD4+Th cells and cytotoxic NK cells. Interestingly, we observed that Stat3 blockage leads to a senescence program, caused by oncogene inactivation, accompanied by the secretion of different pro-inflammatory cytokines. In the present work we studied the effect of supernatant (SN) from senescent breast cancer cells, induced by Stat3 blockage, on human NK cell activation. First, we evaluated whether knock-down of Stat3 expression was able to induce cellular senescence, determined by -galatosidase staining, in human breast cancer cell lines. We observed that MDA-MB-231, JIMT-1 and KPL-4 cell lines underwent senescence after transfection with siRNAs directed to Stat3. MDA-MB-231 cells also showed up-regulation of p21CIP1 expression. On the other hand, we could not detect a senescence phenotype in BT-474 cell line after Stat3 blockage. Then, we cultured NK cells from healthy donors or from breast cancer patients (stage II with nodal metastasis at diagnosis) with a mixture (1:1) of fresh culture media and the SN of MDA-MB231 cells transfected with a Control siRNA (SN-Control) or siRNA to Stat3 (SN-Stat3). Immunofluorescence and flow cytometry analysis showed an increase in the cell activation markers HLA-DR, CD25 and CD7 in NK cells from healthy donors cultured with SN-Stat3 in comparison to NK cells cultured with SN-control (n=5). NK cells from breast cancer patients were able to up-regulate HLA-DR, CD25 and CD7 (n=3) or failed to modulate these markers (n=4) upon treatment with SN-Stat3. These results show that the SN from senescent cells can induce activation of NK cells from healthy donors and from some breast cancer patients. In this sense, exploration of the quali/quantitative composition of the SN from Stat3-blocked senescent cells remains an attractive task for developing a potential adjuvant formulation, useful for antibody or peptide-based IT.