Immunotherapy against breast cancer based on Stat3 blockade

DE MARTINO M, MERCOGLIANO MF,TKACH M, VENTURUTTI L, ELIZALDE PV, SCHILLACI R.

Congreso; AACR 106th Annual Meeting 2015; 2015

The signal transducer and activator of transcription 3 (Stat3) is constitutively active in breast cancer (BC) and contributes to malignant transformation by promoting cell cycle progression, inhibiting apoptosis and mediating tumor immune evasion. Stat3 inhibition induces expression of proinflammatory cytokines and chemokines. On the other hand, it has been shown that oncogene inactivation induces cellular senescence and the secretion of senescence-associated secretome (SAS) in diverse tumor types. We recently described that Stat3 blockade leads to a senescence program in murine BC cells, which is accompanied by the secretion of proinflammatory cytokines. In addition, we demonstrated that immunization of mice with syngeneic Stat3 blocked BC cells induces an antitumoral immune response that involves the participation of CD4+ Th cells and cytotoxic NK cells. In this study our objectives were to study whether immunization with supernatants (SN) produced by Stat3-blocked cells induces an antitumor immune response and to explore the contribution of senescence phenotype in this response. For that purpose we used BC models of ErbB-2-positive, JIMT-1 and KPL-4 cells (human) and C4HD cells (murine), and of triple negative, MDA-MB231 cells (human) and 4T1 cell (murine). Knockdown of Stat3 with siRNA in these cells, induced senescence (assessed by acidic β-galactosidase staining). In human BC cells the senescent phenotype was accompanied by up-regulation of p21cip1 and downregulation of Rb expressions. In mouse BC cells we observed an increased expression of p16ink4a. In addition, simultaneous transfection with siRNAs targeting Stat3 and p16ink4a reverted the senescent phenotype. Then, we used a pre-clinical model in which we embedded the SN of C4HD cells in a slow-delivery depot as an adjuvant of a cellular immunotherapy. We immunized the animals with irradiated C4HD cells together with a depot containing lyophilized SN of C4HD cells transfected in vitro either with Stat3 siRNA (senescent) and Stat3 and p16ink4a siRNA (non-senescent), or a control siRNA. After 3 immunizations, with 15 d of interval between them, animals were challenged with C4HD tumor and tumor growth was monitored for 40 d. We observed that immunization with SN of cells with Stat3 siRNA decreased tumor growth vs. control siRNA group. Interestingly, there was no difference between tumor growth of animals immunized with SN of cells with Stat3 siRNA vs Stat3 and p16ink4a siRNA. We observed in these two groups greater cytotoxic activity of NK cells and an increase in the number of memory CD4+ T cells vs. control. These results suggest that Stat3 blockade drives a senescence program also in human BC cells. The secretome of Stat3-blocked BC cells is an effective adjuvant and could be formulated for immunotherapies independently of the SAS. Defining the protein, peptide, cytokine, chemokine composition of this supernatant opens up new avenues for immunotherapy in cancer patients.