Abstract 1549: Targeting Stat3 induces senescence in breast cancer cells and elicits an immune response inhibiting tumor growth and metastasis
Authors: Tkach Mercedes , Rivas Martin A. , Proietti Cecilia J. , Flaqué María C. Díaz , Frahm Isabel , Charreau Eduardo H. , Elizalde Patricia V. , Schillaci Roxana 
Published: August 2012

Abstract
Having in mind that Stat3 inhibition in tumor cells induces the expression of chemokines and pro-inflammatory cytokines, we proposed the use of Stat3-inhibited breast cancer cells as a source of immunogens to induce an anti-tumor immune response. We have demonstrated that the administration of irradiated breast cancer cells that express a dominant negative (DN) form of Stat3 (Stat3Y705F-breast cancer cells) provides protection against the murine progestin-dependent C4HD tumor, through the activation of CD4+ T cells and cytotoxic natural killer (NK) cells. To extend our results to different breast cancer models, we worked with the hormone independent 4T1 mammary carcinoma cell line that displays constitutive activation of Stat3. Immunization with irradiated Stat3Y705F-4T1 cells prevented wild-type 4T1 tumor development in 50% of the challenged mice (P&lt;0.001), and the tumor-bearing mice displayed tumors of smaller size (81% decrease in tumor volume, P&lt;0.001) when compared to mice injected with pcDNA3.1-4T1 cells. Moreover, the number of metastasis per lung decreased by 90% in Stat3Y705F-4T1-immunized animals (P&lt;0.05). When we analyzed the tumor milieu composition by flow cytometry, we observed that Stat3Y705F-4T1 immunized animals displayed an increase in the percentage of tumor infiltrating NK cells (CD3-DX5+) and a decrease in tumor infiltrating T regulatory lymphocytes (CD4+CD25+FoxP3+), compared to pcDNA3.1-4T1-immunized mice. In order to evaluate if this vaccination may be effective in a therapeutic setting, we immunized mice with irradiated Stat3Y705F-4T1 or pcDNA3.1-4T1 cells, 4, 11 and 18 days after challenging with 4T1 cells. On day 35, we observed a significant decrease on tumor volume and growth rate in Stat3Y705F-4T1 cell-immunized animals, when compared to mice immunized with pcDNA3.1-4T1 cells (37.5%, P&lt;0.05) and a decrease in the number of metastasis per lung (P&lt;0.05). On the other hand, cellular senescence is an important mechanism of tumor regression upon oncogene inactivation that leads to the secretion of pro-inflammatory cytokines that resemble the ones we found after blocking Stat3. Therefore, we wondered whether Stat3 inhibition could drive a senescence program. Inhibition of Stat3 in murine C4HD and 4T1 cells by transfection with Stat3Y705F, or Stat3 silencing by siRNA, resulted in increased senescence-associated-β-galactosidase (SA-α-gal) accumulation and increased expression of the senescence-associated markers p15INK4b and p16INK4a. As cellular senescence is associated to chromatin changes, we studied heterochromatin formation and observed an increase of trimethyl-K4 histone H3 upon Stat3 inhibition. As a whole, our findings indicate that Stat3 inhibition in breast cancer cells induce an increase in immunogenicity capable of eliciting an anti tumor immune response, presumably through the activation of a senescence program.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1549. doi:1538-7445.AM2012-1549