Abstract 1333: CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor apoptosis in breast cancer
Authors: Rosemblit Cinthia , Datta Jashodeep , Berk Erik , Czerniecki Brian J. 
Published: August 2015

Abstract
Background: The therapeutic benefit of blocking HER2/HER3 signaling in breast cancer (BC) has been demonstrated by several studies. We have previously shown that the pleiotropic T-helper type 1 (Th1) cytokines IFN-γ and TNF-α induce senescence in BC cells and that all BC cell lines tested express IFN-γ and TNF-α receptors by western blot analysis. We have also demonstrated an inverse correlation between the HER2 expression level and the senescence induced by the treatment with both cytokines. Moreover, simultaneous HER-2/HER-3 blockade significantly enhanced cytokine-induced senescence. Here, we studied whether these Th1 cytokines induce apoptosis of HER-2 expressing BC cells and assessed the impact of IFN-γ and TNF-α with simultaneous HER-2 and HER-3 blockade on permanent tumor abrogation.
Results: To determine the Th1-mediated effects on HER2high (SK-BR-3, BT-474), HER2intermediate (MCF-7, T-47D), and HER2low (MDA-MB-231) human BC cell lines in vitro, we performed co-culture of increasing number of HER2 Class II peptide-specific CD4+ T-cells (generated by priming CD4+ T cells with HER2 peptide loaded type-1 polarized DCs) with BC cells using a transwell culture system. This resulted in a cell number-dependent apoptosis of SK-BR-3 and MCF-7, but not MDA-MB-231 cells compared with CD4+ T cells primed either with immature dendritic cells (DC) or mature DC plus irrelevant (Class II BRAF) peptide. In addition, SK-BR-3 cells incubated with supernatants from the CD4+ T cells-immature DC or mature DC co-culture demonstrated similar results. Compared with controls, HER2-specific Th1 cells generated a 25-fold increase in SK-BR-3 apoptosis by DAPI staining. Neutralizing antibodies against IFN-γ and TNF-α significantly reduced apoptosis induction. Also, IFN-γ and TNF-α treatment resulted in significant apoptosis of SK-BR-3 and MCF-7, but not MDA-MB-231 cells. However, MDA-MB-231 cells transfected with a wild type HER2 plasmid, were highly susceptible to cytokine-induced apoptosis. Treatment of HER-2-depleted cells (by RNAi) with IFN-γ and TNF-α resulted in an increased apoptotic phenotype. Although the combined treatment of IFN-γ and TNF-α in HER3-depleted cells did not enhanced the apoptosis, the double knock down with HER2 and HER3 RNAi strongly increased apoptosis induction as observed by western blot analysis of active caspase-3 and flow cytometric analysis of Anexin V-PI staining. Interestingly, the double depleted cells treated with IFN-γ alone evidenced slightly higher cleaved caspase-3 levels than TNF-α alone but the combined treatment with both cytokines had a strong synergistic effect.
Conclusions: Our results establish a role for IFN-γ and TNF-α in inducing tumor apoptosis in BC. An effective CD4 Th1 response, combined with HER-2 and HER-3 blockade can significantly drive tumor apoptosis that can be explored as treatment to effectively eliminate residual BC cells and prevent recurrence.
Citation Format: Cinthia Rosemblit, Jashodeep Datta, Erik Berk, Brian J. Czerniecki. CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor apoptosis in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1333. doi:10.1158/1538-7445.AM2015-1333