CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor apoptosis in breast cancer

ROSEMBLIT C ; DATTA J; BERK E; CZERNIECKI BJ

Philadelphia, PA

Congreso; Annual Meeting; 2015

American Association for Cancer Research

Background: The therapeuticbenefit of blocking HER2/HER3 signaling in breast cancer (BC) has been demonstratedby several studies. We have previously shown that the pleiotropic T-helper type1 (Th1) cytokines IFN-γ and TNF-α induce senescence in BC cells and that all BC celllines tested express IFN-γ and TNF-α receptors by westernblot analysis. We have also demonstrated an inverse correlation between theHER2 expression level and the senescence induced by the treatment with both cytokines. Moreover, simultaneous HER-2/HER-3 blockade significantly enhancedcytokine-induced senescence. Here, we studied whether these Th1 cytokinesinduce apoptosis of HER-2 expressing BC cells and assessed the impact of IFN-γ and TNF-α with simultaneous HER-2and HER-3 blockade on permanent tumor abrogation.Results: Todetermine 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, weperformed co-culture of increasing number of HER2 Class II peptide-specific CD4+T-cells (generated by priming CD4+ T cells with HER2 peptide loadedtype-1 polarized DCs) with BC cells using a transwell culture system. This resulted in a cell number-dependent apoptosis of SK-BR-3and MCF-7, but not MDA-MB-231 cells compared with CD4+T cells primed either with immature dendritic cells (DC) or mature DC plusirrelevant (Class II BRAF) peptide. In addition, SK-BR-3 cells incubated withsupernatants from the CD4+ T cells-immature DC or mature DCco-culture demonstrated similar results. Compared with controls,HER2-specific Th1 cells generated a 25-fold increase in SK-BR-3 apoptosis byDAPI staining. Neutralizing antibodies against IFN-γ and TNF-α significantly reduced apoptosisinduction. Also, IFN-γ and TNF-α treatment resulted in significantapoptosis of SK-BR-3 and MCF-7, but not MDA-MB-231 cells. However, MDA-MB-231 cells transfected with a wildtype 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 apoptoticphenotype. Althoughthe combined treatment of IFN-γ and TNF-α inHER3-depleted cells did not enhanced the apoptosis, the double knock down withHER2 and HER3 RNAi strongly increased apoptosis induction as observed bywestern blot analysis of active caspase-3 and flow cytometric analysis of AnexinV-PI staining. Interestingly, the double depleted cells treated with IFN-γ alone evidenced slightlyhigher cleaved caspase-3 levels than TNF-α alone but the combined treatment with bothcytokines had a strong synergistic effect. Conclusions:Our results establish a role for IFN-γ and TNF-α ininducing tumor apoptosis in BC. An effective CD4 Th1 response, combined with HER-2and HER-3 blockade can significantly drive tumor apoptosis that can be exploredas treatment to effectively eliminate residual BC cells and prevent recurrence.