Abstract P4-06-04: Gamma-secretase inhibitors suppress the activation of NFκB and the expression of TNFα, IL-6 and IL-8 in triple negative breast cancer cells

Abstract

Abstract Background: Inflammation mediators, such as NF-kappa B (NFκB) and tumor necrosis factor alpha (TNFα), play major role in breast cancer pathogenesis, progression, and relapse. Triple negative breast cancer (TNBC) is a heterogeneous group of aggressive breast cancers and often has a poor outcome, in which Notch pathways are highly activated. However, role of crosstalk between Notch and inflammation mediators in TNBC progression and recurrence is poorly understood. The present study determines: 1) whether NFκB is highly activated in TNBC cells such as MDA-MB-231 (claudin-low-like) and MDA-MB-468 (basal-like), compared to ER-positive cells (MCF-7); 2) whether TNFα, IL-6 and IL-8 are highly expressed in TNBC cells, compared to MCF-7 cells; 3) whether Notch inhibition by gamma-secretase inhibitors (GSIs) significantly decrease NFκB activation and the expression of TNFα, IL-6 and IL-8 in TNBC cells; 4) whether GSI inhibits TNBC cell migration. Material and Methods: MDA-MB-231, MDA-MB-468, MCF-7 cells were cultured using RPMI 1640 media with 10% FBS. The cells were exposed to GSIs such as DAPT and RO4929097 for 18 hours. Nuclear NFκB activation was determined by using the TransAM NFκB p65 kit (Active Motif). The expressions of TNFα, IL-6, IL-8, and IFNγ were determined by the ELISA kits (R&D Systems). Migration was determined using BD BioCoat Matrigel Invasion Chamber (BD Bioscience Discovery Labware, Sedford, MA). Results: Nuclear NFκB p65 activations (A450) were 0.292±0.015, 0.222±0.005, and 0.132±0.004 in cultured MDA-MB-231, MDA-MB-468, and MCF-7 cells, respectively, in which the negative control was 0.125±0.008 and the difference between the groups were significant (P < 0.01, n=6). TNFα protein level was 266±14 pg/mg in MDA-MB-468 cells, but it was not detected in MCF-7 and MDA-MB-231 cells. IL-8 protein levels were 42.8±3, 30.3±2, and 0.23±0.01 ng/mg in MDA-MB-468, MDA-MB-231, and MCF-7 cells, respectively (P < 0.01 between groups, n=6). IL-6 protein levels were 17.1±0.9, 13.9±0.7, and 0.11±0.02 ng/mg in MDA-MB-468, MDA-MB-231, and MCF-7 cells, respectively (P < 0.01 between groups, n=6). IFNγ was not detected in these three cell lines. DAPT (20 µmol/L) or RO4929097 (10 µmol/L) caused 47.7% or 30.6% reduction in NFκB activation, 34.6% or 32.1% reduction in TNFα expression, and 22.2% or 16.4% reduction in IL-8 expression in MDA-MB-468 cells, compared to DMSO group (P < 0.01, n=6). GSIs had no effect on IL-6 expression. DAPT (20 µmol/L) significantly reduced migration of MDA-MB-468 cells by 23%, compared to DMSO group (P < 0.01, n=6). Discussion: The novel findings are 1) NFκB is highly activated in TNBC cells, compared to ER-positive cells, which is associated with significantly increased the expression of inflammation mediators such as IL-6 and IL-8 in TNBC cells and TNFα in MDA-MB-468; and 2) Notch inhibition by GSIs significantly decrease NFκB activation and the expression of TNFα, and IL-8 in TNBC cells. These results support the hypothesis that the crosstalk between Notch and NFκB lead to activation of inflammation mediators such as TNFα and IL 8, which contribute to TNBC progression and recurrence. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-04.