Abstract LB-261: A novel notch-AKT-NF-KB axis in triple negative breast cancer cells

Abstract

Abstract BACKGROUND: Basal-like and claudin-low are two major subgroups of triple-negative breast cancers (TNBC). The clinical subtype claudin-low is represented by MDA-MB 231 cells. The Notch and NF-κB signaling pathways are known to regulate proliferation, differentiation, and apoptosis by regulating the transcription of target genes. Both pathways are commonly active in TNBC. There is strong evidence for cross-talk between these pathways: We previously published that in cervical cancer cells, Notch-1 can activate NF-kB through nuclear IKKα. We published that Notch-1 can activate the estrogen receptor alpha (ERα), also through nuclear IKKα and the IKK signalosome in T-LL cells. However, the mechanism(s) of IKK downstream of Notch remain unknown. Notch1 is highly expressed in TNBC, and Notch ligands Jagged1 and 2 correlate with poor prognosis. IKKα (CHUK) is expressed in all subtypes of TNBC. We investigated whether Notch-1 can activate NF-kB in TNBC cells of claudin-low and basal like subtypes, whether this requires IKKα and how IKKα is activated. METHODS: We used Retroviral RNAi transduction of MDA-MB231 cells to establish Notch1 siRNA. ChIP (chromatin Immunoprecipitation), CoIP (co-immunoprecipitation), Western blot, luciferase reporter assays, co-culture MDA-MB 231 cells with LTK-JAG mouse fibroblasts, qPCR, xenograft tumors in nude mice. RESULTS: We have determined that phosphorylation of Akt is mediated through a non-canonical Notch signaling pathway and this is dependent on PTEN status. Unlike the canonical Notch pathway, Notch activation by Jagged1-expressing stroma cells leads to phosphorylation of Akt within minutes of contact between Jagged1-expressing stroma cells in PTEN wt, MDA-MB 231 cells through a non-transcriptional mechanism. Our data suggest that Notch complexes with PI3K subunits, p85 and p110, after ligand-mediated activation. Inhibition of Notch activity through GSIs (γ-secretase inhibitors) leads to decreased phosphorylation of Akt. Dominant-negative AKT abolishes IKKα phosphorylation. Additionally, the combination of the AKT inhibitor, perifosine and gamma-secretase inhibitor MRK-003 is synergistic in vitro and highly active in MDA-MB231 xenografts. CONCLUSIONS: Our findings suggest a novel non-canonical cross-talk mechanism including Jagged, Notch, AKT and IKKα connecting three major oncogenic pathways in TNBC: Notch, AKT and NF-kB. Moreover, our findings suggest that Akt inhibitors may be combined with GSI for therapeutic purposes. 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 LB-261. doi:1538-7445.AM2012-LB-261