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Non-canonical Notch signaling activates IL-6/JAK/STAT signaling in breast tumor cells and is controlled by p53 and IKKβ

ABSTRACT: Notch signaling is frequently hyperactivated in breast cancer, but how the enhanced signaling contributes to the tumor process is less well understood. In this report, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a novel Notch target in breast tumor cells. Enhanced Notch signaling upregulated IL-6 expression at the transcriptional level, leading to activation of autocrine and paracrine JAK/STAT signaling. IL-6 upregulation was mediated by non-canonical Notch signaling, as it could be effectuated by a cytoplasmically localized Notch intracellular domain and was independent on the DNA-binding protein CSL. Instead, Notch-mediated IL-6 upregulation was controlled by two other factors: IKKβ, a protein in the NF-kB signaling cascade, and p53. Activation of IL-6 by Notch required IKKβ function, but interestingly, did not engage canonical NF-κB signaling, in contrast to IL-6 activation by inflammatory agents such as tumor necrosis factor, which requires canonical NF-κB signaling. With regard to p53 status, IL-6 expression was upregulated by Notch when p53 was mutated or lost, but restoring wildtype 53 into p53-mutated or -deficient cells abrogated the IL-6 upregulation. Furthermore, Notch-induced genome-wide transcriptomes from p53 wildtype and -mutated breast tumor cell lines differed extensively, and in a subset of genes upregulated by Notch in a p53-mutant cell line, upregulation was reduced by wildtype p53. In conclusion, we identify IL-6 as a novel non-canonical Notch target gene, and reveal roles for p53 and IKKβ in non-canonical Notch signaling in breast cancer and in the generation of cell context-dependent diversity in the Notch signaling output.

ORGANISM(S): Homo sapiens

PROVIDER: GSE36051 | GEO | 2012/06/30

SECONDARY ACCESSION(S): PRJNA151783

REPOSITORIES: GEO

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Project description:Notch signaling is frequently hyperactivated in breast cancer, but how the enhanced signaling contributes to the tumor process is less well understood. In this report, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a novel Notch target in breast tumor cells. Enhanced Notch signaling upregulated IL-6 expression at the transcriptional level, leading to activation of autocrine and paracrine JAK/STAT signaling. IL-6 upregulation was mediated by non-canonical Notch signaling, as it could be effectuated by a cytoplasmically localized Notch intracellular domain and was independent on the DNA-binding protein CSL. Instead, Notch-mediated IL-6 upregulation was controlled by two other factors: IKKÎ², a protein in the NF-kB signaling cascade, and p53. Activation of IL-6 by Notch required IKKÎ² function, but interestingly, did not engage canonical NF-ÎºB signaling, in contrast to IL-6 activation by inflammatory agents such as tumor necrosis factor, which requires canonical NF-ÎºB signaling. With regard to p53 status, IL-6 expression was upregulated by Notch when p53 was mutated or lost, but restoring wildtype 53 into p53-mutated or -deficient cells abrogated the IL-6 upregulation. Furthermore, Notch-induced genome-wide transcriptomes from p53 wildtype and -mutated breast tumor cell lines differed extensively, and in a subset of genes upregulated by Notch in a p53-mutant cell line, upregulation was reduced by wildtype p53. In conclusion, we identify IL-6 as a novel non-canonical Notch target gene, and reveal roles for p53 and IKKÎ² in non-canonical Notch signaling in breast cancer and in the generation of cell context-dependent diversity in the Notch signaling output. 30 microarray samples consisting of MCF7 (ER+, wild-type p53, luminal type B breast cancer) and MDA-MB-231 (ER-, mutated p53, basal breast cancer) cells cultured on immobilized 1 Î¼g/ml JAGGED1-Fc or 1 Î¼g/ml DLL4-Fc or 1 Î¼g/ml Fc control with or without 5 Î¼M DAPT for 6 hours in 3 biological replicates.

Project description:Microglial overactivation is actively involved in the pathogenesis of neurodegenerative diseases. Polo-like kinase 2 (PLK2) is a serine/threonine protein kinase associated with the regulation of synaptic plasticity and centriole duplication. Here, we identified PLK2 as an important early response gene in lipopolysaccharide (LPS)-stimulated microglial cells. Knockdown or inhibition of PLK2 remarkably attenuated LPS-induced expression of pro-inflammatory factors such as IL-1β, IL-6, COX2, TNF-α,and iNOS in microglial cells via suppressing the IKKβ-NF-κB signaling pathway.Notably, overexpression of PLK2 induced expression of pro-inflammatory factors and NF-κB transcriptional activation in the absence of inflammatory stimuli. Mechanistically,co-immunoprecipitation experiments revealed association between PLK2 and IKKβ, whereas GST pull-down assay showed no direct interaction between PLK2 and IKKβ. Proteomic analysis and in vitro kinase assay identified heat shock protein 90 alpha (HSP90α), a regulator of IKKβ activity, as a novel PLK2 substrate. Knockdown or pharmacological inhibition of HSP90α abolished PLK2-mediated activation of NF-κB transcriptional activity and microglial inflammatory activation. Furthermore,phosphoproteomic analysis pinpointed Ser252 and Ser263 on HSP90α as novel phosphorylation targets of PLK2. Subsequent functional studies demonstrated that re-expression of phosphor-dead mutation of these two phosphorylation sites on HSP90α failed to rescue the PLK2-induced activation of the NF-κB signaling. Lastly, conditional knockout of PLK2 in microglial cells dramatically ameliorated neuroinflammation and subsequent dopaminergic neuron loss in an intracranial LPS-induced mouse PD model. The present study revealed, for the first time, that PLK2 promoted microglial activation through the phosphorylation of HSP90α and subsequent activation of the IKKβ-NF-κB signaling pathway. Consequently, PLK2 emerges as a potential therapeutic target for the amelioration of neuroinflammation-related diseases.

Project description:Microglial overactivation is actively involved in the pathogenesis of neurodegenerative diseases. Polo-like kinase 2 (PLK2) is a serine/threonine protein kinase associated with the regulation of synaptic plasticity and centriole duplication. Here, we identified PLK2 as an important early response gene in lipopolysaccharide (LPS)-stimulated microglial cells. Knockdown or inhibition of PLK2 remarkably attenuated LPS-induced expression of pro-inflammatory factors such as IL-1β, IL-6, COX2, TNF-α, and iNOS in microglial cells via suppressing the IKKβ-NF-κB signaling pathway. Notably, overexpression of PLK2 induced expression of pro-inflammatory factors and NF-κB transcriptional activation in the absence of inflammatory stimuli. Mechanistically, co-immunoprecipitation experiments revealed association between PLK2 and IKKβ, whereas GST pull-down assay showed no direct interaction between PLK2 and IKKβ. Proteomic analysis and in vitro kinase assay identified heat shock protein 90 alpha (HSP90α), a regulator of IKKβ activity, as a novel PLK2 substrate. Knockdown or pharmacological inhibition of HSP90α abolished PLK2-mediated activation of NF-κB transcriptional activity and microglial inflammatory activation. Furthermore, phosphoproteomic analysis pinpointed Ser252 and Ser263 on HSP90α as novel phosphorylation targets of PLK2. Subsequent functional studies demonstrated that re-expression of phosphor-dead mutation of these two phosphorylation sites on HSP90α failed to rescue the PLK2-induced activation of the NF-κB signaling. Lastly, conditional knockout of PLK2 in microglial cells dramatically ameliorated neuroinflammation and subsequent dopaminergic neuron loss in an intracranial LPS-induced mouse PD model. The present study revealed, for the first time, that PLK2 promoted microglial activation through the phosphorylation of HSP90α and subsequent activation of the IKKβ-NF-κB signaling pathway. Consequently, PLK2 emerges as a potential therapeutic target for the amelioration of neuroinflammation-related diseases.

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Non-canonical Notch signaling activates IL-6/JAK/STAT signaling in breast tumor cells and is controlled by p53 and IKKβ

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