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Aberrant IKKα and IKKβ cooperatively activate NF-κB and induce EGFR/AP1 signaling to promote survival and migration of head and neck cancer.

Nottingham LK, Yan CH, Yang X, Si H, Coupar J, Bian Y, Cheng TF, Allen C, Arun P, Gius D, Dang L, Van Waes C, Chen Z - Oncogene (2013)

Bottom Line: Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits.Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration.Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity.

View Article: PubMed Central - PubMed

Affiliation: Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD, USA.

ABSTRACT
The inhibitor-κB kinase-nuclear factor-κB (IKK-NF-κB) and epidermal growth factor receptor-activator protein-1 (EGFR-AP1) pathways are often co-activated and promote malignant behavior, but the underlying basis for this relationship is unclear. Resistance to inhibitors of IKKβ or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and β contribute to nuclear activation of canonical and alternate NF-κB/REL family transcription factors, and overexpression of signal components that enhance co-activation of the EGFR-AP1 pathway. We observed that IKKα and IKKβ exhibit increased protein expression, nuclear localization, and phosphorylation in HNSCC tissues and cell lines. Individually, IKK activity varied among different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits. Knock down of both IKKs more effectively inhibited NF-κB activation, broadly modulated gene expression and suppressed cell proliferation and migration. Global expression profiling revealed that NF-κB, cytokine, inflammatory response and growth factor signaling are among the top pathways and networks regulated by IKKs. Importantly, IKKα and IKKβ together promoted the expression and activity of transforming growth factor α, EGFR and AP1 transcription factors cJun, JunB and Fra1. Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration. Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity. Compounds such as wedelactone with dual IKK inhibitory activity and geldanomycins that block IKKα/β and EGFR pathways were more active than IKKβ-specific inhibitors in suppressing NF-κB activation and proliferation and inducing cell death. We conclude that IKKα and IKKβ cooperatively activate NF-κB and EGFR/AP1 networks of signaling pathways and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC.

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Array profiling of gene expression after IKK single or dual knockdowns by siRNA in UM-SCC1 cellsA, Venn-diagram of down-regulated gene expressions and overlaps in IKKα, IKKβ, IKKα & IKKβ knockdown samples compared with control scramble siRNA transfected cells. B, Enriched signaling pathways were annotated among down-regulated genes, and p value (−log) depicted the significance. Ingenuity Pathway Analysis (IPA) constructed networks using the genes involved in the cytokine and NFκB pathways (C), and the growth factor signaling pathways (D).
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Figure 4: Array profiling of gene expression after IKK single or dual knockdowns by siRNA in UM-SCC1 cellsA, Venn-diagram of down-regulated gene expressions and overlaps in IKKα, IKKβ, IKKα & IKKβ knockdown samples compared with control scramble siRNA transfected cells. B, Enriched signaling pathways were annotated among down-regulated genes, and p value (−log) depicted the significance. Ingenuity Pathway Analysis (IPA) constructed networks using the genes involved in the cytokine and NFκB pathways (C), and the growth factor signaling pathways (D).

Mentions: Dual knockdown of IKKs showed significant down-regulation of genes compared to individual IKK knockdown at 48 hours using ~23K human gene profiling (Fig. 4A). Representative down-regulated genes are shown in Supplemental Fig. 1. IPA annotation indicates the genes enriched in cytokine-, immune response-, and NF-κB related pathways (top), and growth factor signaling pathways (bottom, Fig. 4B, Supplemental Table 2). The interactive networks, established using gene components in cytokines and NF-κB related pathways (Fig. 4C), or growth factor signaling pathways (Fig. 4D), reveal potential interactive relationships amongst different molecules and pathways.


Aberrant IKKα and IKKβ cooperatively activate NF-κB and induce EGFR/AP1 signaling to promote survival and migration of head and neck cancer.

Nottingham LK, Yan CH, Yang X, Si H, Coupar J, Bian Y, Cheng TF, Allen C, Arun P, Gius D, Dang L, Van Waes C, Chen Z - Oncogene (2013)

Array profiling of gene expression after IKK single or dual knockdowns by siRNA in UM-SCC1 cellsA, Venn-diagram of down-regulated gene expressions and overlaps in IKKα, IKKβ, IKKα & IKKβ knockdown samples compared with control scramble siRNA transfected cells. B, Enriched signaling pathways were annotated among down-regulated genes, and p value (−log) depicted the significance. Ingenuity Pathway Analysis (IPA) constructed networks using the genes involved in the cytokine and NFκB pathways (C), and the growth factor signaling pathways (D).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3926900&req=5

Figure 4: Array profiling of gene expression after IKK single or dual knockdowns by siRNA in UM-SCC1 cellsA, Venn-diagram of down-regulated gene expressions and overlaps in IKKα, IKKβ, IKKα & IKKβ knockdown samples compared with control scramble siRNA transfected cells. B, Enriched signaling pathways were annotated among down-regulated genes, and p value (−log) depicted the significance. Ingenuity Pathway Analysis (IPA) constructed networks using the genes involved in the cytokine and NFκB pathways (C), and the growth factor signaling pathways (D).
Mentions: Dual knockdown of IKKs showed significant down-regulation of genes compared to individual IKK knockdown at 48 hours using ~23K human gene profiling (Fig. 4A). Representative down-regulated genes are shown in Supplemental Fig. 1. IPA annotation indicates the genes enriched in cytokine-, immune response-, and NF-κB related pathways (top), and growth factor signaling pathways (bottom, Fig. 4B, Supplemental Table 2). The interactive networks, established using gene components in cytokines and NF-κB related pathways (Fig. 4C), or growth factor signaling pathways (Fig. 4D), reveal potential interactive relationships amongst different molecules and pathways.

Bottom Line: Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits.Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration.Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity.

View Article: PubMed Central - PubMed

Affiliation: Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD, USA.

ABSTRACT
The inhibitor-κB kinase-nuclear factor-κB (IKK-NF-κB) and epidermal growth factor receptor-activator protein-1 (EGFR-AP1) pathways are often co-activated and promote malignant behavior, but the underlying basis for this relationship is unclear. Resistance to inhibitors of IKKβ or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and β contribute to nuclear activation of canonical and alternate NF-κB/REL family transcription factors, and overexpression of signal components that enhance co-activation of the EGFR-AP1 pathway. We observed that IKKα and IKKβ exhibit increased protein expression, nuclear localization, and phosphorylation in HNSCC tissues and cell lines. Individually, IKK activity varied among different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits. Knock down of both IKKs more effectively inhibited NF-κB activation, broadly modulated gene expression and suppressed cell proliferation and migration. Global expression profiling revealed that NF-κB, cytokine, inflammatory response and growth factor signaling are among the top pathways and networks regulated by IKKs. Importantly, IKKα and IKKβ together promoted the expression and activity of transforming growth factor α, EGFR and AP1 transcription factors cJun, JunB and Fra1. Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration. Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity. Compounds such as wedelactone with dual IKK inhibitory activity and geldanomycins that block IKKα/β and EGFR pathways were more active than IKKβ-specific inhibitors in suppressing NF-κB activation and proliferation and inducing cell death. We conclude that IKKα and IKKβ cooperatively activate NF-κB and EGFR/AP1 networks of signaling pathways and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC.

Show MeSH
Related in: MedlinePlus