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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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A combinatory effect of dual IKKα and IKKβ knockdown by siRNA in blocking NF-κB signaling molecule expression and reporter activityA, Knockdown efficiency of IKKα and IKKβ individually or in combination, and effects on downstream NF-κB subunits RelA, p-RelA (ser536), p105, p50, RelB, p100, p52, p-p52 and p-IκBα (ser32/36) after knockdown for 48 hours and treatment with TNF-α for 1 hour. β-tubulin and Histone1 served as loading and fractionation controls. B, Knockdown of IKKs affected mRNA expression of NF-κB p100/p52 subunits. UM-SCC1 cells were transfected with siRNA of IKKs under the same experimental condition as shown above, and RNAs were harvested and relative fold-change in mRNA expression was measured via quantitative RT-PCR. Starred (*) values indicate a p-value<0.05 by t-test vs. negative control. p-p100 and p52 subunits in cytoplasmic (C) or nuclear (D) fraction were tested after IKK knockdown for 44 hours and treatment with lymphotoxin beta (α1/β2; 100ng/ml) for 4 hours. E, Greater IκBα luciferase fusion protein was observed in the double IKK knockdown than individual knockdowns. F, Conversely, the greater combinatory inhibitory effect of NF-κB reporter activity was observed in the IKK double knockdown over the single knockdowns in UM-SCC1 (left), UM-SCC11B (right), and UM-SCC6 (data not shown). Statistical significance of t-test, * single knockdowns compared to negative control: p<0.01; ** double knockdowns compared to negative control: p<0.001; ++ dual knockdowns compared to single knockdowns: p<0.001.
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Figure 3: A combinatory effect of dual IKKα and IKKβ knockdown by siRNA in blocking NF-κB signaling molecule expression and reporter activityA, Knockdown efficiency of IKKα and IKKβ individually or in combination, and effects on downstream NF-κB subunits RelA, p-RelA (ser536), p105, p50, RelB, p100, p52, p-p52 and p-IκBα (ser32/36) after knockdown for 48 hours and treatment with TNF-α for 1 hour. β-tubulin and Histone1 served as loading and fractionation controls. B, Knockdown of IKKs affected mRNA expression of NF-κB p100/p52 subunits. UM-SCC1 cells were transfected with siRNA of IKKs under the same experimental condition as shown above, and RNAs were harvested and relative fold-change in mRNA expression was measured via quantitative RT-PCR. Starred (*) values indicate a p-value<0.05 by t-test vs. negative control. p-p100 and p52 subunits in cytoplasmic (C) or nuclear (D) fraction were tested after IKK knockdown for 44 hours and treatment with lymphotoxin beta (α1/β2; 100ng/ml) for 4 hours. E, Greater IκBα luciferase fusion protein was observed in the double IKK knockdown than individual knockdowns. F, Conversely, the greater combinatory inhibitory effect of NF-κB reporter activity was observed in the IKK double knockdown over the single knockdowns in UM-SCC1 (left), UM-SCC11B (right), and UM-SCC6 (data not shown). Statistical significance of t-test, * single knockdowns compared to negative control: p<0.01; ** double knockdowns compared to negative control: p<0.001; ++ dual knockdowns compared to single knockdowns: p<0.001.

Mentions: To define the roles of endogenous IKKα and IKKβ on NF-κB/REL activation, we knocked down both IKK subunits individually and in combination using siRNAs. We first examined the effects of IKK knockdown in UM-SCC1 cells treated with TNF-α, an inducer of canonical signaling which is detected in human HNSCC tumor specimens (28). The efficacy and specificity of the knockdowns were verified in cytoplasmic and nuclear fractions in UM-SCC1 (Fig. 3A). Canonical subunit RELA was modestly inhibited by IKKα or β knockdown, and strongly inhibited by dual IKK knockdown in both the cytoplasm and nucleus. A similar stepwise reduction from individual to dual IKK inhibition was observed for cytoplasmic phospho-RELA (p-RELA) at Serine536, while individual IKK knockdown enhanced nuclear p-RELA relative to basal or dual knockdown. The NF-κB1 subunit involved in the canonical pathway, p105 and its processed product p50, were also reduced after IKKα or β individual knockdown, while the strongest effects were observed after knockdown of both. We detected expression of alternate NF-κB2 p100 protein predominantly localized in the cytoplasm, while processed p52 and RelB predominantly localized in the nuclear fraction (Fig. 3A). Interestingly, expression of the NF-κB subunit p100, and its processed product, p52, were suppressed more by IKKβ knockdown, while dual knockdown was the most effective in inhibiting the nuclear fraction. These protein expression data for p100/p52 can be largely explained by the effects of IKK siRNAs on p100/p52 mRNA, as blocking IKKβ exhibited a stronger inhibitory effect on p100/p52 mRNA expression than knockdown of IKKα, while knockdown of both IKKs exhibited the strongest effects (Fig. 3B). Knockdown of IKKs did not significantly affect RELA gene expression (Fig. 3B). These data are consistent with previous observations that the canonical pathway is reported to regulate transcription of alternate pathway subunits NF-κB2/p100 precursor of p52 (29, 30). In addition, IKKα knockdown preferentially inhibited nuclear RelB along with p-p52 at Ser222 (Fig. 3A), a site implicated in p52 dimer and heterodimer formation recently identified by Perkins and colleagues (31, 32). Further, under TNFα stimulation, we did not detect significant induction of p-p100 (ser866/870), a marker for alternate IKKα pathway signal mediated processing, in the cytoplasm or nucleus (data not shown). p-IκBα, the downstream target of canonical IKK activation, was more strongly inhibited by IKKβ–specific or combinatory knockdowns in the cytoplasmic or nuclear fraction. Together, the greater effects of dual inhibition upon the cytoplasmic-nuclear translocation and phosphorylation of NF-κB subunits and IκBα are consistent with predominantly canonical IKKα/β-dependent activation of these subunits in response to inflammatory signaling by TNF-α.


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)

A combinatory effect of dual IKKα and IKKβ knockdown by siRNA in blocking NF-κB signaling molecule expression and reporter activityA, Knockdown efficiency of IKKα and IKKβ individually or in combination, and effects on downstream NF-κB subunits RelA, p-RelA (ser536), p105, p50, RelB, p100, p52, p-p52 and p-IκBα (ser32/36) after knockdown for 48 hours and treatment with TNF-α for 1 hour. β-tubulin and Histone1 served as loading and fractionation controls. B, Knockdown of IKKs affected mRNA expression of NF-κB p100/p52 subunits. UM-SCC1 cells were transfected with siRNA of IKKs under the same experimental condition as shown above, and RNAs were harvested and relative fold-change in mRNA expression was measured via quantitative RT-PCR. Starred (*) values indicate a p-value<0.05 by t-test vs. negative control. p-p100 and p52 subunits in cytoplasmic (C) or nuclear (D) fraction were tested after IKK knockdown for 44 hours and treatment with lymphotoxin beta (α1/β2; 100ng/ml) for 4 hours. E, Greater IκBα luciferase fusion protein was observed in the double IKK knockdown than individual knockdowns. F, Conversely, the greater combinatory inhibitory effect of NF-κB reporter activity was observed in the IKK double knockdown over the single knockdowns in UM-SCC1 (left), UM-SCC11B (right), and UM-SCC6 (data not shown). Statistical significance of t-test, * single knockdowns compared to negative control: p<0.01; ** double knockdowns compared to negative control: p<0.001; ++ dual knockdowns compared to single knockdowns: p<0.001.
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Figure 3: A combinatory effect of dual IKKα and IKKβ knockdown by siRNA in blocking NF-κB signaling molecule expression and reporter activityA, Knockdown efficiency of IKKα and IKKβ individually or in combination, and effects on downstream NF-κB subunits RelA, p-RelA (ser536), p105, p50, RelB, p100, p52, p-p52 and p-IκBα (ser32/36) after knockdown for 48 hours and treatment with TNF-α for 1 hour. β-tubulin and Histone1 served as loading and fractionation controls. B, Knockdown of IKKs affected mRNA expression of NF-κB p100/p52 subunits. UM-SCC1 cells were transfected with siRNA of IKKs under the same experimental condition as shown above, and RNAs were harvested and relative fold-change in mRNA expression was measured via quantitative RT-PCR. Starred (*) values indicate a p-value<0.05 by t-test vs. negative control. p-p100 and p52 subunits in cytoplasmic (C) or nuclear (D) fraction were tested after IKK knockdown for 44 hours and treatment with lymphotoxin beta (α1/β2; 100ng/ml) for 4 hours. E, Greater IκBα luciferase fusion protein was observed in the double IKK knockdown than individual knockdowns. F, Conversely, the greater combinatory inhibitory effect of NF-κB reporter activity was observed in the IKK double knockdown over the single knockdowns in UM-SCC1 (left), UM-SCC11B (right), and UM-SCC6 (data not shown). Statistical significance of t-test, * single knockdowns compared to negative control: p<0.01; ** double knockdowns compared to negative control: p<0.001; ++ dual knockdowns compared to single knockdowns: p<0.001.
Mentions: To define the roles of endogenous IKKα and IKKβ on NF-κB/REL activation, we knocked down both IKK subunits individually and in combination using siRNAs. We first examined the effects of IKK knockdown in UM-SCC1 cells treated with TNF-α, an inducer of canonical signaling which is detected in human HNSCC tumor specimens (28). The efficacy and specificity of the knockdowns were verified in cytoplasmic and nuclear fractions in UM-SCC1 (Fig. 3A). Canonical subunit RELA was modestly inhibited by IKKα or β knockdown, and strongly inhibited by dual IKK knockdown in both the cytoplasm and nucleus. A similar stepwise reduction from individual to dual IKK inhibition was observed for cytoplasmic phospho-RELA (p-RELA) at Serine536, while individual IKK knockdown enhanced nuclear p-RELA relative to basal or dual knockdown. The NF-κB1 subunit involved in the canonical pathway, p105 and its processed product p50, were also reduced after IKKα or β individual knockdown, while the strongest effects were observed after knockdown of both. We detected expression of alternate NF-κB2 p100 protein predominantly localized in the cytoplasm, while processed p52 and RelB predominantly localized in the nuclear fraction (Fig. 3A). Interestingly, expression of the NF-κB subunit p100, and its processed product, p52, were suppressed more by IKKβ knockdown, while dual knockdown was the most effective in inhibiting the nuclear fraction. These protein expression data for p100/p52 can be largely explained by the effects of IKK siRNAs on p100/p52 mRNA, as blocking IKKβ exhibited a stronger inhibitory effect on p100/p52 mRNA expression than knockdown of IKKα, while knockdown of both IKKs exhibited the strongest effects (Fig. 3B). Knockdown of IKKs did not significantly affect RELA gene expression (Fig. 3B). These data are consistent with previous observations that the canonical pathway is reported to regulate transcription of alternate pathway subunits NF-κB2/p100 precursor of p52 (29, 30). In addition, IKKα knockdown preferentially inhibited nuclear RelB along with p-p52 at Ser222 (Fig. 3A), a site implicated in p52 dimer and heterodimer formation recently identified by Perkins and colleagues (31, 32). Further, under TNFα stimulation, we did not detect significant induction of p-p100 (ser866/870), a marker for alternate IKKα pathway signal mediated processing, in the cytoplasm or nucleus (data not shown). p-IκBα, the downstream target of canonical IKK activation, was more strongly inhibited by IKKβ–specific or combinatory knockdowns in the cytoplasmic or nuclear fraction. Together, the greater effects of dual inhibition upon the cytoplasmic-nuclear translocation and phosphorylation of NF-κB subunits and IκBα are consistent with predominantly canonical IKKα/β-dependent activation of these subunits in response to inflammatory signaling by TNF-α.

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