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

Abstract

The Inhibitor-κB Kinase-Nuclear Factor-κB (IKK-NF-κB) and Epidermal Growth Factor Receptor-Activator Protein-1 (EGFR-AP-1) 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 enhancing 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 amongst different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knockdown of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα, and alternative p52 and RELB subunits. Knockdown 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 TGFα, EGFR, and AP1 transcription factors cJun, JunB, and Fra1. Knockdown 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 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.

NF‐κB inhibition reverses acidic bile‐induced miR‐21, miR‐155, miR‐192, miR‐34a, miR‐375 and miR‐451a deregulations in human hypopharyngeal cells

“…Our novel data demonstrate strong inverted correlations among the BAY 11‐7082‐induced levels of the analysed tumour suppressors” miR‐34a, miR‐375 and miR451a, and NF‐κB‐related genes, such as RELA(p65), STAT3, TNF‐α, IL‐6 and IL‐1β, that previous studies documented as crucial mediators of inflammatory and neoplastic events in head and neck cancer35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 (Figure 7). In line with our recent study,33 our current data suggest that NF‐κB inhibition may reverse acidic bile‐induced molecular events in normal human hypopharyngeal cells that are known to link inflammation to cancer, thereby in a sense shielding HHPC from the effects of bile‐induced oncogenic molecular events.…”

“…Nuclear factor kappa B (NF‐κB) is a key factor that mediates inflammatory and early tumorigenic events in epithelial cells,35 and its importance in initiation and progression of cancer, including head and neck cancer, has been widely supported 23, 36, 37, 38, 39, 40, 41 by its interactions with a complex network of other cancer‐related transcriptional factors, cytokines and growth factors 34, 42, 43, 44, 45, 46. Additionally, Van Waes and Chen recently showed a cluster of genes and miRNA markers that are related to activated NF‐κB and that may contribute to an aggressive phenotype of head and neck cancer 23, 38…”

EGFR and NF-κB: partners in cancer

“…NF-kB subsequently translocates to the nucleus to drive the expression of its target genes. 6 EGFR mRNA and protein levels [43]. In contrast to this NF-kB signaling pathway that positively regulates EGFR signaling, EGFR expression is negatively regulated at the transcriptional level by receptor-interacting kinase (RIPK1), which is typically activated by proinflammatory and NF-kB-activating cytokines such as TNFa [44].…”

“…These studies highlighted the need to design new combinatorial approaches by using a morespecific NF-kB inhibitor than bortezomib, which blocks the degradation of IkBa as well as of numerous other proteins, including key oncogenic products [68]. Interestingly, both IKKa and IKKb are aberrantly activated in HNSCC, act as mediators of NF-kB activation, and enhance EGFR signaling in HNSCC [43]. Consistently, wedelactone, a dual IKKa/b inhibitor, more effectively inhibited NF-kB activation than MLN120b, a specific IKKb inhibitor [43,69].…”

“…Interestingly, both IKKa and IKKb are aberrantly activated in HNSCC, act as mediators of NF-kB activation, and enhance EGFR signaling in HNSCC [43]. Consistently, wedelactone, a dual IKKa/b inhibitor, more effectively inhibited NF-kB activation than MLN120b, a specific IKKb inhibitor [43,69]. Moreover, 17-DMAG, a geldanamycin derivative that inhibits the heat shock protein 90 kDa (HSP90), a molecular chaperone involved in both IKKa/b-and EGFRdependent signaling cascades, was more effective at triggering cell death than MLN120b alone [43,70].…”