Purpose: The aim of this study is to investigate the expression of CK2 subunits and CK2 effects on NF-κB-mediated and TP53-mediated signal activation and gene expression, the malignant phenotype, and chemosensitivity in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo.Experimental Design: Protein expression of CK2 subunits was investigated by Western blot and immunohistochemistry. CK2 subunits were knocked down by small interfering RNA, and NF-κB activation was examined using DNA binding, Western blot, and luciferase reporter assays. Gene expression was measured by quantitative reverse transcription-PCR. Cell growth, survival, motility, and sensitivity to cisplatin were measured by MTT, flow cytometry, and migration assays. In vivo targeting of CK2α/α′ in HNSCC xenograft models was achieved using anti-CK2α/α′ oligodeoxynucleotide encapsulated in sub-50-nm tenfibgen nanocapsules.Results: CK2 subunit proteins were overexpressed in HNSCC lines and tissues. Knockdown of CK2 subunits differentially inhibited IκBα degradation, NF-κB nuclear localization, phosphorylation, DNA binding, and reporter activity. CK2 subunits modulated gene expression and the malignant phenotype involved in cell cycle and migration, whereas CK2α is critical to promote proliferation, antiapoptosis, and cisplatin resistance in vitro. Furthermore, in vivo delivery of anti-CK2α/α′ oligodeoxynucleotide nanocapsules significantly suppressed tumor growth in HNSCC xenograft models, in association with modulation of CK2 and NF-κB regulated molecules, TP53 family proteins, and induction of apoptosis.Conclusions: Our study reveals a novel role of CK2 in coregulating NF-κB activation, TP53/p63 expression, and downstream gene expression. Downregulation of CK2 in HNSCC models in vitro and in vivo shows antitumor effects as well as sensitization to cisplatin.

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.

Purpose: Nuclear factor-nB (NF-nB)/REL transcription factors promote cancer cell survival and progression. The canonical (NF-nB1/RELA or cREL) and alternate (NF-nB2/RELB) pathways require the proteasome for cytoplasmic-nuclear translocation, prompting the investigation of bortezomib for cancer therapy. However, limited clinical activity of bortezomib has been observed in many epithelial malignancies, suggesting this could result from incomplete inhibition of NF-nB/ RELs or other prosurvival signal pathways. Experimental Design:To examine these possibilities, matched biopsies from 24 h posttreatment were obtained from accessible tumors of patients who received low-dose bortezomib (0.6 mg/m 2 ) before reirradiation in a phase I trial for recurrent head and neck squamous cell carcinoma (HNSCC). Effects of bortezomib on apoptosis and proliferation by TUNEL and Ki67 staining were compared with nuclear staining for all five NF-nB subunits, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated signal transducers and activators of transcription 3 (STAT3) in tumor biopsies, and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTP) and DNA binding assay for the five NF-nB subunits in HNSCC cell lines. Results: HNSCC showed increased nuclear staining for all five NF-nB subunits, phosphorylated ERK1/2, and phosphorylated STAT3. Bortezomib treatment significantly enhanced apoptosis with inhibition of nuclear RELA in three of four tumors, but other NF-nB subunits, ERK1/2, and STAT3 were variably or not affected, and tumor progression was observed within 3 months. In HNSCC cell lines, 10 -8 mol/L bortezomib inhibited cell density while inhibiting tumor necrosis factor-aî nduced and partially inhibiting basal activation of NF-nB1/RELA, but not NF-nB2/RELB. Conclusions: Although low-dose bortezomib inhibits activation of subunits of the canonical pathway, it does not block nuclear activation of the noncanonical NF-nB or other prosurvival signal pathways, which may contribute to the heterogeneous responses observed in HNSCC.Nuclear factor-nB (NF-nB) is an injury, pathogen, growth factor, cytokine, and matrix signal-inducible transcription factor that normally functions to regulate cell survival and proliferation and innate and adaptive immune responses (1). NF-nB transcription factors are heterodimers or homodimers composed from two families of REL proteins. Each member of the REL family contains an NH 2 terminal REL homology domain that mediates dimerization and domains mediating nuclear localization and binding to specific nB DNA sequence motifs in target genes (1). The first class, RELA (p65), RELB, and cREL, is synthesized in their mature forms, whereas the second class, consisting of NF-nB1(p105) and NF-nB2(p100), requires proteasome-dependent proteolysis of COOH terminal ankyrin repeats to yield p50 and p52, respectively (2). In the absence of activating stimuli, NF-nB dimers are also bound by a family of ankyrin repeat-containing inhibitor-nB (InB) proteins, sequeste...