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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. Clin Cancer Res; 16(8); 2295–307. ©2010 AACR.

Supplementary Data from CK2 Modulation of NF-κB, TP53, and the Malignant Phenotype in Head and Neck Cancer by Anti-CK2 Oligonucleotides In vitro or In vivo via Sub–50-nm Nanocapsules

The TP53 family member TAp73 has been shown to be pro-apoptotic and enhance chemosensitivity, but is inactivated in head and neck squamous cell carcinoma (HNSCC). We recently found that the inflammatory cytokine, TNFα, induces NF-κB activation and TAp73 nuclear export, which contribute to the suppression of TAp73 pro-apoptotic function. However, the intermediate signaling involved in TNFα regulated TAp73 inactivation and modulation of apoptosis has not been defined. Here, we found that TAp73 exhibits varying levels of phosphorylation in a panel of HNSCC cell lines. An inverse association between phospho- and total TAp73 protein expression was observed, suggesting that signal phosphorylation could contribute to TAp73 turnover. In response to TNFα treatment, phosphorylated TAp73 decreased in the nucleus while increasing in the cytoplasm, consistent with nuclear-cytoplasmic translocation. As we previously showed that protein kinase CK2 subunits are aberrantly overexpressed and promote IKK-NF-κB activation in HNSCC, we examined CK2 as a potential upstream regulator of TAp73 phosphorylation. Both CK2 inhibitor DMAT (2-Dimethy-lamino-4,5,6,7-tetrabromo-1H-benzimidazole) and CK2α siRNA inhibited TAp73 phosphorylation, as well as increased TAp73 mRNA and protein expression. Consequently, DMAT treatment increased the expression of TAp73 downstream pro-apoptotic genes, p21, PUMA and NOXA through increased TAp73 binding to the promoters of these genes. DMAT treatment and mutation of TAp73 phosphorylation sites increased p21 reporter activity. Furthermore, DMAT blocked cell cycle progression and proliferation, and increased apoptosis of HNSCC in vitro. Delivery of anti-CK2α/α′ oligodeoxy-nucleotide nanocapsules into HNSCC xenograft models significantly suppressed tumor growth, concomitant with decreased TAp73 phosphorylation, increased expression of TAp73, and target apoptotic proteins PUMA and NOXA. These results suggest that the aberrant activation of protein kinase CK2 could be one of the upstream signaling molecules modulating phosphorylation of TAp73, TAp73 nuclear exportation, and suppression of apoptosis in HNSCC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4788. doi:10.1158/1538-7445.AM2011-4788