Nuclear NF-κB p65 Phosphorylation at Serine 276 by Protein Kinase A Contributes to the Malignant Phenotype of Head and Neck Cancer

Purpose: Aberrant nuclear activation and phosphorylation of the canonical NF-kappa B subunit RELA/p65 at Serine-536 by inhibitor kappa B kinase is prevalent in head and neck squamous cell carcinoma (HNSCC), but the role of other kinases in NF-kappa B activation has not been well defined. Here, we investigated the prevalence and function of p65-Ser276 phosphorylation by protein kinase A (PKA) in the malignant phenotype and gene transactivation, and studied p65-Ser276 as a potential target for therapy. Experimental Design: Phospho and total p65 protein expression and localization were determined in HNSCC tissue array and in cell lines. The effects of the PKA inhibitor H-89 on NF-kappa B activation, downstream gene expression, cell proliferation and cell cycle were examined. Knockdown of PKA by specific siRNA confirmed the specificity. Results: NF-kappa B p65 phosphorylated at Ser276 was prevalent in HNSCC and adjacent dysplastic mucosa, but localized to the cytoplasm in normal mucosa. In HNSCC lines, tumor necrosis factor-alpha (TNF-alpha) significantly increased, whereas H-89 inhibited constitutive and TNF-alpha-induced nuclear p65 (Ser276) phosphorylation, and significantly suppressed NF-kappa B and target gene IL-8 reporter activity. Knockdown of PKA by small interfering RNA inhibited NF-kappa B, IL-8, and BCL-XL reporter gene activities. H-89 suppressed cell proliferation, induced cell death, and blocked the cell cycle in G(1)-S phase. Consistent with its biological effects, H-89 down-modulated expression of NF-kappa B-related genes Cyclin D1, BCL2, BCL-XL, COX2, IL-8, and VEGF, as well as induced cell cycle inhibitor p21(CIP1/WAF1), while suppressing proliferative marker Ki67 Conclusions: NF-kappa B p65 (Ser276) phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC. (Clin Cancer Res 2009;15(19): 5974-84)