Nuclear NF-kappaB p65 phosphorylation at serine 276 by protein kinase A contributes to the malignant phenotype of head and neck cancer.
OBJECTIVE
Aberrant nuclear activation and phosphorylation of the canonical NF-kappaB subunit RELA/p65 at Serine-536 by inhibitor kappaB kinase is prevalent in head and neck squamous cell carcinoma (HNSCC), but the role of other kinases in NF-kappaB 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.


METHODS
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-kappaB activation, downstream gene expression, cell proliferation and cell cycle were examined. Knockdown of PKA by specific siRNA confirmed the specificity.


RESULTS
NF-kappaB 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-kappaB and target gene IL-8 reporter activity. Knockdown of PKA by small interfering RNA inhibited NF-kappaB, 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-kappaB-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-kappaB p65 (Ser276) phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC.
Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892-0001, USA.
Pattatheyil
P
Arun
Matthew S
MS
Brown
Reza
R
Ehsanian
Zhong
Z
Chen
Carter
C
Van Waes
D002294:Carcinoma, Squamous Cell / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006258:Head and Neck Neoplasms / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D009092:Mucous Membrane / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000378:metabolism* / Q000502:physiology; D014407:Tumor Cells, Cultured
D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D013449:Sulfonamides; D051996:Transcription Factor RelA; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide