Abstract #2758: Nuclear localized NF-kB Rel A is phosphorylated at Serine 276 by protein kinase A and contributes to the malignant phenotype of head and neck cancer.

Aberrant activation of NF-\#954;B is prevalent in head and neck squamous cell carcinoma (HNSCC), mainly through nuclear translocation and phosphorylation of p65 at Ser 536 by the IKK\#945;/\#946;/\#947; complex. However, the role of other kinases in NF-\#954;B p65 phosphorylation in HNSCC has not been delineated. This study underscores the importance of an alternative site of p65 phosphorylation at Ser 276 by Protein Kinase A (PKA) that contributes to NF-\#954;B gene transactivation and the malignant phenotype. Immunohistochemistry and Western blot were performed on human tumor tissue arrays and HNSCC cell lines to detect presence and localization of phosphorylated and total p65. PKA inhibitor, H-89 was used to study effects of PKA inhibition on cell proliferation by MTT assay and cell cycle by flow cytometry. The effects of H-89 and PKA siRNA knockdown on NF-\#954;B activation and downstream gene expression were studied using luciferase reporter assays and by real time RT-PCR. Nuclear localized p65 is phosphorylated at Ser 276 in HNSCC and adjacent mucosal margins, but not in the normal mucosa. In HNSCC cell lines, TNF-\#945; significantly increased nuclear phosphorylated p65 at Ser 276. PKA inhibitor H-89, inhibited constitutive and TNF induced p65 Ser 276 phosphorylation, and significantly suppressed NF-\#954;B and its dependent IL-8 reporter gene activities. Knock down of PKA by siRNA also inhibited NF-\#954;B, IL-8 and Bcl-xL reporter activities. In addition, H-89 suppressed cell proliferation, induced cell death and blocked the cell cycle in G1/S phase. Consistent with its biological effects, H-89 modulated expression of NF-\#954;B related genes, such as cyclin D1, Bcl2, Bcl-xL, Cox2, IL-8, and VEGF, as well as induced p21, which controls cell cycle, and suppressed Ki67, which is a biomarker for proliferation. We have , for the first time, shown that a PKA mediated nuclear phosphorylation of p65 at Ser 276 is observed in HNSCC and premalignant lesions, in contrast to cytoplasmic localization in normal mucosa. HNSCC cell lines showed similar constitutive and TNF-\#945;-inducible nuclear activation. This site specific phosphorylation has been found to be important for NF-\#954;B transactivation and has been shown to regulate expression of genes controlling various components of the cancer phenotype such as cell cycle, apoptosis, inflammation, angiogenesis and proliferation. The study highlights the potential of kinases other than IKK to activate the NF-\#954;B signaling cascade and highlights the fact that inhibition of these kinases or their effects on site specific phosphorylation may need to be addressed in future prevention and treatment strategies. The aberrant activation of p65(Ser 276) at the tumor margins and in dysplastic epithelium may hold promise as a biomarker to predict the behavior of premalignant lesions of head and neck and to assess efficacy of chemopreventive interventions. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2758.