Purpose: As an approach to evaluate the expression pattern and status of activation of signaling pathways in clinical specimens from head and neck squamous cell carcinoma (HNSCC) patients, we established the Head and Neck Cancer Tissue Array Initiative, an international consortium aimed at developing a high-density HNSCC tissue microarray, with a high representation of oral squamous cell carcinoma. Experimental Design: These tissue arrays were constructed by acquiring cylindrical biopsies from multiple individual tumor tissues and transferring them into tissue microarray blocks. From a total of 1,300 cases, 547 cores, including controls, were selected and used to build the array. Results: Emerging information by the use of phosphospecific antibodies detecting the activated state of signaling molecules indicates that the Akt-mammalian target of rapamycin (mTOR) pathway is frequently activated in HNSCC, but independently from the activation of epidermal growth factor receptor or the detection of mutant p53. Indeed, we identified a large group of tissue samples displaying active Akt and mTOR in the absence of epidermal growth factor receptor activation. Furthermore, we have also identified a small subgroup of patients in which the mTOR pathway is activated but not Akt, suggesting the existence of an Akt-independent signaling route stimulating mTOR. Conclusions:These findings provide important information about the nature of the dysregulated signaling networks in HNSCC and may also provide the rationale for the future development of novel mechanism-based therapies for HNSCC patients.

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DNA-PK is a nuclear protein with serine/threonine kinase activity and forms a complex consisting of the DNA-PKcs and a heterodimer of Ku70 and Ku80 proteins. Recent laboratory experiments have demonstrated that the DNA-PK complex formation is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. In this study, we evaluated the relationship between expression levels of DNA-PKcs, Ku70 and Ku80 proteins and radiation sensitivity in oral squamous cell carcinoma (OSCC) cell lines and in OSCC patients treated with preoperative radiation therapy. The OSCC cell lines greatly differed in their response to irradiation, as assessed by a standard colony formation assay. However, the expression levels of the DNA-PK complex proteins were all similar, and there was no association between the magnitude of their expression and the tumor radiation sensitivity. Expression of DNA-PK complex proteins increased after radiation treatment, and the in- he DNA-PK complex is one of the major pathways by which cells respond to DNA double-strand breaks (DSBs). The DNA-PK complex consists of a heterodimer comprising 70-and 80-kDa proteins termed Ku and a 465-kDa serine/ threonine protein kinase catalytic subunit termed DNA-PKcs. 1)The Ku (p70/p80) component functions as an activator of the catalytic subunit, and also represents the major double-stranded DNA binding protein.1, 2) DNA-PK plays an important role in the repair of DSBs and in V(D)J recombination.3) Tumor cell lines defective in the expression of either Ku or DNA-PKcs exhibit marked radiation sensitivity. Cells lacking DNA-PK activity because of defects in DNA-PK components, such as human malignant glioma M059J cells and cells derived from scid mice, show hypersensitivity to ionizing radiation.3-7) These previous laboratory findings suggested that DNA-PK is a candidate as a predictor of cellular radiation sensitivity. There is, however, little information on the expression of DNA-PK in primary human tumors and the correlation, if any, with radiation sensitivity, though the results are not definitive. [8][9][10][11] Therefore, the aim of this study is to evaluate the relationship between expression levels of DNA-PK complex proteins and radiation sensitivity. Materials and MethodsCell culture. All SCC cell lines were grown in Ham/F12: DMEM (1:1) supplemented with 10% fetal bovine serum, 24 µg/ml adenine, 0.4 µg/ml hydrocortisone and 50 units/ml penicillin and streptomycin. The HSC2, HSC3 and HSC 4 cell lines were provided by Japanese Collection Research Bioresources. The SCC15, SCC25, SCC66 and SCC111 cell lines were provided by Dr.