Results: The expression score of MMP-2 correlated with that of MMP-9 (r ‫؍‬ 0.291; P ‫؍‬ 0.036), MT1-MMP (r ‫؍‬ 0.286; P ‫؍‬ 0.039), and TIMP-2 (r ‫؍‬ 0.257; P ‫؍‬ 0.050). Patients who developed regional lymph node and/or distant metastasis showed significantly higher scores in the expressions of MMP-9 and TIMP-2 than patients without any tumor metastases (P ‫؍‬ 0.036 and P ‫؍‬ 0.043, respectively). Kaplan-Meier analyses as well as univariate analyses using the Cox proportional hazards model showed that expression of MMP-9 (P ‫؍‬ 0.0143 and P ‫؍‬ 0.0418, respectively) and marked expression of TIMP-2 (P < 0.0001 and P ‫؍‬ 0.0004, respectively) correlated with worse-cause-specific survival. Multivariate analysis confirmed that marked expression of TIMP-2 was the only independent factor for cause-specific death (hazard ratio, 7.543; confidence interval, 1.693-33.610; P ‫؍‬ 0.0080).Conclusions: Expressions of MMP-9 and TIMP-2 have predictive value for tumor metastases and cause-specific survival. High expression of TIMP-2 is the most independent factor for worse prognosis in early-stage oral SCC.

Purpose: The functional expression of CXCR4, which plays roles in cell migration and proliferation in response to its unique ligand stromal cell^derived factor-1 (SDF-1), has been reported in variety of carcinomas. However, CXCR4 expression and its functional role in head and neck squamous cell carcinomas (HNSCC) remain unclear. In this study, we investigated CXCR4 expression and analyzed its functions in HNSCC cell lines. We also attempted to regulate CXCR4 expression using cytokines, such as interleukin-1B , tumor necrosis factor-A, and IFN-;. Finally, we investigated correlation between CXCR4 expression and clinical features in patients with HNSCC. Experimental Design: Six HNSCC cell lines were used in this study. Reverse transcription-PCR and flow cytometry analysis were shown for CXCR4 expressions with or without stimulations of cytokines. SDF-1-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. The SDF-1-mediated cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The SDF-1-mediated signaling pathways were analyzed by Western blot analysis. Biopsy specimens from 56 patients with HNSCC were used for immunohistologic analysis. Results: The significant CXCR4 expression was found in HSQ-89, IMC-3, and Nakamura cells. The SDF-1-mediated cell migration and proliferation were observed in CXCR4-positive cells. SDF-1 also promoted rapid phosphorylation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways in CXCR4-positive cells. The SDF-1-mediated cell migration and proliferation of CXCR4-positive cells were inhibited by neutralization of CXCR4. Among three cytokines tested, IFN-; significantly reduced CXCR4 expression and SDF-1-induced cell migration and proliferation of CXCR4-positive cells. Immunohistologic analysis revealed that patients with advanced neck status and patients who developed distant metastases showed significantly higher CXCR4 expression, and the cause-specific survival of patients with CXCR4-expression was significantly shorter. Furthermore, multivariate analysis confirmed that CXCR4 positive was the independent factor for cause-specific death. Conclusion: Our results may provide an insight into future therapeutic agent that inhibits tumor metastasis and progression via down-regulating CXCR4 expression in patients with HNSCC.

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.

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