Membrane type 3 matrix metalloproteinase (MT3-MMP), an activator for the zymogen of MMP-2 (proMMP-2, or progelatinase A), is known to be expressed in human placenta, brain, lung and rat vascular smooth muscle cells, but information about its biochemical properties is limited. In the present study, we expressed and purified a truncated form of MT3-MMP lacking the transmembrane and intracytoplasmic domain (DMT3) and characterized the enzyme biochemically. DMT3 digested type III collagen into characteristic 3/4-and 1/4-fragments by cleaving the Gly781-Ile782 and Gly784-Ile785 bonds of a1(III) chains. Although DMT3 did not have such an activity against type I collagen, it attacked the Gly4-Ile5 bond of the triple helical portion of a2(I) chains, leading to removal of the crosslink containing N-terminal telopeptides. By quantitative analyses of the activities of DMT3 and a similar deletion mutant of MT1-MMP (DMT1), DMT3 was approximately fivefold more efficient at cleaving type III collagen. DMT3 also digested cartilage proteoglycan, gelatin, fibronectin, vitronectin, laminin-1, a1-proteinase inhibitor and a2-macroglobulin into almost identical fragments to those given by DMT1, although carboxymethylated transferrin digestion by DMT3 generated some extra fragments. The activity of DMT3 was inhibited by tissue inhibitor of metalloproteinases-2 (TIMP-2) and TIMP-3 in a 1 : 1 stoichiometry, but not by TIMP-1. ProMMP-2 was partially activated by DMT3 to give the intermediate form. These results indicate that, like MT1-MMP, MT3-MMP exhibits proteolytic activities against a wide range of extracellular matrix molecules. However, differences in the proMMP-2 activation and tissue distribution suggest that MT3-MMP and MT1-MMP play different roles in the pathophysiological digestion of extracellular matrix.Keywords: membrane type 3 matrix metalloproteinase; membrane type 1 matrix metalloproteinase; gelatinase A; inhibition by tissue inhibitors of metalloproteinases; degradation of matrix macromolecules. -2 [3,5,7]. In contrast, we and other groups [8±10] have recently demonstrated that MT1-MMP and MT2-MMP exhibit proteolytic activities against many ECM macromolecules, although MT1-MMP differs from MT2-MMP in its ability to degrade fibrillar collagens. Because of the structural homology of the catalytic domain of MT3-MMP to those of MT1-MMP and MT2-MMP, MT3-MMP is expected to possess enzymic activity towards the ECM components. In fact, culture media containing a secretable form of rat MT3-MMP exhibit gelatinolytic and caseinolytic activities on the zymography [11]. Recent studies [12] also reported that a soluble variant of human MT3-MMP, formed by alternatively spliced mRNA, has collagenolytic activity. However, this splice variant is different from MT3-MMP, as the catalytic domain is attached by a novel sequence of 50 amino acids at the C-terminus and its expression is observed only in human ovary [12]. Thus, the substrate specificity of human MT3-MMP against ECM macromolecules, differences in the activity from those of oth...

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27-activated NK cells to kill SCCVII cells in an Fc; receptor IIIdependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity.

RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyperrecombination and repair. In this study, we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas, which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence, we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly, siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover, combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably, we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers. Cancer Res; 71(13); 4598-607. Ó2011 AACR.

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05 HNSCC is promoted by CCL22 in an autocrine or M2-like macrophage-dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.Head and neck squamous cell carcinoma (HNSCC) has a global incidence of 630,000 cases and causes 350,000 deaths annually. 1 Recent advances in multimodal combined therapy have improved loco-regional control of HNSCC.2 However, over the last decade, the overall 5-year survival rate of HNSCC has been only moderately improved, in part due to metastasis.2-4 Lymph nodes are the primary sites of metastasis of HNSCC before distant metastasis occurs.3-5 The presence of lymph node metastasis is strongly associated with poor clinical outcome in patients with HNSCC. 4,[6][7][8] Therefore, it is essential to elucidate the mechanisms underlying lymph node metastasis of HNSCC for improvement of diagnostic and therapeutic strategies for patients with HNSCC.Chemokines have recently been implicated in organspecific metastasis of cancer cells. The role of ectopic Key words: head and neck cancer, lymph node metastasis, CCR4, CCL22, macrophage Abbreviations: a/iLN: axilla and inguinal lymph nodes; ADCC: antibody dependent cellular cytotoxicity; cLN: cervical lymph nodes; FBS: fetal bovine serum; GFP: green fluorescent protein; HNSCC: head and neck squamous cell carcinoma; mAb: monoclonal antibody; pAb: polyclonal antibody; RT-PCR: reverse transcriptase-polymerase chain reaction; SD: standard deviations; siRNA: short-interfering RNA Additional Supporting Information may be found in the online version of this article.