Kanemitsu Shirasuna

Expression of MMPs, MT-MMP, and TIMPs in squamous cell carcinoma of the oral cavity: Correlations with tumor invasion and metastasis

Matrix metalloproteinases (MMPs) that degrade the extracellular matrices (ECMs) have been thought to play an important role in both the invasion and metastasis of tumors. However, the detailed role of MMPs and TIMPs (tissue inhibitors of MMP) on the biological behavior of tumor cells has yet to be elucidated in vivo. The aim of the present study was thus to determine whether expression of MMPs on tumor cells is associated with such clinicopathological features as the invasive and metastatic potential.

High expression levels of nuclear factor κB, IκB kinase α and akt kinase in squamous cell carcinoma of the oral cavity

BACKGROUND It has been reported that the transcription factor nuclear factor κB (NF-κB) is involved in the growth, invasion, and antiapoptotic activity of cultured tumor cells. METHODS The authors used immunohistochemistry to examine the expression of NF-κB and the signaling molecules leading to NF-κB activation in 36 untreated biopsy specimens from patients with squamous cell carcinoma (SCC) and in 15 specimens from patients with epithelial dysplasia of the oral cavity. RESULTS Among the molecules examined, the p65 subunit of NF-κB (p65) and IκB kinase α (IKKα) were expressed highly in almost all SCC specimens examined, whereas the samples of normal squamous epithelia adjacent to tumors as well as epithelial dysplasia specimens were negative in for immunohistochemical staining. The invasiveness and metastasis of SCC seemed to correlate with the degree of staining degree in the molecules. Moreover, phosphorylated Akt kinase, which may be associated with antiapoptosis signaling of NF-κB, was detected in the same areas where IKKα existed in large amounts. CONCLUSIONS The results suggest that high expression levels of p65 and IKKα contribute to malignant behavior and antiapoptotic activity in SCC of the oral squamous epithelium. Cancer 2001;92:3037–44.

Gelatinolytic activity of matrix metalloproteinase in tumor tissues correlates with the invasiveness of oral cancer

We examined whether or not the gelatinolytic activity in tumor tissue was associated with the invasion and metastasis of oral squamous cell carcinoma (OSCC). Tissue homogenates were prepared from 57 biopsy specimens of OSCC. The gelatinolytic activities in the homogenates were measured by gelatin zymography and its densitometric analysis. The Immunoblot findings revealed the major gelatinolytic activities to be due to matrix metalloproteinase (MMP)-2 and -9. The zymography-detected gelatinolytic activities of MMP-2 and MMP-9 in the tissue specimens significantly correlated with the degree of immunohistochemical staining detected in frozen sections of the same biopsy specimens. According to a histopathological analysis of the mode of invasion, highly invasive cases showed the increased gelatinolytic activities of MMP-2 as well as MMP-9 in the tissue specimens. Although no significant differences were observed in the gelatinase activities between the metastatic cases and the non-metastatic cases, the levels of tissue inhibitor of MMP (TIMP)-1 in the tumor tissue specimens were higher in the non-metastatic cases than in the metastatic cases. The cases with the high levels of MMPs and low levels of TIMP-1 thus seemed to have a high potential to metastasize. As a result, the zymographic measurement of the gelatinolytic activity in biopsy tissue specimens may therefore be useful in predicting the behavior and prognosis of OSCC.

Immunohistochemical study of desmosomes in oral squamous cell carcinoma: Correlation with cytokeratin and E-cadherin staining, and with tumour behaviour

Reduction or loss of the intercellular junctions known as desmosomes may contribute to the invasive and metastatic behaviour of various carcinomas. Previous studies have shown that metastasis of oral squamous cell carcinomas of the head and neck correlates with a reduction in immunohistochemical staining for desmoplakin and desmoglein at the invasion front. The primary aim of the present study was to extend these observations to include a third component of desmosomes, the glycoprotein desmocollin. An additional aim was to determine whether the differentiation status of tumours is reflected in their staining for cytokeratins 1, 13, and 19, and, if so, whether these parameters correlate with desmosomal staining and/or metastasis. The study included 54 primary tumours of which 28 showed lymph node metastases. The results of this investigation show that tumours can be divided into three groups according to whether they have lost staining for no, one or more than one desmosomal component. A statistically significant correlation was found between the number of desmosomal components lost and metastasis. Tumours could also be divided into five groups according to their staining for different combinations of cytokeratins. Furthermore, differentiation status as indicated both histologically and by cytokeratin staining correlated with reduced desmosomal staining and metastasis. Tumours were also examined for intensity of staining for the adhesion molecule E‐cadherin. Reduction in E‐cadherin staining was correlated with mode of invasion and with reduction in desmosomal staining, but not with poor differentiation as indicated by cytokeratin staining. The results of this extensive study reinforce the view that adhesive junctions and adhesion molecules contribute to the suppression of tumour invasion and metastasis.

Fibroblast Growth Factor-2 Gene Transfer Can Stimulate Hepatocyte Growth Factor Expression Irrespective of Hypoxia-Mediated Downregulation in Ischemic Limbs

Abstract— Hepatocyte growth factor (HGF) is a potent angiogenic polypeptide that stimulates angiogenesis. Transcriptional regulation of HGF, however, has not been fully defined, with the exception of the hypoxia-mediated downregulation in cultured cells. In the present study, we report that angiogenic growth factors, including HGF, were upregulated in a murine model of critical limb ischemia in vivo, a finding that was in conflict with previous in vitro data. Mice deficient in basic fibroblast growth factor-2 (FGF-2) showed reduced induction of HGF protein in ischemic muscles, and overexpression of FGF-2 via gene transfer stimulated endogenous HGF, irrespective of the presence of ischemia. In culture, FGF-2 rapidly stimulated HGF mRNA, and a sustained expression was evident in the time course in vascular smooth muscle cells and fibroblasts. FGF-2–mediated induction of HGF was fully dependent on the mitogen-activated protein kinase pathway yet was not affected by either hypoxia or a protein kinase A inhibitor. In the early expression, FGF-2 directly stimulated HGF mRNA without the requirement of new protein synthesis, whereas sustained induction of HGF in the later phase was partly mediated by platelet-derived growth factor-AA. Furthermore, in vivo overexpression of FGF-2 significantly improved the blood perfusion, and the effect was abolished by systemic blockade of HGF in ischemic limbs. This is the first demonstration of a regulational mechanism of HGF expression via FGF-2 that was independent of the presence of hypoxia. The harmonized therapeutic effects of FGF-2, accompanied with the activity of endogenous HGF, may provide a beneficial effect for the treatment of limb ischemia.

Oral involvement in chronic graft-versus-host disease after allogeneic bone marrow transplantation

We examined 37 patients who had undergone an allogeneic bone marrow transplantation and compared their oral findings to their systemic involvement with chronic graft-versus-host disease. Among the clinical signs and symptoms in their oral region, only the presence of oral lichenoid lesions had a statistically significant relationship to the diagnosis of chronic graft-versus-host disease. The histologic findings in the labial salivary glands and buccal mucosa closely reflected the status of chronic graft-versus-host disease. Statistically, the presence of diffuse and periductal lymphocytic infiltration in labial salivary glands, subepithelial lymphocytic infiltration and epithelial changes in buccal mucosa also showed a significant relationship to the diagnosis of chronic graft-versus-host disease. The present study suggests that a systematic oral examination, especially pathologic examination of the labial salivary glands and buccal mucosa, is useful in evaluating the status of chronic graft-versus-host disease.

Involvement of proteasomes in migration and matrix metalloproteinase-9 production of oral squamous cell carcinoma

We investigated whether proteasomes were involved in the invasiveness of oral squamous cell carcinoma (SCC) cells. The migration of SCC cells through a gelatin‐coated membrane was enhanced with tumor necrosis factor α (TNFα), which was strongly inhibited by a peptide aldehyde, N‐acetyl‐Leu‐Leu‐norleucinal (ALLN), but not by its structurally related compound, N‐acetyl‐Leu‐Leu‐methioninal (ALLM). Since ALLN is a more potent inhibitor against proteasomal proteolysis than ALLM, cell migration inhibited by ALLN may thus likely depend on proteasomes. The TNFα‐induced migration through gelatin appeared to be associated with the gelatinolytic activity from the cells, since TNFα strongly enhanced the production of matrix metalloproteinase (MMP)‐9/gelatinase B in the SCC cells, as detected by gelatin zymography. The production of MMP‐9 was also inhibited by pretreatment with ALLN, but not ALLM, in a dose‐dependent manner. Moreover, ALLN could block the activation and nuclear translocation of a transcription‐activating factor, NF‐κB, which is known to regulate MMP‐9 expression in TNFα‐stimulated SCC cells. The TNFα‐induced degradation of IκBα was also suppressed by ALLN treatment, thus implying that the molecule linking proteasome to MMP‐9 production should be IκBα. We finally reconfirmed the involvement of proteasomes in the invasive behavior of oral SCC using lactacystin, a specific proteasome inhibitor, which could prevent TNFα from enhancing MMP‐9 production, NF‐κB activation, induction of MMP‐9 mRNA and cell migration. Int. J. Cancer 77:578–585, 1998.

Novel Translational Control through an Iron-Responsive Element by Interaction of Multifunctional Protein YB-1 and IRP2

ABSTRACT The eukaryotic Y-box-binding protein YB-1 functions in various biological processes, including DNA repair, cell proliferation, and transcriptional and translational controls. To gain further insight into how human YB-1 plays its role in pleiotropic functions, we here used two-hybrid screenings to identify partners of this protein; the results showed that YB-1 itself, iron-regulatory protein 2 (IRP2), and five ribosomal proteins each served as partners to YB-1. We then examined the biological effect of the interaction of YB-1 and IRP2 on translational regulation. Both in vitro binding and coimmunoprecipitation assays showed the direct interaction of YB-1 and IRP2 in the presence of a high concentration of iron. RNA gel shift assays showed that YB-1 reduced the formation of the IRP2-mRNA complex when the iron-responsive element of the ferritin mRNA 5′ untranslated region (UTR) was used as a probe. By using an in vitro translation assay using luciferase mRNA ligated to the ferritin mRNA 5′UTR as a reporter construct, we showed that both YB-1 and IRP2 inhibited the translation of the mRNA. However, coadministration of YB-1 and IRP2 proteins abrogated the inhibition of protein synthesis by each protein. An In vivo coimmunoprecipitation assay showed that IRP2 bound to YB-1 in the presence of iron and a proteasome inhibitor. The direct interaction of YB-1 and IRP2 provides the first evidence of the involvement of YB-1 in the translational regulation of an iron-related protein.

A novel function of CD82/KAI-1 on E-cadherin-mediated homophilic cellular adhesion of cancer cells.

In this study, we analyzed the effect of the metastasis suppressor CD82/KAI-1, a member of the tetraspanin superfamily, on intercellular adhesion on cancer cells. The newly established invasion assay and the cell aggregation assay revealed that CD82 strengthens E-cadherin-mediated intercellular adhesion. Interestingly, ectopic expression of CD82 stabilized E-cadherin/beta-catenin complex formation. Furthermore, CD82 reduced tyrosine phosphorylation of beta-catenin on HGF stimulation. Taken together, CD82 may stabilize or strengthen E-cadherin-dependent intercellular adhesion by regulating beta-catenin-mediated signal transduction on cancer cells, and consequently, prevent cancer cells from seceding from the primary tumor site.

Regulation of c-Met signaling by the tetraspanin KAI-1/CD82 affects cancer cell migration

It has been proposed that the metastasis suppressor CD82/KAI‐1, which is a member of the tetraspanin superfamily, regulates biological activity by associating with cell surface receptors or proteins. We show a novel association between CD82 and the hepatocyte growth factor (HGF) receptor c‐Met. Although ectopic expression of CD82 in nonsmall cell lung carcinoma cells did not affect the tyrosine phosphorylation of c‐Met, these cells showed significant suppression of HGF‐induced lamellipodial protrusion and cell migration. CD82 selectively attenuated c‐Met signaling via the Ras‐Cdc42/Rac and the phosphatidylinositol 3‐kinase/Cdc42/Rac pathways. In contrast, another c‐Met signaling pathway that involves phosphatidylinositol 3‐kinase/Akt and phosphatidylinositol 3‐kinase/mitogen activated protein kinase was not affected by CD82. Signaling adapter proteins for c‐Met, such as Grb2 and p85, exhibited reduced association with c‐Met in cells that ectopically expressed CD82. These results indicate that the CD82‐c‐Met complex inhibits HGF‐induced cancer cell migration by the inactivation of small GTP‐binding proteins of the Rho family via c‐Met adapter proteins.