Tomokazu Yoshizaki

Cleavage of Syndecan-1 by Membrane Type Matrix Metalloproteinase-1 Stimulates Cell Migration

The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promoted syndecan-1 shedding, and concentration of cell-associated syndecan-1 was reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interfered with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment was inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 was also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein was shown to be cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), which express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes was significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induced accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduced shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration.

Induction of cyclooxygenase-2 by Epstein–Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IκBα(S32A/S36A), which is not phosphorylated and prevents NF-κB activation, with LMP1 showed that NF-κB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-κB is involved in LMP1-induced cox-2 promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-κB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E2 in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.

Epstein-Barr Virus Latent Membrane Protein 1 Induces Synthesis of Hypoxia-Inducible Factor 1α

ABSTRACT Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β that is the central regulator of responses to hypoxia. The specific binding of HIF-1 to the hypoxia-responsive element (HRE) induces the transcription of genes that respond to hypoxic conditions, including vascular endothelial growth factor (VEGF). Here we report that expression of HIF-1α is increased in diverse Epstein-Barr virus (EBV)-infected type II and III cell lines, which express EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as other latency proteins, but not in the parental EBV-negative cell lines. We show first that transfection of an LMP1 expression plasmid into Ad-AH cells, an EBV-negative nasopharyngeal epithelial cell line, induces synthesis of HIF-1α protein without increasing its stability or mRNA level. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 markedly reduces induction of HIF-1α by LMP1. Catalase, an H2O2 scavenger, strongly suppresses LMP1-induced production of H2O2, which results in a decrease in the expression of HIF-1α induced by LMP1. Inhibition of the NF-κB, c-jun N-terminal kinase, p38 MAPK, and phosphatidylinositol 3-kinase pathways did not affect HIF-1α expression. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1α protein expression, which is controlled by p42/p44 MAPK activity and H2O2. The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1α along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.

Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC), an EBV-associated malignancy, is highly metastatic compared with other head and neck tumors, perhaps because of its viral link. Here, we show that the principal EBV oncoprotein, latent membrane protein 1 (LMP1), induces epithelial-mesenchymal transition (EMT) via Twist, a master transcriptional regulator in embryogenesis and newly implicated in metastasis, which, in turn, are likely to contribute to the highly metastatic character of NPC. LMP1 could induce EMT and its associated cell motility and invasiveness in a cell culture model, whereas expression of Twist small interfering RNA reversed LMP1-induced EMT. In diverse EBV-infected cell lines, expression of Twist correlates with expression of LMP1. Dominant-negative LMP1 could suppress Twist expression in EBV-positive cells, whereas LMP1 could induce Twist in EBV-negative nasopharyngeal cells. LMP1 signals through the nuclear factor-kappaB pathway, and an IkappaB superrepressor inhibited induction of Twist by LMP1. Finally, in human NPC tissues, expression of Twist and LMP1 is directly correlated and expression of Twist is associated with metastasis clinically. These results suggest that induction of Twist by a human viral oncoprotein LMP1 directly contributes to the metastatic nature of NPC.

Expression of tissue inhibitor of matrix metalloproteinase-2 correlates with activation of matrix metalloproteinase-2 and predicts poor prognosis in tongue squamous cell carcinoma

Matrix metalloproteinase 2 (MMP‐2) plays a crucial role in invasion and metastasis of malignant tumors. Membrane type 1‐MMP (MT1‐MMP) was originally identified as an activator of MMP‐2. Tissue inhibitor of MMP‐2 (TIMP‐2) was identified as an inhibitor of MMP‐2 and MT1‐MMP. However, TIMP‐2 was reported to be essential for cell‐mediated activation of MMP‐2 and thus, the contribution of TIMP‐2 to tumor invasion has remained controversial. This study was designed to analyze the role of TIMP‐2 for activation of MMP‐2 and its prognostic value in tongue squamous cell carcinoma (SCC). Expression of MMP‐2, MT1‐MMP and TIMP‐2 protein was analyzed by immunohistochemistry, and their association with clinical factors was evaluated in 51 patients treated surgically for tongue SCC. Expression of MMP‐2, MT1‐MMP and TIMP‐2 was significantly correlated with local and distant metastatic tumor recurrence and poor prognosis (MMP‐2 and MT1‐MMP, p < 0.0001; TIMP‐2, p = 0.0002). Activation of MMP‐2, analyzed by gelatin zymography in 17 fresh specimens, was remarkably associated with expression of MMP‐2 (r = 0.779, p < 0.0001), MT1‐MMP (r = 0.674, p < 0.0022) and TIMP‐2 (r = 0.858, p < 0.0001). Increased expression of TIMP‐2, as well as MMP‐2 and MT1‐MMP, was an important prognostic factor in patients with tongue SCC.

Exosomal HIF1α supports invasive potential of nasopharyngeal carcinoma-associated LMP1-positive exosomes.

It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However, whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1α (HIF1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein–Barr virus (EBV), latent membrane protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF1α is detectable in exosomes and that LMP1 significantly increases levels of HIF1α in exosomes. HIF1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We also show that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with epithelial–mesenchymal transition (EMT). In addition, we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, as exosome-mediated delivery of active and inactive forms of HIF1α results in reciprocal changes in the expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.

EBV Latent Membrane Protein 1 Up-regulates Hypoxia-Inducible Factor 1α through Siah1-Mediated Down-regulation of Prolyl Hydroxylases 1 and 3 in Nasopharyngeal Epithelial Cells

Hypoxia-inducible factor 1 (HIF1) is up-regulated in most malignant tumors usually via interruption of ubiquitination and proteasomal degradation of its subunit alpha. Recently, we have shown that the principal EBV oncoprotein, latent membrane protein 1 (LMP1), activates HIF1alpha and subsequently expression of HIF1-responsive genes in epithelial cells. Here, we explore the mechanism for HIF1alpha activation by LMP1 in nasopharyngeal epithelial cells: LMP1 up-regulates the level of Siah1 E3 ubiquitin ligase by enhancing its stability, which subsequently induces proteasomal degradation of prolyl HIF-hydroxylases 1 and 3 that normally mark HIF1alpha for degradation. As a result, LMP1 prevents formation of von Hippel-Lindau/HIF1alpha complex, as shown by coimmunoprecipitation analyses. Thus, Siah1 is implicated in the regulation of HIF1alpha and is involved in a recently appreciated aspect of EBV-mediated tumorigenesis, namely, the angiogenesis process triggered by LMP1.

Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has a significant role in initiating EBV-associated lymphoproliferative disease and EBV-related malignancies. In view of clinical features related to the type of EBV latency, LMP1 may influence invasiveness of EBV associated tumors categorized as types II and III as represented on nasopharyngeal carcinoma (NPC). To screen for genes associated with invasion of epithelial cells transformed by LMP1, Madin-Darby canine kidney (MDCK) epithelial cells were transformed by LMP1. Stable transfection of a LMP1 gene into MDCK cells induced morphological change from cobblestone to a long spindle-shape, reduced cell-cell adhesion and caused high cell motility. Parental MDCK cells, which form spherical cysts in three-dimensional collagen gel matrix, form branching tubules following exposure to hepatocyte growth factor (HGF). MDCK cells transformed by LMP1 showed invasive growth to form branching tubules into collagen gel without HGF-treatment. mRNA differential display and Northern hybridization identified plasminogen activator inhibitor-1 (PAI-1), urokinase type plasminogen activator (uPA) and ets1 as genes upregulated during transformation by LMP1. Expression of a dominant negative type of Ets1 in LMP1-transformed cells downregulated uPA expression and cell motility. Deletion of LMP1 cytoplasmic carboxy-terminal activating region 1 (CTAR1) domain abolished transformation, but a deletion mutant lacking CTAR2 domain still retained transforming and uPA-inducing ability. Expression of Ets1 was immunolocalized in tumor cells of NPC tissue which frequently express LMP1. Taken together, it is suggested that LMP1 induces expression of Ets1 which may contribute to invasion of NPC by stimulating cell motility and uPA expression.

Induction of c-Met Proto-Oncogene by Epstein-Barr Virus Latent Membrane Protein-1 and the Correlation with Cervical Lymph Node Metastasis of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is distinctive in head and neck carcinomas for its close association with Epstein-Barr virus and its highly metastatic nature. Up-regulation of cell motility is essential for enhancement of metastatic potential. The expression of c-Met proto-oncogene, a high-affinity receptor for hepatocyte growth factor/scatter factor, has been reported to correlate with metastatic ability of the tumor cell. We observed close association of c-Met expression with cervical lymph node metastasis (P = 0.0272) in 39 NPC specimens studied immunohistochemically. Epstein-Barr virus-encoding latent membrane protein-1 (LMP-1) is a primary oncogene and is suggested to enhance the metastatic property of NPC. Previously, we reported that LMP-1 enhanced the motility of Madin-Darby canine kidney (MDCK) epithelial cells that was mediated by activation of Ets-1 transcription factor. Therefore, we examined the interrelationships of LMP-1, Ets-1, and c-Met. In immunohistochemical studies, the expression of LMP-1, Ets-1, and c-Met correlated significantly with each other in NPC (LMP-1 versus Ets-1, P < 0.0001; Ets-1 versus c-Met, P = 0.0012; LMP-1 versus Met, P = 0.0005). Transfection of LMP-1-expressing plasmid in MDCK cells induced c-Met protein expression. The c-Met protein was also induced by Ets-1 expression, and induction of c-Met by LMP-1 was suppressed by introducing a dominant-negative form of Ets-1 in LMP-1-expressing MDCK cells. These results suggest that LMP-1 induces c-Met through the activation of Ets-1, which may contribute in part to the highly metastatic potential of NPC.

Prognostic Value of EGFR and TGF-α in Early Laryngeal Cancer Treated With Radiotherapy

The purpose of this study was to determine the overexpression of both epidermal growth factor receptor (EGFR) and transforming growth factoralpha(TGF‐α) (a ligand of EGFR) in early laryngeal squamous cell carcinoma. In addition, we attempted to evaluate the prognostic values of our findings.