Search

Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) has been linked to Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. In addition to endothelial cells and B lymphocytes, KSHV also has been shown to infect epithelial cells and keratinocytes. The transmembrane glycoprotein K1, encoded by the first open reading frame of KSHV, is a signaling protein capable of eliciting B-cell activation. We show that KSHV K1 can induce expression and secretion of vascular endothelial growth factor (VEGF) in epithelial and endothelial cells. Up-regulation of VEGF was mediated at the transcriptional level because expression of K1 resulted in VEGF promoter activation. We also show that K1 induces expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. Additional analyses with K1 mutant proteins revealed that the SH2 binding motifs present in the K1 cytoplasmic tail are necessary for VEGF secretion and MMP-9 induction. These results indicate that K1 signaling may contribute to KSHV-associated pathogenesis through a paracrine mechanism by promoting the secretion of VEGF and MMP-9 into the surrounding matrix.

DNA methyltransferase 1 (DNMT1) is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Its expression is frequently up-regulated in human tumors, including hepatocellular carcinoma, but the mechanism of overexpression and its biological significance remain unclear. Here, we show that hepatitis B virus X protein (HBx) activates DNMT1 expression via a regulatory circuit involving the p16INK4a-cyclin D1-cyclin-dependent kinase (CDK) 4/6-retinoblastoma protein (pRb)-E2F1 pathway. HBx induced DNA hypermethylation of p16INK4a promoter to repress its expression, which subsequently led to activation of G1-CDKs, phosphorylation of pRb, activation of E2F1, and finally transcriptional activation of DNMT1. Inhibition of DNMT1 activity by either treatment with 5′-Aza-2′dC or introduction of DNMT1 small interfering RNA not only abolished the DNA methylation-mediated p16INK4a repression but also impaired DNMT1 expression itself, suggesting a cross-talk between DNMT1 and p16INK4a. The up-regulation of cyclin D1 by HBx is likely to serve as an initiative impulse for the circuit because it was absolutely required for the activation of DNMT1 expression. We also observed that accumulated DNMT1 via this pathway inactivates E-cadherin expression through promoter hypermethylation. Considering that the pRb-E2F1 pathway is commonly activated in human tumors, activation of this circuit might be widespread and a potential therapeutic target. [Cancer Res 2007;67(12):5771–8]

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-κB pathway, and an IκB 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. [Cancer Res 2007;67(5):1970–8]

Hypoxia-inducible factor 1 (HIF1) is up-regulated in most malignant tumors usually via interruption of ubiquitination and proteasomal degradation of its subunit α. Recently, we have shown that the principal EBV oncoprotein, latent membrane protein 1 (LMP1), activates HIF1α and subsequently expression of HIF1-responsive genes in epithelial cells. Here, we explore the mechanism for HIF1α 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 HIF1α for degradation. As a result, LMP1 prevents formation of von Hippel-Lindau/HIF1α complex, as shown by coimmunoprecipitation analyses. Thus, Siah1 is implicated in the regulation of HIF1α and is involved in a recently appreciated aspect of EBV-mediated tumorigenesis, namely, the angiogenesis process triggered by LMP1. (Cancer Res 2006; 66(20): 9870-7)

We have shown that the EBV oncoprotein, latent membrane protein 1 (LMP1), induces a constellation of tumor-invasiveness factors. Fibroblast growth factor (FGF)-2 is angiogenic as well mitogenic. Although FGF-2 does not contain a hydrophobic signal sequence for secretion, FGF-2 is released extracellularly. However, the mechanism by which FGF-2 is released is unclear. Here we show first that LMP1 induces in epithelial cells the expression of FGF-2 mRNA and protein through both LMP1 COOH-terminal activation domains, CTAR 1 and CTAR 2, which can activate nuclear factor (NF)-kappaB signaling and also the p38 mitogen-activated protein kinase pathway. Coexpression of IkappaBalpha (S32A/S36A), which cannot be phosphorylated and prevents NF-kappaB activation, with LMP1 inhibited induction of FGF-2 by LMP1, which suggests that LMP1 induces FGF-2 via NF-kappaB signaling. Moreover, unlike phorbol 12-myristate 13-acetate LMP1 also induced the release of the M(r) 18,000 isoform of FGF-2 protein. Transfection of Ad-AH cells with LMP1 deletion mutants lacking either CTAR 1 or CTAR 2 also induced the release of the protein. Secretion was confirmed in 293 cells, which do not contain detectable endogenous FGF-2 protein, cotransfected with FGF-2 and LMP1. Finally, Na(+)/K(+)-ATPase participates in FGF-2 release, independently of the classical endoplasmic reticulum/Golgi pathway. In this study, the release of M(r) 18,000 FGF-2 protein was partially suppressed by ouabain, which inhibits the activity of Na(+)/K(+)-ATPase alpha1 subunit, but not by Brefeldin A, which inhibits the endoplasmic reticulum/Golgi-dependent secretory pathway. In contrast, the release of M(r) 18,000 FGF-2 protein was almost completely inhibited by IkappaBalpha (S32A/S36A). These results suggest that FGF-2 release is independently mediated by NF-kappaB signaling, not simply a consequence of induction itself. Thus, NF-kappaB signaling is involved in induction of expression and release of FGF-2 by LMP1.

Nasopharyngeal carcinoma (NPC) is universally associated with EBV infection. We have shown that the phosphonated nucleoside analog, (S)-1-[3-hydroxy-2-(phosphonylmethoxy)-propyl]cytosine (HPMPC) strongly inhibits growth of NPC xenografts in nude mice by causing apoptosis (J. Neyts et al., Cancer Res., 58, 384-388, 1998). We, therefore, tested two additional members of this drug family that have different degrees of antiviral activity, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-2-(R)-(phosphonomethoxy)propyladenine (PMPA). Intratumoral injection of PMEA (75 microl of 2% solution) in C15 NPC xenografts, which are latently infected with EBV, slowed tumor growth moderately, whereas PMPA (75 microl of 2% solution) slowed tumor growth only marginally. Compared with the previous results showing complete regression of tumor, PMEA had less antitumoral effect than HPMPC, and PMPA had the least. After 4 weeks of preventive treatment, tumors formed in 12.5, 50, and 100% of mice treated with HPMPC, PMEA, and PMPA, respectively, in contrast to the development of tumors in all of the PBS-treated control mice. We also investigated the effect of each drug on the EBV-positive epithelial cell line NPC-KT in vitro. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed inhibition of growth of NPC-KT cells by HPMPC and PMEA, but not by PMPA, which correlates with the results observed in tumor xenografts. Growth inhibition was attributable to induction of apoptosis in NPC-KT cells as indicated by a DNA fragmentation assay. Cleavage of poly(ADP-ribose) polymerase after treatment of NPC-KT cells with HPMPC was observed, which suggested that the apoptosis may be mediated by caspase(s). The apoptotic effects of the drugs are independent of any effects on EBV DNA polymerase, which is not expressed in these latently infected NPCs. These results suggest that HPMPC as well as PMEA could provide an adjunctive treatment for NPC.

The effect of the antiviral agent (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (cidofovir) on the EBV-associated tumor nasopharyngeal carcinoma (NPC) was evaluated in NPC xenografts in athymic mice. Intratumoral injection arrested tumor growth within 1 week, and by 4 weeks, tumors regressed to 8-75% (39 +/- 33%) of the original size, whereas control tumors injected with PBS grew to 282 +/- 25% of the original size. Ganciclovir slowed but did not arrest or cause regression of tumor growth. A striking antitumor effect was also produced by systemic administration; at 4 weeks, tumors were 79 +/- 49% of the original size, compared with 635 +/- 91% for the controls. Widespread apoptosis was detected after treatment for 2-6 days in C15 as well as two other NPC xenografts, C17 and C18; the latter NPCs have mutations in the p53 gene. These data indicate that cidofovir induces rapid cell death through apoptosis in EBV-transformed epithelial cells.

Human ectocervical explant cultures were grown in medium with D-valine substituted for L-valine. Pure epithelial cell monolayers were obtained with both dialyzed and undialyzed fetal calf serum. Epithelial cell explant colonies grown in D-valine medium supplemented with undialyzed serum could routinely be subcultured if plated at a density of 1.5 x 10(4) cells/sq cm or higher. Such cultures could be passaged at least three times and could yield up to 21 population doublings per culture lifetime. Contaminating fibroblastic colonies were never detected in these cultures, which were free of surface-associated fibronectin as revealed by immunofluorescent tests. Both primary and passaged epithelial colonies retained many characteristic morphological features of ectocervical epithelium when examined by light and electron microscopy. Such cultures may be of use in investigating the action of viral and chemical carcinogenic agents upon epithelial cells in vitro.

A fatal disseminated polyclonal malignant lymphoproliferative disorder of B-cells (immunoblastic sarcoma) developed shortly after a second thymic epithelial peritoneal implant in a 5-yr-old girl with combined immunodeficiency. The immunodeficiency was characterized by low T-cell numbers and function, very low levels of thymic hormone, dysgammaglobulinemia, and an inability to mount a primary antibody or cell-mediated response to new antigens. At necropsy, the thymus fulfilled morphological criteria for thymic dysplasia. Epstein-Barr virus (EBV) antigen and DNA were identified in neoplastic infiltrates in the lymph nodes and thymus by immunofluorescence for the EBV nuclear antigen and by EBV-specific complementary RNA/DNA hybridization. No antibodies to nuclear antigen, early antigen, or viral capsid antigen of EBV were identified in the serum. The concurrence of these events suggests that the thymic epithelial implant itself may have been instrumental in the pathogenesis of this neoplasm. It is proposed that the thymus may have provided factors which indirectly potentiated the proliferation of EBV-infected B-cells, possibly by induction of nonspecific T-helper cells and perhaps through other thymic humoral factors. It is suggested that some forms of immunoblastic sarcoma, even when polyclonal, and especially those which arise in immunocompromised hosts, may, in some instances, represent an opportunistic form of EBV-induced B-cell neoplasia.