Mechanistic insights into the activation of the IKK kinase complex by the Kaposi's sarcoma herpes virus oncoprotein vFLIP.

Constitutive activation of the canonical NF-κB signaling pathway is a major factor in Kaposi's sarcoma-associated herpes virus pathogenesis where it is essential for the survival of primary effusion lymphoma. Central to this process is persistent upregulation of the inhibitor of κB kinase (IKK) complex by the virally encoded oncoprotein vFLIP. Although the physical interaction between vFLIP and the IKK kinase regulatory component essential for persistent activation, IKKγ, has been well characterized, it remains unclear how the kinase subunits are rendered active mechanistically. Using a combination of cell-based assays, biophysical techniques, and structural biology, we demonstrate here that vFLIP alone is sufficient to activate the IKK kinase complex. Furthermore, we identify weakly stabilized, high molecular weight vFLIP-IKKγ assemblies that are key to the activation process. Taken together, our results are the first to reveal that vFLIP-induced NF-κB activation pivots on the formation of structurally specific vFLIP-IKKγ multimers which have an important role in rendering the kinase subunits active through a process of autophosphorylation. This mechanism of NF-κB activation is in contrast to those utilized by endogenous cytokines and cellular FLIP homologues.

GRWD1-WDR5-MLL2 Epigenetic Complex Mediates H3K4me3 Mark and Is Essential for Kaposi's Sarcoma-Associated Herpesvirus-Induced Cellular Transformation.

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is causally associated with numerous cancers. The mechanism of KSHV-induced oncogenesis remains unclear. By performing a CRISPR-Cas9 screening in a model of KSHV-induced cellular transformation of primary cells, we identified epigenetic regulators that were essential for KSHV-induced cellular transformation. Examination of TCGA data sets of the top 9 genes, including glutamate-rich WD repeat containing 1 (GRWD1), a WD40 family protein upregulated by KSHV, that had positive effects on cell proliferation and survival of KSHV-transformed cells (KMM) but not the matched primary cells (MM), uncovered the predictive values of their expressions for patient survival in numerous types of cancer. We revealed global epigenetic remodeling including H3K4me3 epigenetic active mark in KMM cells compared to MM cells. Knockdown of GRWD1 inhibited cell proliferation, cellular transformation, and tumor formation and caused downregulation of global H3K4me3 mark in KMM cells. GRWD1 interacted with WD repeat domain 5 (WDR5), the core protein of H3K4 methyltransferase complex, and several H3K4me3 methyltransferases, including myeloid leukemia 2 (MLL2). Knockdown of WDR5 and MLL2 phenocopied GRWD1 knockdown, caused global reduction of H3K4me3 mark, and altered the expression of similar sets of genes. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses further identified common and distinct cellular genes and pathways that were regulated by GRWD1, WDR5, and MLL2. These results indicate that KSHV hijacks the GRWD1-WDR5-MLL2 epigenetic complex to regulate H3K4me3 methylation of specific genes, which is essential for KSHV-induced cellular transformation. Our work has identified an epigenetic complex as a novel therapeutic target for KSHV-induced cancers. IMPORTANCE By performing a genome-wide CRISPR-Cas9 screening, we have identified cellular epigenetic regulators that are essential for KSHV-induced cellular transformation. Among them, GRWD1 regulates epigenetic active mark H3K4me3 by interacting with WDR5 and MLL2 and recruiting them to chromatin loci of specific genes in KSHV-transformed cells. Hence, KSHV hijacks the GRWD1-WDR5-MLL2 complex to remodel cellular epigenome and induce cellular transformation. Since the dysregulation of GRWD1 is associated with poor prognosis in several types of cancer, GRWD1 might also be a critical driver in other viral or nonviral cancers.

Alternative lengthening of telomeres phenotype in malignant vascular tumors is highly associated with loss of ATRX expression and is frequently observed in hepatic angiosarcomas.

Alternative lengthening of telomeres (ALT) is a mechanism using homologous recombination to maintain telomere length and sustain limitless replicability of cancer cells. Recently, ALT has been found to be associated with inactivation of either α-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated (DAXX) protein. In this study, 119 tumors (88 angiosarcomas, 11 epithelioid hemangioendotheliomas, and 20 Kaposi sarcomas) were analyzed to determine the ALT status, its relationship to loss of ATRX/DAXX expression, and the clinicopathological features. In addition, the mutation status in the telomerase reverse transcriptase gene (TERT) promoter was also studied. Loss of ATRX expression was observed in 21% (16/77) of the primary angiosarcomas and 9% (1/11) of epithelioid hemangioendotheliomas. DAXX expression was intact in all but 2 ATRX-deficient angiosarcomas. Telomere-specific fluorescence in situ hybridization assay showed 28% (17/61) of the primary angiosarcomas were ALT positive. Remarkably, ALT was highly associated with loss of ATRX expression: all but 2 ALT-positive angiosarcomas were ATRX deficient. Notably, hepatic angiosarcomas were frequently ATRX deficient (8/13) and/or ALT positive (8/12). None of the secondary angiosarcomas were ATRX/DAXX deficient or ALT positive. The only ATRX-deficient epithelioid hemangioendothelioma was positive for ALT. Forty-seven angiosarcomas were tested for TERT promoter mutation. Despite the fact that angiosarcoma occurs most commonly in sun-damaged skin, mutation was detected in only 1 radiation-associated angiosarcoma (2%). We conclude that ALT is an important telomere maintenance mechanism in primary angiosarcomas. This feature is highly associated with loss of ATRX expression and is frequently observed in hepatic angiosarcomas.

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit ß by EPR Spectroscopy.

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/ß subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKß is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.

Lipoxins exert antiangiogenic and anti-inflammatory effects on Kaposi's sarcoma cells.

Lipoxin A4 (LXA4) is an endogenously produced host molecule with anti-inflammatory resolution effects. Previous studies demonstrated it to be involved in anti-vascular endothelial growth factor (VEGF)-mediated angiogenesis and in a possible anticancer role via interaction with its receptor, lipoxin A 4 receptor (ALXR). Here, we examined the effects of LXA4 and its epimer 15-epi-LXA4 in inhibiting proinflammatory and angiogenic functions in a human Kaposi's sarcoma tumor-derived cell line (KS-IMM). KS-IMM cells expressed increased levels of inflammatory cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LO) pathway enzymes when compared with human microvascular dermal endothelial cells (HMVEC-d). KS-IMM cells secreted high levels of prostaglandin E2 (PGE2) and chemotactic leukotriene B4 (LTB4). Treatment with LXA4 or 15-epi-LXA4 effectively reduced the levels of COX-2, 5-LO proteins, and secretion of PGE2 and LTB4 in KS-IMM cells. LXA4 or 15-epi-LXA4 treatment also decreased secretion of proinflammatory interleukin 6 (IL-6) and IL-8 cytokines but induced the secretion of anti-inflammatory IL-10. LXA4 treatment reduced the phosphorylation of VEGF receptor (VEGFR) and ephrin family receptor tyrosine kinases. LXA4 treatment effectively induced dephosphorylation of multiple cellular kinases such as Focal Adhesion Kinase, Protein kinase B, nuclear factor kappa-light-chain-enhancer of activated B cells, and Extracellular signal-regulated kinases (ERK)1/2, and reduced angiogenic factor VEGF-C secretion in KS cells. LX treatment drastically induced the Src-homology 2 domain-containing phosphatase tyrosine (Y542) phosphatase and reduced VEGFR-2 phosphorylation at sites Y1059, Y1175, and Y1212. Treatment of KS-IMM cells with LXA4 resulted in selective localization of VEGFR-2 in nonlipid raft (non-LR) and ALXR to LR fractions. These results demonstrated that LXA4 or 15-epi-LXA4 induce anti-inflammatory and antiangiogenic effects in KS cells and suggest that treatment with LXs is an attractive novel strategy against KS.

Stability of structured Kaposi's sarcoma-associated herpesvirus ORF57 protein is regulated by protein phosphorylation and homodimerization.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 plays an essential role in KSHV lytic infection by promoting viral gene expression at the posttranscriptional level. Using bioinformatic and biochemical approaches, we determined that ORF57 contains two structurally and functionally distinct domains: a disordered nonstructural N-terminal domain (amino acids [aa] 1 to 152) and a structured α-helix-rich C-terminal domain (aa 153 to 455). The N-terminal domain mediates ORF57 interaction with several RNA-protein complexes essential for ORF57 to function. The N-terminal phosphorylation by cellular casein kinase II (CKII) at S21, T32, and S43, and other cellular kinases at S95 and S97 residues in proximity of the caspase-7 cleavage site, 30-DETD-33, inhibits caspase-7 digestion of ORF57. The structured C-terminal domain mediates homodimerization of ORF57, and the critical region for this function was mapped carefully to α-helices 7 to 9. Introduction of point mutations into α-helix 7 at ORF57 aa 280 to 299, a region highly conserved among ORF57 homologues from other herpesviruses, inhibited ORF57 homodimerization and led to proteasome-mediated degradation of ORF57 protein. Thus, homodimerization of ORF57 via its C terminus prevents ORF57 from degrading and allows two structure-free N termini of the dimerized ORF57 to work coordinately for host factor interactions, leading to productive KSHV lytic infection and pathogenesis. IMPORTANCE: KSHV is a human oncogenic virus linked to the development of several malignancies. KSHV-mediated oncogenesis requires both latent and lytic infection. The KSHV ORF57 protein is essential for KSHV lytic replication, as it regulates the expression of viral lytic genes at the posttranscriptional level. This report provides evidence that the structural conformation of the ORF57 protein plays a critical role in regulation of ORF57 stability. Phosphorylation by CKII on the identified serine/threonine residues at the N-terminal unstructured domain of ORF57 prevents its digestion by caspase-7. The C-terminal domain of ORF57, which is rich in α-helices, contributes to homodimerization of ORF57 to prevent proteasome-mediated protein degradation. Elucidation of the ORF57 structure not only enables us to better understand ORF57 stability and functions but also provides an important tool for us to modulate ORF57's activity with the aim to inhibit KSHV lytic replication.

Short-chain fatty acids from periodontal pathogens suppress histone deacetylases, EZH2, and SUV39H1 to promote Kaposi's sarcoma-associated herpesvirus replication.

UNLABELLED: Periodontal pathogens such as Porphyromonas gingivalis and Fusobacterium nucleatum produce five different short-chain fatty acids (SCFAs) as metabolic by-products. We detect significantly higher levels of SCFAs in the saliva of patients with severe periodontal disease. The different SCFAs stimulate lytic gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) dose dependently and synergistically. SCFAs inhibit class-1/2 histone deacetylases (HDACs) and downregulate expression of silent information regulator-1 (SIRT1). SCFAs also downregulate expression of enhancer of zeste homolog2 (EZH2) and suppressor of variegation 3-9 homolog1 (SUV39H1), which are two histone N-lysine methyltransferases (HLMTs). By suppressing the different components of host epigenetic regulatory machinery, SCFAs increase histone acetylation and decrease repressive histone trimethylations to transactivate the viral chromatin. These new findings provide mechanistic support that SCFAs from periodontal pathogens stimulate KSHV replication and infection in the oral cavity and are potential risk factors for development of oral Kaposi's sarcoma (KS). IMPORTANCE: About 20% of KS patients develop KS lesions first in the oral cavity, while other patients never develop oral KS. It is not known if the oral microenvironment plays a role in oral KS tumor development. In this work, we demonstrate that a group of metabolic by-products, namely, short-chain fatty acids, from bacteria that cause periodontal disease promote lytic replication of KSHV, the etiological agent associated with KS. These new findings provide mechanistic support that periodontal pathogens create a unique microenvironment in the oral cavity that contributes to KSHV replication and development of oral KS.

Phase II trial of imatinib in AIDS-associated Kaposi's sarcoma: AIDS Malignancy Consortium Protocol 042.

PURPOSE: Kaposi's sarcoma (KS) is a disease of multifocal vascular proliferation that requires infection with KS herpes virus (KSHV/HHV-8). Activation of the c-kit and platelet-derived growth factor (PDGF) receptors by autocrine/paracrine mechanisms follows endothelial cell KSHV infection. In a pilot study, imatinib, a c-kit/PDGF-receptor inhibitor, induced partial regression of AIDS-associated KS (AIDS-KS) in five of 10 patients. PATIENTS AND METHODS: This multicenter phase II study was designed to estimate the response rate to imatinib in AIDS-KS. Secondary objectives included investigation of predictors of response and imatinib pharmacokinetics in patients on antiretrovirals. Patients received imatinib 400 mg/day by mouth for up to 12 months with dose escalation up to 600 mg/day at 3 months if their disease was stable. RESULTS: Thirty patients were treated at 12 AIDS Malignancy Consortium sites. Ten patients (33.3%) achieved partial response, six (20%) had stable disease, and seven (23.3%) exhibited KS progression. Nine patients completed 52 weeks of imatinib therapy. The median treatment duration was 22.5 weeks. Only five patients (16.7%) discontinued therapy owing to adverse events. Antiretroviral regimens did not significantly alter imatinib metabolism. Activating mutations in PDGF-R and c-kit were not found at baseline or at disease progression. We found no correlation with response with changes in any of the candidate cytokines. CONCLUSION: Imatinib has activity in AIDS-KS. Pharmacokinetic interactions with antiretroviral drugs did not correlate with toxicity. Thirty percent of patients showed long-term clinical benefit and remained on imatinib for the entire year. These results suggest imatinib is well tolerated and may be an alternative therapy for some patients with AIDS-KS.

Kaposi's sarcoma-associated herpesvirus induces rapid release of angiopoietin-2 from endothelial cells.

Kaposi sarcoma-associated herpesvirus (KSHV) stimulates proliferation, angiogenesis, and inflammation to promote Kaposi sarcoma (KS) tumor growth, which involves various growth factors and cytokines. Previously, we found that KSHV infection of human umbilical vein endothelial cells (HUVECs) induces a transcriptional induction of the proangiogenic and proinflammatory cytokine angiopoietin-2 (Ang-2). Here, we report that KSHV induces rapid release of Ang-2 that is presynthesized and stored in the Weibel-Palade bodies (WPB) of endothelial cells upon binding to its integrin receptors. Blocking viral binding to integrins inhibits Ang-2 release. KSHV binding activates the integrin tyrosine kinase receptor signaling pathways, leading to tyrosine phosphorylation of focal adhesion kinase (FAK), the tyrosine kinase Src, and the Calα2 subunit of the l-type calcium channel to trigger rapid calcium (Ca(2+)) influx. Pretreatment of endothelial cells with specific inhibitors of protein tyrosine kinases inhibits KSHV-induced Ca(2+) influx and Ang-2 release. Inhibition of Ca(2+) mobilization with calcium channel blockers also inhibits Ang-2 release. Thus, the interaction between KSHV and its integrin receptors plays a key role in regulating rapid Ang-2 release from endothelial cells. This finding highlights a novel mechanism of viral induction of angiogenesis and inflammation, which might play important roles in the early event of KS tumor development.

KSHV-initiated notch activation leads to membrane-type-1 matrix metalloproteinase-dependent lymphatic endothelial-to-mesenchymal transition.

Kaposi sarcoma (KS), an angioproliferative disease associated with Kaposi sarcoma herpesvirus (KSHV) infection, harbors a diversity of cell types ranging from endothelial to mesenchymal cells of unclear origin. We developed a three-dimensional cell model for KSHV infection and used it to demonstrate that KSHV induces transcriptional reprogramming of lymphatic endothelial cells to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT). KSHV-induced EndMT was initiated by the viral proteins vFLIP and vGPCR through Notch pathway activation, leading to gain of membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive properties and concomitant changes in viral gene expression. Mesenchymal markers and MT1-MMP were found codistributed with a KSHV marker in the same cells from primary KS biopsies. Our data explain the heterogeneity of cell types within KS lesions and suggest that KSHV-induced EndMT may contribute to KS development by giving rise to infected, invasive cells while providing the virus a permissive cellular microenvironment for efficient spread.

What has the study of the K3 and K5 viral ubiquitin E3 ligases taught us about ubiquitin-mediated receptor regulation?

Cells communicate with each other and the outside world through surface receptors, which need to be tightly regulated to prevent both overstimulation and receptor desensitization. Understanding the processes involved in the homeostatic control of cell surface receptors is essential, but we are not alone in trying to regulate these receptors. Viruses, as the ultimate host pathogens, have co-evolved over millions of years and have both pirated and adapted host genes to enable viral pathogenesis. K3 and K5 (also known as MIR1 and MIR2) are viral ubiquitin E3 ligases from Kaposi's Sarcoma Associated Herpesvirus (KSHV) which decrease expression of a number of cell surface receptors and have been used to interrogate cellular processes and improve our understanding of ubiquitin-mediated receptor endocytosis and degradation. In this review, we summarize what has been learned from the study of these viral genes and emphasize their role in elucidating the complexity of ubiquitin in receptor regulation.

Phosphatase and tensin homolog on chromosome 10 is phosphorylated in primary effusion lymphoma and Kaposi's sarcoma.

Primary effusion lymphoma (PEL) is a non-Hodgkin's B-cell lymphoma driven by Kaposi's sarcoma-associated herpesvirus. It is uniquely sensitive to mTOR, PI3K, and Akt inhibitors; however, the basis of this requirement for the mTOR pathway remains to be elucidated. The phosphatase and tensin homolog gene (PTEN) on chromosome 10 controls the first step in the phosphatidylinositol 3 kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway and is genetically inactivated in many solid tumors. We find an absence of PTEN deletions, mutations, or protein mislocalization in PEL. However, we find consistent hyperphosphorylation at serine 380 of PTEN, which is an inactivating modification, in PEL cell lines and in tumor xenografts. We also evaluated a large tissue microarray of Kaposi's sarcoma biopsies and observed concordant high levels of phospho-PTEN, phospho-Akt, and phospho-S6 ribosomal protein. Reintroduction of PTEN into PEL inhibited colony formation in soft agar, verifying the functional dependence of PEL on PI3K signaling. This was also true for PEL cell lines that carried mutant p53 and for KS-like cell lines. Activating PTEN in these cancers may yield a new treatment strategy for PEL, KS, and similar PTEN wild-type lymphomas.

Matrix metalloproteinases 2 and 9, and extracellular matrix in Kaposi's sarcoma.

Matrix metalloproteinases (MMPs) are associated with Kaposi's sarcoma (KS) tumorigenesis and may contribute to the mechanism of KS invasive growth. To date, only a few MMPs have been studied in KS lesions, and exactly which MMPs are involved in KS development and progression remains unanswered. However, MMPs 2 and 9 have been associated with different phases of angiogenesis, but their role in the proteolytic modification of the extracellular matrix has not been investigated. The results of this study confirm that MMPs, specifically MMP-2 and MMP-9, can contribute to angiogenesis by disrupting the vessel basement membrane and other extracellular matrix barriers, and enabling endothelial cells migration through the surrounding tissues.

Kaposi's sarcoma associated herpes virus (KSHV) induced COX-2: a key factor in latency, inflammation, angiogenesis, cell survival and invasion.

Kaposi's sarcoma (KS), an enigmatic endothelial cell vascular neoplasm, is characterized by the proliferation of spindle shaped endothelial cells, inflammatory cytokines (ICs), growth factors (GFs) and angiogenic factors. KSHV is etiologically linked to KS and expresses its latent genes in KS lesion endothelial cells. Primary infection of human micro vascular endothelial cells (HMVEC-d) results in the establishment of latent infection and reprogramming of host genes, and cyclooxygenase-2 (COX-2) is one of the highly up-regulated genes. Our previous study suggested a role for COX-2 in the establishment and maintenance of KSHV latency. Here, we examined the role of COX-2 in the induction of ICs, GFs, angiogenesis and invasive events occurring during KSHV de novo infection of endothelial cells. A significant amount of COX-2 was detected in KS tissue sections. Telomerase-immortalized human umbilical vein endothelial cells supporting KSHV stable latency (TIVE-LTC) expressed elevated levels of functional COX-2 and microsomal PGE2 synthase (m-PGES), and secreted the predominant eicosanoid inflammatory metabolite PGE2. Infected HMVEC-d and TIVE-LTC cells secreted a variety of ICs, GFs, angiogenic factors and matrix metalloproteinases (MMPs), which were significantly abrogated by COX-2 inhibition either by chemical inhibitors or by siRNA. The ability of these factors to induce tube formation of uninfected endothelial cells was also inhibited. PGE2, secreted early during KSHV infection, profoundly increased the adhesion of uninfected endothelial cells to fibronectin by activating the small G protein Rac1. COX-2 inhibition considerably reduced KSHV latent ORF73 gene expression and survival of TIVE-LTC cells. Collectively, these studies underscore the pivotal role of KSHV induced COX-2/PGE2 in creating KS lesion like microenvironment during de novo infection. Since COX-2 plays multiple roles in KSHV latent gene expression, which themselves are powerful mediators of cytokine induction, anti-apoptosis, cell survival and viral genome maintainence, effective inhibition of COX-2 via well-characterized clinically approved COX-2 inhibitors could potentially be used in treatment to control latent KSHV infection and ameliorate KS.

Imatinib interferes with survival of multi drug resistant Kaposi's sarcoma cells.

Multi drug resistance (MDR) is defined as the ability of tumor cells to become resistant to unrelated drugs. Tyrosine kinase inhibitor imatinib has been demonstrated to be effective in the treatment of certain tumors. In particular, imatinib inhibits Bcr-Abl kinase activity, c-kit and the phosphorylation of platelet-derived growth factor (PDGF) receptors. In this work, we show that imatinib inhibits PDGF phosphorylation not only in wt Kaposi sarcoma (KS) but also in multi drug resistant KS cells. This was associated with an increased apoptosis in wt cells and an increased autophagy in MDR-KS cells. These data add new insights to the possible use of imatinib in the overcoming of MDR in KS cells.

Notch inhibition in Kaposi's sarcoma tumor cells leads to mitotic catastrophe through nuclear factor-kappaB signaling.

Kaposi's sarcoma (KS) is the most common neoplasm in untreated AIDS patients and accounts for significant morbidity and mortality worldwide. We have recently reported that Notch signaling (which plays an important role in cell proliferation, apoptosis, and oncogenesis) is constitutively activated in KS tumor cells. Blockade of this activity using gamma-secretase inhibitors resulted in apoptosis of SLK cells, a KS tumor cell line; however, this apoptosis was preceded by a prolonged G(2)-M cell cycle arrest. This result led us to hypothesize that the cells were undergoing mitotic catastrophe, an abnormal mitosis that leads to eventual cell death. Here, we show that Notch inhibition in KS tumor cells using gamma-secretase inhibitors or Notch-1 small interfering RNA resulted in G(2)-M cell cycle arrest and mitotic catastrophe characterized by the presence of micronucleated cells and an increased mitotic index. Interestingly, Notch inhibition led to a sustained increase in nuclear cyclin B1, a novel observation suggesting that Notch signaling can modulate expression of this critical cell cycle protein. Further analysis showed the induction of cyclin B1 was due, at least in part, to increased nuclear factor-kappaB (NF-kappaB) activity, which was also required for the G(2)-M growth arrest after Notch inhibition. Taken together, these studies suggest that Notch inhibition can initiate aberrant mitosis by inducing NF-kappaB activity that inappropriately increases cyclin B1 resulting in cell death via mitotic catastrophe.

Evidence of COX-1 and COX-2 expression in Kaposi's sarcoma tissues.

Cyclooxygenases (COXs) are enzymes catalysing prostaglandin synthesis and are implicated in the carcinogenesis of some cancer types. In addition, an important role of these enzymes in herpesvirus infections was demonstrated and it has recently been proposed that COX-2 may participate in herpesvirus-induced neoplasia such as Kaposi's sarcoma (KS). To date no immunohistochemical study has been performed to determine the identification of COX-1 and COX-2 in KS. We have investigated 35 cases of classic KS and 27 cases of epidemic KS form in order to study the distribution and localisation of COXs. We have examined by immunohistochemistry the expression of COX-1 and COX-2 in classic and epidemic forms of KS also in relationship to the characteristic morphological phases (patch, plaque and nodular stage) of KS and cell localisation by double immunostaining. Moreover, we have obtained COX-1 and COX-2 expression by Western blot analysis. Our results establish that (a) COX-1 and COX-2 are overexpressed significantly in classic and epidemic KS compared with control skin tissues (P<0.01 and P>0.03, respectively, for COX-1; P<0.01 and P>0.03, respectively, for COX-2); (b) the extent and intensity staining for both COXs were higher in classic than in epidemic form of KS. Our data support the hypothesis that both COXs may be involved in the pathogenesis of KS.

Kaposi's sarcoma-associated herpesvirus infection promotes invasion of primary human umbilical vein endothelial cells by inducing matrix metalloproteinases.

Matrix metalloproteinases (MMPs) play important roles in cancer invasion, angiogenesis, and inflammatory infiltration. Kaposi's sarcoma is a highly disseminated angiogenic tumor of proliferative endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we showed that KSHV infection increased the invasiveness of primary human umbilical vein endothelial cells (HUVEC) in a Matrigel-based cell invasion assay. KSHV-induced cell invasion was abolished by an inhibitor of MMPs, BB-94, and occurred in both autocrine- and paracrine-dependent fashions. Analysis by zymography and Western blotting showed that KSHV-infected HUVEC cultures had increased secretion of MMP-1, -2, and -9. KSHV increased the secretion of MMP-2 within 1 h following infection without upregulating its mRNA expression level. In contrast, the secretion of MMP-1 and -9 was not increased until 6 h after KSHV infection and was correlated with the upregulation of their mRNA expression levels. Promoter analysis by reporter assays and electrophoretic mobility shift assays identified an AP-1 cis-element as the dominant KSHV-responsive site in the MMP-1 promoter. Together, these results suggest that KSHV infection modulates the production of multiple MMPs to increase cell invasiveness and thus contributes to the pathogenesis of KSHV-induced malignancies.