Kaposi sarcoma-associated herpesvirus promotes tumorigenesis by modulating the Hippo pathway.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the culprit behind the human disease Kaposi sarcoma (KS), an AIDS-defining malignancy. KSHV encodes a viral G-protein-coupled receptor (vGPCR) critical for the initiation and progression of KS. In this study, we identified that YAP/TAZ, two homologous oncoproteins inhibited by the Hippo tumor suppressor pathway, are activated in KSHV-infected cells in vitro, KS-like mouse tumors and clinical human KS specimens. The KSHV-encoded vGPCR acts through Gq/11 and G12/13 to inhibit the Hippo pathway kinases Lats1/2, promoting the activation of YAP/TAZ. Furthermore, depletion of YAP/TAZ blocks vGPCR-induced cell proliferation and tumorigenesis in a xenograft mouse model. The vGPCR-transformed cells are sensitive to pharmacologic inhibition of YAP. Our study establishes a pivotal role of the Hippo pathway in mediating the oncogenic activity of KSHV and development of KS, and also suggests a potential of using YAP inhibitors for KS intervention.

Kaposi sarcoma-associated herpesvirus and other viruses in human lymphomagenesis.

Kaposi sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is associated with a specific subset of lymphoproliferative disorders. These include two main categories. The first is primary effusion lymphomas and related solid variants. The second is multicentric Castleman disease, from which KSHV-positive plasmablastic lymphomas can arise. KSHV contributes to lymphomagenesis by subverting the host cell molecular signaling machinery to deregulate cell growth and survival. KSHV expresses a selected set of genes in the lymphoma cells, encoding viral proteins that play important roles in KSHV lymphomagenesis. Deregulation of the NF-kappaB pathway is an important strategy used by KSHV to promote lymphoma cell survival, and the viral protein vFLIP is essential for this process. Two other viruses that are well documented to be causally associated with lymphoid neoplasia in humans are Epstein-Barr virus (EBV/HHV-4) and human T-cell lymphotropic virus (HTLV-1). Both of these are similar to KSHV in their use of viral proteins to promote cell survival by deregulating the NF-kappaB pathway. Here we review the basic information and recent developments that have contributed to our knowledge of lymphomas caused by KSHV and other viruses. The understanding of the mechanisms of viral lymphomagenesis should lead to the identification of novel therapeutic targets and to the development of rationally designed therapies.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor promotes endothelial cell survival through the activation of Akt/protein kinase B.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV-GPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma, playing a central role in the promotion of vascular endothelial growth factor (VEGF)-driven angiogenesis and spindle cell proliferation. We previously have shown that KSHV-GPCR has oncogenic potential when overexpressed in fibroblasts and is responsible for the expression and secretion of VEGF through the regulation of different intracellular signaling pathways (A. Sodhi et al., Cancer Res., 60: 4873-4880, 2000; C. Bais et al., Nature, 391: 86-89, 1998). Here, we describe that this constitutively active G protein-coupled receptor is able to promote cell survival in primary human umbilical vein endothelial cells and that this effect is independent of its ability to secrete VEGF because it is not prevented by the expression of antisense constructs for VEGF or the addition of VEGF-blocking antibodies. Instead we found that ectopic expression of KSHV-GPCR potently induces the kinase activity of Akt/protein kinase B in a dose-dependent manner and triggers its translocation to the plasma membrane. This signaling pathway requires the function of phosphatidylinositol 3'-kinase and is dependent on betagamma subunits released from both pertussis toxin-sensitive and -insensitive G proteins. Furthermore, we found that KSHV-GPCR is able to protect human umbilical vein endothelial cells from the apoptosis induced by serum deprivation and that both wortmannin and the expression of a kinase-deficient Akt K179M mutant are able to block this effect. Finally, we observed that the Akt K179M protein also inhibits the activation of nuclear factor-KB induced by KSHV-GPCR, suggesting that this transcription factor may represent one of the putative downstream targets for Akt in the survival-signaling pathway. These results provide further knowledge in the elucidation of the signal transduction pathways activated by KSHV-GPCR and support its key role in promoting the survival of viral-infected cells. Moreover, the present findings also emphasize the importance of this G protein-coupled receptor in the development of KSHV-related neoplasias.

Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma.

The t(4;14) translocation occurs frequently in multiple myeloma (MM) and results in the simultaneous dysregulated expression of 2 potential oncogenes, FGFR3 (fibroblast growth factor receptor 3) from der(14) and multiple myeloma SET domain protein/Wolf-Hirschhorn syndrome candidate gene 1 from der(4). It is now shown that myeloma cells carrying a t(4;14) translocation express a functional FGFR3 that in some cases is constitutively activated by the same mutations that cause thanatophoric dysplasia. As with activating mutations of K-ras and N-ras, which are reported in approximately 40% of patients with MM, activating mutations of FGFR3 occur during tumor progression. However, the constitutive activation of ras and FGFR3 does not occur in the same myeloma cells. Thus the activated forms of these proteins appear to share an overlapping role in tumor progression, suggesting that they also share the signaling cascade. Consistent with this prediction, it is shown that activated FGFR3-when expressed at levels similar to those seen in t(4;14) myeloma-is an oncogene that acts through the MAP kinase pathway to transform NIH 3T3 cells, which can then generate tumors in nude mice. Thus, FGFR3, when overexpressed in MM, may be not only oncogenic when stimulated by FGF ligands in the bone marrow microenvironment, but is also a target for activating mutations that enable FGFR3 to play a ras-like role in tumor progression.

The Kaposi's sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1alpha.

The elucidation of the molecular mechanisms governing the transition from a nonangiogenic to an angiogenic phenotype is central for understanding and controlling malignancies. Viral oncogenes represent powerful tools for disclosing transforming mechanisms, and they may also afford the possibility of investigating the relationship between transforming pathways and angiogenesis. In this regard, we have recently observed that a constitutively active G protein-coupled receptor (GPCR) encoded by the Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus 8 is oncogenic and stimulates angiogenesis by increasing the secretion of vascular endothelial growth factor (VEGF), which is a key angiogenic stimulator and a critical mitogen for the development of Kaposi's sarcoma. Here we show that the KSHV GPCR enhances the expression of VEGF by stimulating the activity of the transcription factor hypoxia-inducible factor (HIF)-1alpha, which activates transcription from a hypoxia response element within the 5'-flanking region of the VEGF promoter. Stimulation of HIF-1alpha by the KSHV GPCR involves the phosphorylation of its regulatory/inhibitory domain by the p38 and mitogen-activated protein kinase (MAPK) signaling pathways, thereby enhancing its transcriptional activity. Moreover, specific inhibitors of the p38 (SKF86002) and MAPK (PD98059) pathways are able to inhibit the activation of the transactivating activity of HIF-1alpha induced by the KSHV GPCR, as well as the VEGF expression and secretion in cells overexpressing this receptor. These findings suggest that the KSHV GPCR oncogene subverts convergent physiological pathways leading to angiogenesis and provide the first insight into a mechanism whereby growth factors and oncogenes acting upstream from MAPK, as well as inflammatory cytokines and cellular stresses that activate p38, can interact with the hypoxia-dependent machinery of angiogenesis. These results may also help to identify novel targets for the development of antiangiogenic therapies aimed at the treatment of Kaposi's sarcoma and other neoplastic diseases.

Kaposi's sarcoma-associated herpesvirus-encoded G protein-coupled receptor activation of c-jun amino-terminal kinase/stress-activated protein kinase and lyn kinase is mediated by related adhesion focal tyrosine kinase/proline-rich tyrosine kinase 2.

The Kaposi's sarcoma-associated herpesvirus (KSHV) (also known as human herpesvirus 8) has been implicated in the pathogenesis of Kaposi's sarcoma and B cell primary effusion lymphomas. KSHV encodes a G protein-coupled receptor (GPCR) that acts as an oncogene and constitutively activates two protein kinases, c-Jun amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase. It also induces the production of vascular endothelial growth factor. These processes are believed to be important in KSHV-GPCR-related oncogenesis. We have characterized the signaling pathways mediated by KSHV-GPCR in a reconstituted 293T cell model in which the related adhesion focal tyrosine kinase (RAFTK) was ectopically expressed. RAFTK has been shown to play an important role in growth factor signaling in endothelium and in B cell antigen receptor signaling in B lymphocytes. KSHV-GPCR induced the tyrosine phosphorylation of RAFTK. Expression of wild-type RAFTK enhanced GPCR-mediated JNK/SAPK activation, whereas dominant-negative mutant constructs of RAFTK, such as K457A (which lacks kinase activity) and Y402F (a Src-binding mutant), inhibited KSHV-GPCR-mediated activation of JNK/SAPK. RAFTK also mediated the KSHV-GPCR-induced activation of Lyn, a Src family kinase. However, RAFTK did not mediate the activation of p38 mitogen-activated protein kinase induced by KSHV-GPCR. Human interferon gamma-inducible protein-10, which is known to inhibit KSHV-GPCR activity, was found to reduce RAFTK phosphorylation and JNK/SAPK activation. These results suggest that in cells expressing RAFTK/proline-rich tyrosine kinase 2, such as endothelial and B cells, RAFTK can act to enhance KSHV-GPCR-mediated downstream signaling to transcriptional regulators such as JNK/SAPK.

Virus-associated lymphomas.

Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, and human T-cell lymphotrophic virus are viruses that are implicated in lymphoid neoplasia in humans. Their association with specific subsets of lymphomas suggests that they play an important, although not sufficient, etiologic role in their development. Current knowledge suggests that these viruses contribute to lymphomagenesis by subverting the host-cell molecular machinery to deregulate cell growth and survival. In this article, the basic information and recent developments that have contributed to our understanding of viral lymphomagenesis are reviewed.

Inhibition of constitutive signaling of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor by protein kinases in mammalian cells in culture.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, which is consistently present in tissues of patients with Kaposi's sarcoma and primary effusion lymphomas, contains a gene that encodes a G protein-coupled receptor (KSHV-GPCR). We recently showed that KSHV-GPCR exhibits constitutive signaling via activation of phosphoinositide-specific phospholipase C and stimulates cell proliferation and transformation. In this study, we determined whether normal cellular mechanisms could inhibit constitutive signaling by KSHV-GPCR and thereby KSHV-GPCR-stimulated proliferation. We show that coexpression of GPCR-specific kinases (GRKs) and activation of protein kinase C inhibit constitutive signaling by KSHV-GPCR in COS-1 monkey kidney cells and in mouse NIH 3T3 cells. Moreover, GRK-5 but not GRK-2 inhibits KSHV-GPCR-stimulated proliferation of rodent fibroblasts. These data provide evidence that cell regulatory pathways of receptor desensitization may be therapeutic targets in human diseases involving constitutively active receptors.

G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is a gamma-2 herpesvirus that is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs). KSHV infects malignant and progenitor cells of Kaposi's sarcoma and PEL, it encodes putative oncogenes and genes that may cause Kaposi's sarcoma pathogenesis by stimulating angiogenesis. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposi's sarcoma lesions and in PEL and stimulates signalling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signalling by this KSHV G-protein-coupled receptor leads to cell transformation and tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor, an angiogenesis and Kaposi's-spindle-cell growth factor. We find that this receptor can activate two protein kinases, JNK/SAPK and p38MAPK, by triggering signalling cascades like those induced by inflammatory cytokines that are angiogenesis activators and mitogens for Kaposi's sarcoma cells and B cells. We conclude that the KSHV G-protein-coupled receptor is a viral oncogene that can exploit cell signalling pathways to induce transformation and angiogenesis in KSHV-mediated oncogenesis.

Establishment and characterization of a primary effusion (body cavity-based) lymphoma cell line (BC-3) harboring kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus.

The recently identified Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), has been found to be consistently associated with an unusual subset of acquired immunodeficiency syndrome-related lymphomas, the so-called body cavity-based lymphomas (BCBL) or primary effusion lymphomas (PEL). These lymphomas are characterized by a unique spectrum of morphologic and molecular characteristics, and grow as lymphomatous effusions without an identifiable contiguous tumor mass. Until now, efforts to delineate the role of KSHV in the pathogenesis of PELs have been hampered by the lack of appropriate model systems and the concomitant presence of Epstein-Barr virus (EBV) in nearly all cases examined, and in all previously established cell lines. We now report the establishment and characterization of a novel PEL cell line, BC-3, which is KSHV+ by polymerase chain reaction (PCR) but EBV- as assessed by a variety of methods including PCR for EBER, EBNA-2, and EBNA-3C. This cell line was established from a lymphomatous effusion obtained from an HIV- patient, and has immunophenotypic and molecular features consistent with the diagnosis of PEL, including an indeterminate immunophenotype with a B-cell immunogenotype and lack of c-myc proto-oncogene rearrangements. Pulsed-field gel electrophoresis shows an intact KSHV genome of about 170 kb both in the cell line and in the viral isolate, whereas herpesvirus-like capsids are visible by electron microscopy. Consequently, the BC-3 cell line represents an invaluable tool as a source of KSHV, for both the evaluation of the pathogenic potential of this virus and the mechanistic characterization of its role in the development of Kaposi's sarcoma and malignant lymphoma.

Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus is a new transmissible virus that infects B cells.

Herpesviral DNA fragments isolated from AIDS-associated Kaposi's sarcoma (KS) tissue (KSHV-DNA) share homology with two lymphotropic oncogenic gamma-herpesviruses, Epstein-Barr virus and Herpesvirus saimiri, and are present in the lesions of more than 95% of HIV and non-HIV-associated forms of KS, AIDS-related body cavity-based lymphomas, and AIDS-related multicentric Castleman's disease. Here we show that BC-1, a KSHV-DNA-positive, body cavity-based lymphoma cell line, produces infective herpesviral particles carrying a linear 270-kb genome that specifically transmits KSHV-DNA to CD19+ B cells. Transmission of KSHV-DNA is dependent upon a biologically active, replicating virus, since it is blocked by UV irradiation and foscarnet, an inhibitor of viral DNA-polymerase. This study represents the first isolation and transmission of the human herpesvirus-8/KS-associated herpesvirus.

Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice.

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific angiogenic growth factor. VEGF gene transfer strategies to stimulate focal angiogenesis could be used to ameliorate myocardial ischemia. To induce angiogenesis in vivo, we have constructed a replication-defective herpes simplex virus type 1 (HSV-1) amplicon vector that places the human VEGF-165 cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter (HSVhvegf). Transduction of NIH 3T3 fibroblasts with HSVhvegf resulted in the secretion of high levels of biologically active VEGF, as assayed by microvascular endothelial mitogenesis. By use of an ex vivo protocol, BLK-CL4 fibroblasts were transduced with HSVhvegf or control HSVlac virus (expressing Escherichia coli beta-galactosidase), resuspended in basement membrane extract (matrigel), and coinjected subcutaneously into syngeneic C57BL/6 mice. One week later, the matrigel plugs with HSVhvegf showed a strong angiogenic response, in contrast to the plugs with HSVlac-transduced fibroblasts. These data indicate that transduction with HSVhvegf virus can induce an angiogenic response in vivo and suggest that this is a viable gene therapy approach for tissue ischemia.

The heparin-binding domain of heparin-binding EGF-like growth factor can target Pseudomonas exotoxin to kill cells exclusively through heparan sulfate proteoglycans.

Heparin-binding EGF-like growth factor (HB-EGF) is a smooth muscle cell mitogen composed of both EGF receptor and heparin-binding domains. To better understand the function of its domains, intact HB-EGF or its heparin-binding (HB) domain (amino acids 1-45) were fused to a mutant Pseudomonas exotoxin with an inactivated cell-binding domain. The resulting chimeric toxins, HB-EGF-PE* and HB-PE*, were tested on tumor cells, proliferating smooth muscle cells and a mutant Chinese hamster ovary cell line deficient in heparan sulfate proteoglycans (HSPGs). Two targets were found for HB-EGF-PE*. Cells were killed mainly through EGF receptors, but the HB domain was responsible for killing via HSPGs. HB-PE* did not bind to the EGF receptor and thus was cytotoxic by interacting exclusively with HSPGs. We conclude that the HB domain of HB-EGF is able to mediate internalization through HSPGs, without requiring the EGF receptor.

Cytotoxic effects of vascular smooth muscle cells of the chimeric toxin, heparin binding TGF alpha-Pseudomonas exotoxin.

OBJECTIVE: Smooth muscle cell proliferation appears to be very important in restenosis after angioplasty. A chimeric toxin created by genetically fusing the gene encoding TGF alpha (targets the EGF receptor) to the gene encoding Pseudomonas exotoxin (PE) preferentially kills rapidly proliferating smooth muscle cells. Recently, a heparin binding EGF-like growth factor (HB-EGF) has been identified. The HB domain enhances the mitogenic activity for smooth muscle cells. The purpose of this study was to design a new chimeric toxin, having both heparin binding and EGF receptor binding function, and to determine whether it is more cytotoxic to smooth muscle cells. METHODS: By recombinant DNA techniques, a new chimeric toxin, HB-TGF alpha-PE4EKDEL, was synthesised. Cytotoxic assays were performed by assessing the capacity to inhibit protein synthesis of rat vascular smooth muscle cells. RESULTS: The toxin preferentially killed rapidly proliferating smooth muscle cells (p < 0.025). The HB domain increased the cytotoxicity of the molecule when compared to the other chimeric toxins tested against smooth muscle cells. The cytotoxic effect of the new molecule was significantly decreased by exogenously added heparin (p < 0.05). CONCLUSIONS: The presence of a heparin binding domain increases the smooth muscle cell cytotoxicity of the TGF alpha fusion toxin, perhaps because HB-TGF alpha-PE4EKDEL functions as a molecule with two ligands. It will be important to determine whether the greater smooth muscle cell cytotoxicity that exists in vitro will facilitate the specific targeting and killing of rapidly proliferating cells in vivo.

Heparin-binding transforming growth factor alpha-Pseudomonas exotoxin A. A heparan sulfate-modulated recombinant toxin cytotoxic to cancer cells and proliferating smooth muscle cells.

TGF alpha-PE40, a recombinant toxin in which transforming growth factor alpha (TGF alpha) is fused to a mutant form of Pseudomonas exotoxin, is selectively cytotoxic to cells bearing epidermal growth factor (EGF) receptors. Heparin binding EGF-like growth factor is a potent mitogen for smooth muscle cells capable of binding to both the EGF receptor and to immobilized heparin (Higashiyama, S., Abraham, J., Miller, J., Fiddes, J., and Klagsbrun, M. (1991) Science 251, 936-938). To study the effect of the heparin-binding domain in a chimeric toxin targeted to the EGF receptor, we fused the DNA sequence corresponding to the putative NH2-terminal heparin-binding (HB) domain of HB-EGF to chimeric toxins composed of TGF alpha and two different recombinant forms of Pseudomonas exotoxin (PE). One of these is a truncated form of PE devoid of the binding domain (TGF alpha-PE38); another is a mutant form of full-length toxin containing inactivating mutations in the binding domain and an altered carboxyl terminus (TGF alpha-PE4EKDEL). The resulting chimeric toxins HB-TGF alpha-PE38 and HB-TGF alpha-PE4EKDEL were expressed in Escherichia coli as inclusion bodies, refolded, and purified by heparin affinity chromatography. Both of the toxins were eluted from heparin at 0.8 M NaCl, in contrast to their respective TGF alpha toxins which were eluted at 0.15 M. Binding studies on A431 cells showed that the HB-TGF alpha toxins bound to the EGF receptor with an affinity similar to that of the TGF alpha toxins. However, cell killing studies on a panel of malignant cell lines showed that cytotoxicity was strongly affected by the presence of the HB domain. Cell lines expressing high numbers of EGF receptors such as A431 and KB were less sensitive to toxins containing the HB domain. Cells with low number of EGF receptors had similar responses to both types of toxins (MCF-7 and LNCaP) or were more sensitive to the toxin with the added HB domain (HEP-G2). HB-TGF alpha-PE4EKDEL was over 10-fold more cytotoxic against proliferating vascular smooth muscle cells (VSMC) than to quiescent VSMC. Moreover, HB-TGF alpha-PE4EKDEL was 6-fold more potent than TGF alpha-PE4EKDEL to proliferating VSMC. Competition studies with EGF and/or heparin showed that heparin blocks the cytotoxicity of HB-TGF toxins and the inhibitory action of heparin is stronger in cells expressing lower number of EGF receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Major Trypanosoma cruzi antigenic determinant in Chagas' heart disease shares homology with the systemic lupus erythematosus ribosomal P protein epitope.

A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease.