Productively Infected Murine Kaposi's Sarcoma-Like Tumors Define New Animal Models for Studying and Targeting KSHV Oncogenesis and Replication

Kaposi''s sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). KS tumors are composed of KSHV-infected spindle cells of vascular origin with aberrant neovascularization and erythrocyte extravasation. KSHV genes expressed during both latent and lytic replicative cycles play important roles in viral oncogenesis. Animal models able to recapitulate both viral and host biological characteristics of KS are needed to elucidate oncogenic mechanisms, for developing targeted therapies, and to trace cellular components of KS ontogeny. Herein, we describe two new murine models of Kaposi''s sarcoma. We found that murine bone marrow-derived cells, whether established in culture or isolated from fresh murine bone marrow, were infectable with rKSHV.219, formed KS-like tumors in immunocompromised mice and produced mature herpesvirus-like virions in vivo. Further, we show in vivo that the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) enhanced viral lytic reactivation. We propose that these novel models are ideal for studying both viral and host contributions to KSHV-induced oncogenesis as well as for testing virally-targeted antitumor strategies for the treatment of Kaposi''s sarcoma. Furthermore, our isolation of bone marrow-derived cell populations containing a cell type that, when infected with KSHV, renders a tumorigenic KS-like spindle cell, should facilitate systematic identification of KS progenitor cells.     

Activation of the B Cell Antigen Receptor Triggers Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus in B Cells

Kaposi''s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus and the cause of Kaposi''s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman''s disease. Latently infected B cells are the main reservoir of this virus in vivo, but the nature of the stimuli that lead to its reactivation in B cells is only partially understood. We established stable BJAB cell lines harboring latent KSHV by cell-free infection with recombinant virus carrying a puromycin resistance marker. Our latently infected B cell lines, termed BrK.219, can be reactivated by triggering the B cell receptor (BCR) with antibodies to surface IgM, a stimulus imitating antigen recognition. Using this B cell model system we studied the mechanisms that mediate the reactivation of KSHV in B cells following the stimulation of the BCR and could identify phosphatidylinositol 3-kinase (PI3K) and X-box binding protein 1 (XBP-1) as proteins that play an important role in the BCR-mediated reactivation of latent KSHV.     

Global Metabolic Profiling of Infection by an Oncogenic Virus: KSHV Induces and Requires Lipogenesis for Survival of Latent Infection

Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposi''s Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi''s Sarcoma (KS), the most common tumor of AIDS patients. Approximately one-third of the nearly 200 measured metabolites were altered following latent infection of endothelial cells by KSHV, including many metabolites of anabolic pathways common to most cancer cells. KSHV induced pathways that are commonly altered in cancer cells including glycolysis, the pentose phosphate pathway, amino acid production and fatty acid synthesis. Interestingly, over half of the detectable long chain fatty acids detected in our screen were significantly increased by latent KSHV infection. KSHV infection leads to the elevation of metabolites involved in the synthesis of fatty acids, not degradation from phospholipids, and leads to increased lipid droplet organelle formation in the infected cells. Fatty acid synthesis is required for the survival of latently infected endothelial cells, as inhibition of key enzymes in this pathway led to apoptosis of infected cells. Addition of palmitic acid to latently infected cells treated with a fatty acid synthesis inhibitor protected the cells from death indicating that the products of this pathway are essential. Our metabolomic analysis of KSHV-infected cells provides insight as to how oncogenic viruses can induce metabolic alterations common to cancer cells. Furthermore, this analysis raises the possibility that metabolic pathways may provide novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors.     

Kaposi's Sarcoma-Associated Herpesvirus-Positive Primary Effusion Lymphoma Tumor Formation in NOD/SCID Mice Is Inhibited by Neomycin and Neamine Blocking Angiogenin's Nuclear Translocation

Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi''s sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG''s nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV⁺ PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.     

Do viral chemokines modulate Kaposi's sarcoma?

Kaposi's sarcoma (KS) is an angiogenic tumor of mixed cellularity most commonly found in homosexual men infected with HIV. Both molecular and epidemiologic evidence has linked a newly described herpesvirus to this disease. This virus, Kaposi's sarcoma-associated herpesvirus (KSHV), encodes a number of cellular homologues, including two genes that share remarkable similarity to the human chemokine macrophage inhibitory factor-1α. Recently, studies have begun to shed light on the roles these viral chemokines (vMIP-I and vMIP-II) may play in the complex pathogenesis of KS.^1–3 The vMIP peptides may contribute to the formation of new blood vessels (neovascularization), inhibit infection by certain strains of HIV-1 and modify the cellular immune response. BioEssays 20 :367–370, 1998.© 1998 John Wiley & Sons Inc.     

Suppression of KSHV-induced angiopoietin-2 inhibits angiogenesis,infiltration of inflammatory cells,and tumor growth

Kaposi's sarcoma (KS) is a highly angiogenic and inflammatory neoplasia. The angiogenic and inflammatory cytokine angiopoietin-2 (Ang-2) is strongly expressed in KS due to Kaposi's sarcoma-associated herpesvirus (KSHV) infection. In the present study, we determined how Ang-2 contributes to development of KS by using telomerase-immortalized human umbilical vein endothelial cells (TIVE) as a model, which become malignantly transformed and express increased levels of Ang-2 following KSHV infection. Ang-2 released from TIVE-KSHV cells induces tyrosine phosphorylation of Tie-2 receptor from both human and mouse endothelial cells and promotes angiogenesis in nude mice. Functional inhibition or expressional “knock-down” of Ang-2 in these cells blocks angiogenesis and inhibits tumor growth. Ang-2 suppression also reduces the numbers of infiltrating monocytes/macrophages in tumors. In transwell-based cell migration assays, Ang-2 indeed enhances migration of human monocytes in a dose-dependent manner. These results underscore a pivotal role of KSHV-induced Ang-2 in KS tumor development by promoting both angiogenesis and inflammation. Our data also suggest that selective drug targeting of Ang-2 may be used for treatment of KS.     

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene

Kaposi''s sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G₁-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G₁-S transition, possibly providing an intracellular milieu favorable for its replication.     

Oncogenic Herpesvirus KSHV Hijacks BMP-Smad1-Id Signaling to Promote Tumorigenesis

Kaposi''s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi''s sarcoma (KS), a malignancy commonly found in AIDS patients. Whether KS is a true neoplasm or hyperplasia has been a subject of intensive debate until recently when KSHV is unequivocally shown to efficiently infect, immortalize and transform rat primary mesenchymal precursor cells (MM). Moreover, KSHV-transformed MM cells (KMM) efficiently induce tumors with hallmark features of KS when inoculated into nude mice. Here, we showed Smad1 as a novel binding protein of KSHV latency-associated nuclear antigen (LANA). LANA interacted with and sustained BMP-activated p-Smad1 in the nucleus and enhanced its loading on the Id promoters. As a result, Ids were significantly up-regulated in KMM cells and abundantly expressed in human KS lesions. Strikingly, genetic and chemical inhibition of the BMP-Smad1-Id pathway blocked the oncogenic phenotype of KSHV-transformed cells in vitro and in vivo. These findings illustrate a novel mechanism by which a tumor virus hijacks and converts a developmental pathway into an indispensable oncogenic pathway for tumorigenesis. Importantly, our results demonstrate the efficacy of targeting the BMP-Smad1-Id pathway for inhibiting the growth of KSHV-induced tumors, and therefore identify the BMP pathway as a promising therapeutic target for KS.     

The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) causes Kaposi''s sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that proline-directed modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1''s effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.     

Reactive oxygen species hydrogen peroxide mediates Kaposi's sarcoma-associated herpesvirus reactivation from latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the host following an acute infection. Reactivation from latency contributes to the development of KSHV-induced malignancies, which include Kaposi''s sarcoma (KS), the most common cancer in untreated AIDS patients, primary effusion lymphoma and multicentric Castleman''s disease. However, the physiological cues that trigger KSHV reactivation remain unclear. Here, we show that the reactive oxygen species (ROS) hydrogen peroxide (H₂O₂) induces KSHV reactivation from latency through both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic replication, and KSHV reactivation from latency induced by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines are mediated by H₂O₂. Mechanistically, H₂O₂ induction of KSHV reactivation depends on the activation of mitogen-activated protein kinase ERK1/2, JNK, and p38 pathways. Significantly, H₂O₂ scavengers N-acetyl-L-cysteine (NAC), catalase and glutathione inhibit KSHV lytic replication in culture. In a mouse model of KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and significantly prolongs the lifespan of the mice. These results directly relate KSHV reactivation to oxidative stress and inflammation, which are physiological hallmarks of KS patients. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs could be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies.