Tumorigenesis by human herpesvirus 8 vGPCR is accelerated by human immunodeficiency virus type 1 Tat

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma (KS) herpesvirus, can cause KS but is inefficient. Untreated human immunodeficiency virus type 1 (HIV-1) coinfection is a powerful risk factor. The HHV-8 chemokine receptor, vGPCR (ORF74), activates NF-kappaB and NF-AT, and their levels of activation are synergistically increased by HIV-1 Tat. Transgenic vGPCR mice develop KS-like tumors. A cell line derived from one such tumor expresses vGPCR and forms tumors in nude mice. Here we show that transfection of DNA encoding HIV-1 tat (but not a transactivation-defective mutant) into these tumor cells increases NF-kappaB and NF-AT activation levels and accelerates tumor formation. Tumorigenesis was also accelerated when Tat DNA was transfected into normal cells and the transfected cells were mixed with the tumor cells and injected into a single site. Tumorigenesis was also increased when the two cell types were injected at separate sites, suggesting that tumorigenesis is accelerated by Tat through soluble factors.     

Kaposi's sarcoma-associated herpesvirus induces sustained levels of vascular endothelial growth factors A and C early during in vitro infection of human microvascular dermal endothelial cells: biological implications

Kaposi's sarcoma (KS), a vascular tumor associated with human immunodeficiency virus type 1 infection, is characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth and angiogenic factors, and angiogenesis. KS spindle cells are believed to be of the lymphatic endothelial cell (LEC) type. Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) is etiologically linked to KS, and in vitro KSHV infection of primary human dermal microvascular endothelial cells (HMVEC-d) is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, sustained induction of NF-κB and several cytokines, and growth and angiogenic factors. KSHV induced robust vascular endothelial growth factor A (VEGF-A) and VEGF-C gene expression as early as 30 min postinfection (p.i.) in serum-starved HMVEC-d, which was sustained throughout the observation period of 72 h p.i. Significant amounts of VEGF-A and -C were also detected in the culture supernatant of infected cells. VEGF-A and -C were also induced by UV-inactivated KSHV and envelope glycoprotein gpK8.1A, thus suggesting a role for virus entry stages in the early induction of VEGF and requirement of KSHV viral gene expression for sustained induction. Exogenous addition of VEGF-A and -C increased KSHV DNA entry into target cells and moderately increased latent ORF73 and lytic ORF50 promoter activation and gene expression. KSHV infection also induced the expression of lymphatic markers Prox-1 and podoplanin as early as 8 h p.i., and a paracrine effect was seen in the neighboring uninfected cells. Similar observations were also made in the pure blood endothelial cell (BEC)-TIME cells, thus suggesting that commitment to the LEC phenotype is induced early during KSHV infection of blood endothelial cells. Treatment with VEGF-C alone also induced Prox-1 expression in the BEC-TIME cells. Collectively, these studies show that the in vitro microenvironments of KSHV-infected endothelial cells are enriched, with VEGF-A and -C molecules playing key roles in KSHV biology, such as increased infection and gene expression, as well as in angiogenesis and lymphangiogenesis, thus recapitulating the microenvironment of early KS lesions.     

Emerging targets and novel strategies in the treatment of AIDS-related Kaposi's sarcoma: bidirectional translational science

Through the mentorship process, Dr. Arthur Pardee emphasized the critical importance of bidirectional translational research-not only advancing drug development from bench to bedside, but also bringing back precious clinical material to the laboratory to assess the biologic effects of therapeutic agents on their targets. This mini-review focuses on the signal transduction pathways of Kaposi's sarcoma (KS) and on how the knowledge of such pathways has led to the rational development of molecularly targeted pathogenesis-driven therapies. Acquired immune deficiency syndrome (AIDS) related-KS results from co-infection with human immunodeficiency virus and KS herpesvirus/human herpesvirus-8 (KSHV/HHV8), which leads to the development of an angiogenic-inflammatory state that is critical in the pathogenesis of KS. KS is driven by KSHV/HHV8-specific pathways, which include viral G protein-coupled receptor (vGPCR), viral interleukin-6 (vIL-6), and viral chemokine homologues. In addition, cellular growth/angiogenic pathways, such as vascular endothelial growth factor (VEGF), insulin-like growth factor, platelet-derived growth factor (PDGF), angiopoietin and matrix metalloproteinases (MMPs) are "pirated" by KSHV/HHV8. As a very tangible example of how translational research has led to a marked improvement in patient outcome, the signal transduction inhibitor imatinib (a tyrosine kinase inhibitor of c-kit and PDGF) was administered to patients with KS whose tumors were serially biopsied. Not only did the patients' tumors regress, but also the regression was correlated with the inhibition of PDGF receptor (PDGFR) in the biopsy samples. Recent and future clinical trials of molecularly targeted therapy for the treatment of KS are a prelude to a shift in the paradigm of how KS is managed.     

Inhibition of heme oxygenase-1 interferes with the transforming activity of the Kaposi sarcoma herpesvirus-encoded G protein-coupled receptor

Heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in the heme catabolism, is expressed in AIDS-Kaposi sarcoma (KS) lesions. Its expression is up-regulated by the Kaposi sarcoma-associated herpesvirus (KSHV) in endothelial cells, but the mechanisms underlying KSHV-induced HO-1 expression are still unknown. In this study we investigated whether the oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR), one of the key KSHV genes involved in KS development, activated HO-1 expression. Here we show that vGPCR induces HO-1 mRNA and protein levels in fibroblasts and endothelial cells. Moreover, targeted knock-down gene expression of HO-1 by small hairpin RNA and chemical inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPP), impaired vGPCR-induced survival, proliferation, transformation, and vascular endothelial growth factor (VEGF)-A expression. vGPCR-expressing cells implanted in the dorsal flank of nude mice developed tumors with elevated HO-1 expression and activity. Chronic administration of SnPP to the implanted mice, under conditions that effectively blocked HO-1 activity and VEGF-A expression in the transplanted cells, strikingly reduced tumor growth, without apparent side effects. On the contrary, administration of the HO-1 inducer cobalt protoporphyrin (CoPP) further enhanced vGPCR-induced tumor growth. These data postulate HO-1 as an important mediator of vGPCR-induced tumor growth and suggest that inhibition of intratumoral HO-1 activity by SnPP may be a potential therapeutic strategy.     

Kaposi's sarcoma-associated herpesvirus K7 induces viral G protein-coupled receptor degradation and reduces its tumorigenicity

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome encodes a G protein-coupled receptor (vGPCR). vGPCR is a ligand-independent, constitutively active signaling molecule that promotes cell growth and proliferation; however, it is not clear how vGPCR is negatively regulated. We report here that the KSHV K7 small membrane protein interacts with vGPCR and induces its degradation, thereby dampening vGPCR signaling. K7 interaction with vGPCR is readily detected in transiently transfected human cells. Mutational analyses reveal that the K7 transmembrane domain is necessary and sufficient for this interaction. Biochemical and confocal microscopy studies indicate that K7 retains vGPCR in the endoplasmic reticulum (ER) and induces vGPCR proteasomeal degradation. Indeed, the knockdown of K7 by shRNA-mediated silencing increases vGPCR protein expression in BCBL-1 cells that are induced for KSHV lytic replication. Interestingly, K7 expression significantly reduces vGPCR tumorigenicity in nude mice. These findings define a viral factor that negatively regulates vGPCR protein expression and reveal a post-translational event that modulates GPCR-dependent transformation and tumorigenicity.     

The Galpha12/13 family of heterotrimeric G proteins and the small GTPase RhoA link the Kaposi sarcoma-associated herpes virus G protein-coupled receptor to heme oxygenase-1 expression and tumorigenesis

Heme oxygenase-1 (HO-1), an inducible enzyme that metabolizes the heme group, is highly expressed in human Kaposi sarcoma lesions. Its expression is up-regulated by the G protein-coupled receptor from the Kaposi sarcoma-associated herpes virus (vGPCR). Although recent evidence shows that HO-1 contributes to vGPCR-induced tumorigenesis and vascular endothelial growth factor (VEGF) expression, the molecular steps that link vGPCR to HO-1 remain unknown. Here we show that vGPCR induces HO-1 expression and transformation through the Galpha(12/13) family of heterotrimeric G proteins and the small GTPase RhoA. Targeted small hairpin RNA knockdown expression of Galpha(12), Galpha(13), or RhoA and inhibition of RhoA activity impair vGPCR-induced transformation and ho-1 promoter activity. Knockdown expression of RhoA also reduces vGPCR-induced VEFG-A secretion and blocks tumor growth in a murine allograft tumor model. NIH-3T3 cells expressing constitutively activated Galpha(13) or RhoA implanted in nude mice develop tumors displaying spindle-shaped cells that express HO-1 and VEGF-A, similarly to vGPCR-derived tumors. RhoAQL-induced tumor growth is reduced 80% by small hairpin RNA-mediated knockdown expression of HO-1 in the implanted cells. Likewise, inhibition of HO-1 activity by chronic administration of the HO-1 inhibitor tin protoporphyrin IX to mice reduces RhoAQL-induced tumor growth by 70%. Our study shows that vGPCR induces HO-1 expression through the Galpha(12/13)/RhoA axes and shows for the first time a potential role for HO-1 as a therapeutic target in tumors where RhoA has oncogenic activity.     

Amplification of the angiogenic signal through the activation of the TSC/mTOR/HIF axis by the KSHV vGPCR in Kaposi's sarcoma

Background

Kaposi''s sarcoma (KS) is a vascular neoplasm characterized by the dysregulated expression of angiogenic and inflammatory cytokines. The driving force of the KS lesion, the KSHV-infected spindle cell, secretes elevated levels of vascular endothelial growth factor (VEGF), essential for KS development. However, the origin of VEGF in this tumor remains unclear.

Methodology/Principal Findings

Here we report that the KSHV G protein-coupled receptor (vGPCR) upregulates VEGF in KS through an intricate paracrine mechanism. The cytokines secreted by the few vGPCR-expressing tumor cells activate in neighboring cells multiple pathways (including AKT, ERK, p38 and IKKβ) that, in turn, converge on TSC1/2, promoting mTOR activation, HIF upregulation, and VEGF secretion. Conditioned media from vGPCR-expressing cells lead to an mTOR-dependent increase in HIF-1α and HIF-2α protein levels and VEGF upregulation. In a mouse allograft model for KS, specific inhibition of the paracrine activation of mTOR in non-vGPCR-expressing cells was sufficient to inhibit HIF upregulation in these cells, and abolished the ability of the vGPCR-expressing cells to promote tumor formation in vivo. Similarly, pharmacologic inhibition of HIF in this model blocked VEGF secretion and also lead to tumor regression.

Conclusions/Significance

Our findings provide a compelling explanation for how the few tumor cells expressing vGPCR can contribute to the dramatic amplification of VEGF secretion in KS, and further provide a molecular mechanism for how cytokine dysregulation in KS fuels angiogenesis and tumor development. These data further suggest that activation of HIF by vGPCR may be a vulnerable target for the treatment of patients with KS.     

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor contains an immunoreceptor tyrosine-based inhibitory motif that activates Shp2

The Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) is a constitutively active, highly angiogenic homologue of the interleukin-8 (IL-8) receptors that signals in part via the cytoplasmic protein tyrosine phosphatase Shp2. We show that vGPCR contains a bona fide immunoreceptor tyrosine-based inhibitory motif (ITIM) that binds and constitutively activates Shp2.     

Cellular tropism and viral interleukin-6 expression distinguish human herpesvirus 8 involvement in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease

Human herpesvirus 8 (HHV-8) infection has been implicated in the etiology of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), three diseases that frequently develop in immunocompromised, human immunodeficiency virus-positive individuals. One hypothesis that would account for different pathological manifestations of infection by the same virus is that viral genes are differentially expressed in heterogeneous cell types. To test this hypothesis, we analyzed the localization and levels of expression of two viral genes expressed in latent and lytic infections and the viral homologue of interleukin-6 (vIL-6). We show that PEL parallels KS in the pattern of latent and lytic cycle viral gene expression but that the predominant infected cell type is a B cell. We also show that MCD differs from KS not only in the infected cell type (B-cell and T-cell lineage) but also in the pattern of viral gene expression. Only a few cells in the lesion are infected and all of these cells express lytic-cycle genes. Of possibly greater significance is the fact that in a comparison of KS, PEL, and MCD, we found dramatic differences in the levels of expression of vIL-6. Interleukin-6 is a B-cell growth and differentiation factor whose altered expression has been linked to plasma cell abnormalities, as well as myeloid and lymphoid malignancies. Our findings support the hypothesis that HHV-8 plays an important role in the pathogenesis of PEL and MCD, in which vIL-6 acts as an autocrine or paracrine factor in the lymphoproliferative processes common to both.     

The Biology of Kaposi＇s Sarcoma-Associated Herpesvirus and the Infection of Human Immunodeficiency Virus

Kaposi sarcoma-associated herpesvirus (KSHV),also known as human herpesvirus 8 (HHV-8),is discovered in 1994 from Kaposi's sarcoma (KS) lesion of an acquired immunodeficiency syndrome (AIDS)patient.In addition to its association with KS,KSHV has also been implicated as the causative agent of two other AIDS-associated malignancies:primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD).KSHV is a complex DNA virus that not only has the ability to promote cellular growth and survival for tumor development,but also can provoke deregulated angiogenesis,inflammation,and modulate the patient's immune system in favor of tumor growth.As KSHV is a necessary but not sufficient etiological factor for KS,human immunodeficiency virus (HIV) is a very important cofactor.Here we review the basic information about the biology of KSHV,development of pathogenesis and interaction between KSHV and HIV.     

Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.     

KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFκB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.