Kaposi's sarcoma (KS) is the most frequent AIDS-associated malignancy, etiologically linked to the infection with the human herpesvirus 8 (HHV-8/KSHV). This member of the γ-herpesviridae family encodes 81 open reading frames, several bearing oncogenic potential. A constitutively active virally encoded G protein–coupled receptor (vGPCR) readily induces KS-like lesions when expressed in endothelial cells in vivo, and unmasks the oncogenic potential of other HHV-8 genes in a paracrine fashion. How vGPCR causes endothelial cell transformation is still not fully understood. Using full-genome microarray analysis we show here that the expression of nuclear factor-κB (NF-κB)-regulated genes is a prominent feature triggered by vGPCR in cells expressing this viral oncogene and in cells exposed to vGPCR-induced secretions, thus mimicking its paracrine effect. Indeed, vGPCR activates the NF-κB pathway potently, and NF-κB activation is a hallmark of both human and experimental KS. Of interest, whereas constitutive NF-κB signaling is not sufficient to promote endothelial cells transformation, NF-κB function is strictly required for vGPCR-induced direct and paracrine neoplasia. Taken together, these results strongly support the role of NF-κB regulated genes in KS pathogenesis, thus providing the rationale for the development of novel mechanism-based therapies for this angioproliferative disease.
Kaposi's sarcoma (KS) is an angioproliferative tumor characterized by an almost invariable presence of human herpesvirus 8 (HHV-8, also termed KSHV) infection. KS was considered a rare disease affecting elderly Mediterranean and young African men (classic KS), but its incidence changed radically during the last few decades due to the widespread appearance of KS cases in the sub-Saharan region in Africa (endemic KS), in transplanted patients undergoing immunosuppressive therapy (iatrogenic KS) and due to its tragic association with AIDS (epidemic KS) (Morris, 2003). Although the incidence of KS has been greatly reduced since the introduction of the triple antiretroviral therapy (HAART) (Bower et al., 2006), KS remains the most prevalent cancer in children in Africa, and it is still more prevalent among minority groups and in developing countries. In addition, there is a risk of reemergence of KS due to the increasing prevalence of asymptomatic KSHV-8-infected individuals, particularly among HIV-infected patients (Rezza et al., 1999).
KSHV is a γ-herpesvirus that is the etiologic agent of KS and two B-cell type lymphomas known as pleural effusion lymphoma and Castleman's diseases (reviewed in Cesarman and Mesri, 2007). Several KSHV genes exhibiting transforming potential have been identified, and their contribution to KS pathogenesis is currently under intense investigation. Among them, the open reading frame 74 (ORF-74), which encodes a G protein–coupled receptor (GPCR) that resembles the chemokine receptor CXCR2 (Arvanitakis et al., 1997), acts as a potent oncogene both in vitro and in vivo (Bais et al., 1998; Yang et al., 2000; Montaner et al., 2003). Virally encoded G protein–coupled receptor (vGPCR) expression is also required for the sarcomagenic activity of endothelial-derived cells expressing the entire KSHV genome (Mutlu et al., 2007). Persistent expression and activity of vGPCR is required for tumor maintenance (Montaner et al., 2006). Thus, vGPCR and its regulated signaling pathways may represent suitable candidates for KS-treatment.
Surprisingly, few cells express vGPCR in human as well as in experimental KS (Yang et al., 2000; Montaner et al., 2003). In this regard, accumulating evidence suggest that vGPCR can promote the paracrine growth of endothelial-derived cells, and particularly those expressing latent KSHV genes (Montaner et al., 2003). The molecular mechanism underlying this vGPCR-initiated paracrine transformation is still poorly understood. Here, we provide evidence that vGPCR stimulates a nuclear factor-κB (NF-κB)-related gene expression signature in endothelial cells, either when expressing vGPCR or when exposed to vGPCR-induced secretions. Furthermore, we observed that these two processes are highly interrelated, as vGPCR promotes the NF-κB-dependent expression of cytokines and chemokines that can activate NF-κB in endothelial cells not expressing this KSHV oncogene. In turn, NF-κB activation is required for vGPCR-induced transformation. The expression of cytokines by endothelial cells exposed to vGPCR-induced secretions may contribute to propagate the vGPCR-initiated NF-κB paracrine/autocrine signaling network to the surrounding and even distant endothelial cells, thereby contributing to their unrestricted growth.
An NF-κB gene expression signature as a hallmark of vGPCR direct and paracrine signaling
We used a murine genome-wide long oligonucleotide gene array platform to explore the pattern of gene expression caused by the expression of vGPCR in endothelial cells and by the exposure of endothelial cells to supernatants from vGPCR-expressing cells, thus mimicking their paracrine effect. Analysis of multiple independent RNA isolates from murine endothelial cells (SVEC), SVECs stably expressing vGPCR (SVEC-vGPCR) and SVECs exposed to supernatants from SVEC-vGPCR (SVEC-CM) revealed a consistent pattern of gene expression (Figure 1). Genes whose expression was induced or repressed by at least twofold by vGPCR with a very stringent false discovery rate (FDR) ⩽1% were identified by significance analysis of microarrays (SAM) (Figure 1a and Supplementary Tables 1A and B). Surprisingly, when gene cluster analysis was performed, we observed that SVEC-CM grouped with SVEC-vGPCR rather than with control SVEC cells (Figure 1b). Of those genes significantly different between SVECs and SVEC-vGPCR, 63% were similarly found to be up- or downregulated in SVEC-CM when compared to SVECs.
Because SVEC-CM induced a gene expression pattern similar to that of vGPCR-transformed cells, we explored whether vGPCR expression and the exposure to vGPCR-induced secretions may elicit a shared pro-proliferative mechanism. As an approach, we performed gene set enrichment analysis on the list of SVEC-vGPCR and SVEC-CM-shared regulated genes (Table 1). Surprisingly, NF-κB signaling gene sets appeared as two independently generated lists, suggesting that dysregulation of NF-κB signaling pathway represents a prominent feature of both vGPCR expression and its paracrine effects.
vGPCR activates NF-κB potently in endothelial cells
Based on these results and prior reports documenting NF-κB activation by vGPCR in model cell lines (Montaner et al., 2001; Schwarz and Murphy, 2001), we asked whether NF-κB contributes to vGPCR-induced transforming in endothelial cells. vGPCR stimulated an NF-κB luciferase reporter in SVECs and provoked the accumulation of active NF-κB p65 subunit (Figures 2a and b), as judged by the increase in p65 binding activity and the accumulation of NF-κB in the nucleus, using tumor necrosis factor α (TNFα) as a control. Interestingly, while vGPCR-expressing cells exhibited a markedly nuclear localization of p65, cells adjacent to those expressing vGPCR also exhibited nuclear localized NF-κB, in line with the ability of vGPCR to stimulate this transcription factor in neighboring cells in a paracrine fashion. The constitutive activation of NF-κB caused by vGPCR likely results from the persistent activation of IKKα/β (Figure 2e), two protein kinases acting upstream of NF-κB that promote the phosphorylation-dependent degradation of IκB (Karin, 2006). Indeed, increased levels of phospho-IκB (pIκB) and both pIKKs were observed in SVEC-vGPCR cells, which could not be further increased by treatment with TNFα, suggesting that vGPCR can converge on NF-κB with a distinct pathway activated by TNFα.
p65 overexpression and activity is a common feature of both human and experimental KS
We next explored the status of activation of NF-κB in human and experimental KS samples. Human KS lesions present spindle-shaped cells surrounded by abundant blood vessels and erythrocyte-replete vascular slits, all characteristic features defining KS (Figure 3). Expression of the viral marker latent nuclear antigen 1 (LANA1) (right, upper panel) revealed cells latently infected by HHV-8 showing a generalized pattern of expression, mainly in spindle-shaped cells, but not in every cell within the tumor lesions. Experimental KS resembled human nodular KS (Montaner et al., 2003), but in spite of these tumors arising from vGPCR-expressing cells, the pattern of expression of the tagged vGPCR was restricted to a limited number of dispersed cells within the tumor. This observation mimics the clinical situation in which vGPCR expression in KS lesions involves 1–10% of the tumor cells (Yang et al., 2000). However, the nuclear localization of p65 RelA, as a marker of NF-κB activation, showed a broad cellular distribution in both human and experimental KS.
NF-κB activation is not sufficient but strictly required to transform endothelial cells
As NF-κB activation is a shared feature in clinical and experimental KS, we overexpressed the p65 NF-κB subunit, RelA, to explore whether NF-κB is sufficient to initiate the direct or paracrine transformation of endothelial cells. The activation of NF-κB was confirmed by monitoring the expression of proteins whose expression is regulated by NF-κB, including COX-2, cyclin D1 and PKCδ (Figure 4a). However, p65 RelA-overexpressing cells failed to cause the tumoral growth of endothelial cells even after prolonged observation (>6 months) (Figure 4b). Although we cannot rule the possibility that higher levels of activation of NF-κB may provoke cellular transformation, constitutive activation of NF-κB at levels similar to or greater than those provoked by vGPCR appears not sufficient to initiate sarcomagenesis.
To address whether NF-κB activation contributes to tumor induction by vGPCR, we expressed green fluorescent protein (GFP) fussed to a nonphosphorylatable version of its physiological inhibitor, IκBα, (GFP-IκBsr). Co-transfection of vGPCR with GFP-IκBsr resulted in a dose-dependent inhibition of NF-κB activation and its nuclear translocation in vGPCR-expressing cells (Supplementary Figures 1A–D). SVEC and SVEC-vGPCR cells infected with lentiviruses expressing GFP or GFP-IκBsr were then tested for their ability to form tumors in nude mice. While SVEC-vGPCR cells expressing GFP produced tumors in a time frame similar to that observed in uninfected cells (Figure 4b), SVEC controls and SVEC-vGPCR cells expressing GFP-IκBsr failed to induce tumors. These findings suggest that NF-κB signaling, although not likely sufficient, is required for vGPCR-induced tumorigenesis in endothelial cells.
Identification of NF-κB-dependent genes stimulated by vGPCR in endothelial cells
To identify NF-κB-regulated genes expressed by SVEC-vGPCR cells, we performed Pavlidis Template Matching analysis, which revealed genes up- or downregulated in SVEC-vGPCR cells with respect to SVEC cells whose expression was affected in an opposite fashion upon expression of the NF-κB repressor or GFP as a control. This analysis led to the identification of numerous putatively NF-κB-regulated genes with a P<0.01 (not shown), which were then compared against the list of SVEC-vGPCR and SVEC-CM-shared regulated genes. This resulted in the identification of a subset of 60 NF-κB-controlled genes regulated in endothelial cells by both vGPCR- and vGPCR-conditioned media (Tables 2a and 2b). Among them, we identified numerous genes involved in cell proliferation and survival (that is, c-myc, Bcl2) as well as genes encoding cytokines and chemokines (that is, VEGF-A, IL-10, CXCL-1, CXCL-12, CCL-2, CCL-7 and CXCL-9) that are likely involved in autocrine and paracrine transformation.
vGPCR requires NF-κB for its autocrine and paracrine secretion
As vGPCR promotes the expression of numerous cytokines in an NF-κB-dependent manner, and NF-κB activation is the cause and effect of cytokine secretion and signaling, we next analysed whether NF-κB inhibition in vGPCR-expressing cells prevented their ability to stimulate NF-κB in other cells in a paracrine fashion. Medium from vGPCR-expressing cells potently activated NF-κB in SVECs, but that was abolished when vGPCR was coexpressed with GFP-IκBsr (Figure 5). These observations supported the emerging concept that the activation of NF-κB by vGPCR is required to induce the secretion of molecules that, in turn, stimulate NF-κB signaling in other cells in a paracrine fashion.
Tumor-causing viruses have evolved to subvert normal cell regulating circuitries to promote viral replication and dissemination, and thus represent valuable tools for disclosing key intracellular regulation mechanisms controlling normal and aberrant cell proliferation. This is nicely exemplified by the early discovery that v-Rel, an oncoprotein encoded by the turkey retrovirus REV-T, is a homologue of the mammalian p65 NF-κB DNA binding subunit, which provided the first glimpse of the central role of NF-κB in human cancer development (reviewed in Karin, 2006). Other viral oncoproteins, such as the Epstein–Barr virus (EBV) latent infection membrane protein 1, the human T-cell leukemia virus protein Tax and the KSHV-encoded v-FLIP also activate NF-κB (reviewed by Hiscott et al., 2006). The ability of v-FLIP to stimulate NF-κB is strictly required for tumor cell survival in KSHV-associated lymphomas (Guasparri et al., 2004). However, v-FLIP fails to transform endothelial cells or to induce tumor formation in animal models (Montaner et al., 2003), in line with our observation that NF-κB activation is not alone sufficient to promote the tumoral growth of endothelial cells. Thus, whereas hematopoietic cells may be particularly susceptible to NF-κB-induced transformation, prior studies and our current findings support the emerging notion that vGPCR requires at least two distinct mechanisms to induce tumorigenesis in endothelial cells; one initiated by the direct activation of pro-proliferative pathways, including AKT-mTOR, small GTPases of the Rho family and MAPK signaling pathways (Sodhi et al., 2004), and another involving an autocrine and paracrine mechanism elicited through the NF-κB-dependent release of pro-survival and growth-promoting cytokines.
These direct and autocrine/paracrine mechanisms may be highly interrelated. Indeed, paracrine stimulation may not be restricted to NF-κB, as the release of cytokines and chemokines such as VEGF and CXCL8 (IL-8) can cause the activation of Akt and MAPKs in vGPCR-expressing cells as well as in the surrounding cells (Sodhi et al., 2004). On the other hand, our gene array analysis suggests that NF-κB also plays a role in the direct transformation of endothelial cells. For example, vGPCR stimulates the expression of c-myc, which plays a central role in cell proliferation and tumorigenesis (Pelengaris et al., 2002). Similarly, the NF-κB-dependent upregulation of the potent pro-survival protein Bcl2 (Danial and Korsmeyer, 2004), may explain why vGPCR may not require the expression of the KSHV viral homologue of Bcl2, vBcl2, to promote the transformation of endothelial cells (Montaner et al., 2003). As c-myc and Bcl2 are expressed by both vGPCR-transformed cells and cells exposed to vGPCR-induced secretions, these genes may also participate in the paracrine transformation caused by vGPCR.
Because endothelial cells expressing vGPCR cells are exposed to their own secretions, it is difficult to separate strictly direct- from autocrine-gene regulation. In this regard, the ability to use bioinformatic tools to identify genes expressed in SVEC-vGPCR cells that are not stimulated by their conditioned media revealed the existence of a number of genes whose expression was induced by vGPCR independently of paracrine signaling. Among them, Semaphorin 4B was the most potently induced gene. A highly related semaphorin, Semaphorin 4D, is an axon-guiding molecule that also acts as a potent proangiogenic factor by stimulating its receptor, Plexin B1, on endothelial cells (Basile et al., 2004). Thus, the likely possibility that Semaphorin 4B and other vGPCR-induced gene products, including the VEGF receptor Flt-1, may contribute to the proangiogenic and transforming effects of vGPCR warrant further investigation.
The most prominent NF-κB-regulated genes stimulated by vGPCR include growth factors, chemokines and cytokines, such as VEGF-A, CCL7, IL-10, CXCL12 (SDF-1), CXCL9, CXCL1 (KC) and CCL2. Several of these molecules have been found to be highly expressed in KS and other angioproliferative and inflammatory diseases (Ensoli et al., 1989; Balkwill, 2004). In particular for VEGF-A, which is one of the most potent angiogenic factors, it is highly induced by vGPCR and plays a fundamental role in KS development and progression, likely by promoting angioproliferation, permeability and endothelial cell recruitment (Bais et al., 1998). Other potent angiogenic and chemoattractant factors induced by vGPCR include CXCL12/SDF-1 and the proinflammatory cytokine CXCL1, which are likely to promote the recruitment, survival and proliferation of mature and progenitor endothelial cells in a paracrine fashion (Balkwill, 2004; Dorsam and Gutkind, 2007). The increased expression of proinflammatory cytokines, such as CCL7 and CCL2, may also help create a permissive environment for KS development and progression. For example, the broad spectrum cytokine CCL7/MCP-3 binds and activates a large number of CC and CXC family receptor members, leading to the recruitment of monocytes, which may contribute to spread the KSHV-infected cells by helping degrade extracellular matrix proteins (Balkwill, 2004). Similarly, CCL2 has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis and atherosclerosis, and is largely secreted by cultured KS-derived cells (Sciacca et al., 1994). Other cytokines may play a role in immune system evasion, such as IL-10 and cytokine synthesis inhibitory factor, which inhibits the synthesis of interferon-γ, IL-2, IL-3, TNF-α and granulocyte-macrophage colony-stimulating factor by macrophages and Type 2 T-helper cells thereby preventing the organisms from mounting an effective immune response (Hamilton et al., 2002). Whereas the direct role of IL-10 in KS development is still unclear, its importance for viral-induced pathogenesis is underscored by the high level of secretion from KSHV-infected PEL lymphocytes (Asou et al., 1998) and the presence of a viral homolog, vIL-10, in the EBV genome (Kanegane et al., 1997).
In summary, the microarray analysis of vGPCR-expressing endothelial cells and cells stimulated with vGPCR-induced secretions revealed that a shared NF-κB-related gene expression signature may underlie the direct and paracrine mechanisms deployed by this potent viral oncoprotein. Interestingly, the activation of NF-κB by vGPCR can promote the release of a number of chemokines, including CXCL1 that can activate NF-κB in vGPCR-expressing endothelial cells by acting on vGPCR itself as well as on endogenously expressed CXCR2 and CXCR1 receptors, among others (Heidemann et al., 2003). This seemingly feed-forward autocrine mechanism may amplify the signal initiated by vGPCR, which may explain its high oncogenic potential. The resulting secretion of numerous proangiogenic cytokines may in turn act in a paracrine fashion, facilitating the recruitment and proliferation of local endothelial cells, their circulating progenitors and/or latently KSHV-infected cells, thereby supporting their tumoral growth. In this regard, our emerging findings also indicate that vGPCR secretions can stimulate NF-κB in these tumoral cells, thus propagating the vGPCR-initiated NF-κB-activation throughout the tumor tissue through a cytokine-driven signaling circuitry. Ultimately, this dependence on NF-κB activation for vGPCR-induced neoplasia and the recent development of clinically relevant inhibitors of the NF-κB pathway can now provide new opportunity for the molecular-targeted approaches for the treatment of KS and other KSHV-related malignancies.
Materials and methods
See Supplementary Materials for an extended description of the experimental approaches.
Cell culture, cloning and lentiviral expression vector
Lentiviruses expressing a dominant interfering mutant of IκB, IκB S32A/S36A was generated by the Gateway LR reaction between pWPI GW, a modified pWPI lentivector see and an IκBsr entry clone (see Supplementary Methods). An expression vector for an EE-tagged form of the p65 subunit of NF-κB, pcEFL2 p65-EE was kindly provided by James Lyons (University of Sydney, Australia). Nontumorigenic SV40-immortalized murine endothelial cells (SVEC4-10) were transfected using ExGen 500 (Fermentas, Hanover, MD, USA) and selected in 750 μg ml−1 of G418 (Calbiochem, La Jolla, CA, USA).
Immunohistochemistry, immunofluorescence and immunoblot assays
AU5 antibody was purchased from Covance (Vienna, VA, USA), ORF73 (LANA1) from Advanced Biotechnologies Inc. (Columbia, MD, USA), and p65 from Neomarkers (Fremont, CA, USA). Antibodies p65, cyclin D1 and anti-Tubulin were purchased from Santa Cruz (Santa Cruz, CA, USA), p-IκB, p-IKK (α/β) were from Cell Signaling Technology, Inc. (Danvers, MA, USA), COX2 was from Stressgen (Victoria, BC, Canada) and PKCδ was from Zymed Laboratories Inc. (San Francisco, CA, USA).
Animal studies were carried out according to NIH-approved protocols. SVEC cells lines were used to induce endothelial tumor xenografts in athymic mice as described previously (Montaner et al., 2003).
Microarrays and statistical analysis
Total mRNA extraction, cDNA synthesis, labeling, hybridization and analysis are described in Supplementary Material and methods. Analysis of variance followed by the Tukey t-test was used to analyse the differences between experimental groups. Data analysis was done using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). P<0.05 was considered statistically significant.
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This research was supported by a National Institutes of Health Intramural AIDS Targeted Antiviral Program grant and the National Institute of Dental and Craniofacial Research.
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Journal of Virology (2019)
PLOS Pathogens (2018)
Journal of Medical Virology (2018)
PLOS Pathogens (2018)
Frontiers in Microbiology (2017)