The role of Kaposi Sarcoma-associated Herpesvirus in the pathogenesis of

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Kaposi Sarcoma Silvia Gramolelli

Thomas F. Schulz

Affiliation: Institute of Virology, Hannover Medical School, Carl Neuberg Strasse 1, 30625 Hannover, Germany and German Centre for Infection Research, Hannover-Braunschweig site. Corresponding author: Thomas F Schulz, Institute of Virology, Hannover Medical School, Carl Neuberg Strasse 1, 30625 Hannover, Germany, E-mail address: [email protected] Conflict of interest: no conflict of interest ABSTRACT Kaposi sarcoma is an unusual vascular tumour caused by an oncogenic γ-herpesvirus, Kaposi Sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV 8). Kaposi Sarcoma (KS) lesions are characterized by an abundant inflammatory infiltrate, the presence of KSHV-infected endothelial cells that show signs of aberrant differentiation, as well as faulty angiogenesis/ vascularisation. Here we discuss the molecular mechanisms that lead to the development of these histological features of KS with an emphasis on the viral proteins that are responsible for their development.

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Keywords: Kaposi sarcoma, KSHV, atypical differentiation, inflammation, aberrant

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angiogenesis, oncogenesis, invasiveness. From Kaposi Sarcoma to Kaposi sarcoma herpesvirus: epidemiological observations pave the way to virus discovery In 1872 the Hungarian physician Moritz Kaposi described 5 cases of a lethal and previously unknown “idiopathic multiple pigmented sarcoma of the skin” [1]. Named ‘Kaposi sarcoma’ (KS), it was considered a rare tumour affecting elderly men. In epidemiological reports of the 1950s less than 500 KS cases were counted in Europe and America [2]. However, in Equatorial Africa, this disease represented 9% of all cancers observed in indigenous populations [3]. The peculiar geographical distribution of KS was compatible with an infectious aetiology involving a pathogen with an uneven geographic distribution. This concept was also supported by the work of Giraldo and colleagues [4], who observed, by electron microscopy, herpes-like virus particles in cell cultures from KS biopsies. Several infectious agents (CMV, HBV, HHV 6, HTLV, and HIV) were then suspected to be the causal agent of KS, but none of them showed a significant association with the disease [5-8]. Additional strands of evidence in favour of an aetiological link between a viral agent and KS emerged during the AIDS epidemic. The number of KS cases grew exponentially in gay and bisexual AIDS patients, who have a much higher risk of developing KS than patients with haemophilia who contracted HIV through contaminated factor VIII preparations [9, 10]. A careful epidemiological analysis of the risk factor for AIDS KS yielded very strong evidence for a sexually transmissible agent, distinct from HIV, as the cause of KS [11, 12]. Motivated by these epidemiological observations, Chang and colleagues identified, in 1994, Herpesvirus-like DNA sequences in KS tissue [13]. This observation led to the identification This article is protected by copyright. All rights reserved.

of Kaposi sarcoma-associated herpesvirus (KSHV), the eighth human member of the

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herpesviridae family, which is therefore also known as human herpesvirus 8 (HHV 8). Subsequently viral DNA sequences were identified in virtually all KS patients, but not in healthy donors and many prospective cohort studies demonstrated a strong association between the detection of KSHV DNA by PCR, or of antibodies to KSHV, and the subsequent development of KS [14-16]. Successively, KSHV was aetiologically linked to two B-cell malignancies, primary effusion lymphoma (PEL) [17] and multicentric Castleman disease (MCD) [18]. These investigations, together with accumulating laboratory data pointing to the oncogenic properties of KSHV, led to the classification of KSHV as a class I carcinogen by the International Agency for Research on Cancer (IARC) [19]. Kaposi Sarcoma is an unusual tumour, which is driven by an oncogenic virus that affects the differentiation of the latently infected endothelial cell, but is not apparently the result of a malignant transformation. In this review, we will give an overview on KS pathogenesis focusing on the four key features that characterize this process: aberrant differentiation, inflammation, proliferation and angiogenesis. KS: four epidemiological variants with similar histopathology features reflecting the border between hyperplasia and cancer It is possible to identify 4 different epidemiological variants of Kaposi sarcoma. ‘Classical KS’, the tumour described by M. Kaposi, affects mainly elderly males of Mediterranean or eastern European origin [1]. Endemic KS is clinically more aggressive and occurs mainly in men in Eastern and Central Africa; a lymphoadenopathic sub-variant is found in children [3, 20]. The iatrogenic variant occurs as a consequence of the immunosuppressive therapy following organ transplantation and normally regresses upon immune reconstitution [21]. The fourth and most aggressive clinical variant is found in AIDS patients and frequently resolves

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upon treatment of HIV by antiretroviral combination therapy (HAART) and the resulting

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immune reconstitution [6, 9, 10, 22]. Although these four clinical forms differ with regard to geographic distribution, aggressiveness and localization, all display common clinical features and are histologically indistinguishable [23]. The cellular composition of a KS lesion is highly heterogeneous and, in addition to the main proliferating element, the so-called spindle cells (SCs), the inflammatory infiltrate is abundant and surrounded by abnormal vessels. At early stages, KS lesions appear as flat red patches, in which the main component is the inflammatory infiltrate and few SCs are present [24]. Patches develop into plaques and SCs become predominant. During the advanced stage SC growth leads to the formation of macroscopically visible nodules [25]. SCs are either arranged around vascular spaces containing red blood cells or can form fascicles streaming in different directions (Figure 1) [24]. Atypical differentiation of endothelial cells induced by KSHV The origins of SCs have long been debated as these peculiar cells express markers of both lymphatic and blood vessel endothelium (podoplanin, VEGFR3, VEGF C and D, CXCR4, DLL4, VEGFR1, CXCL12, CD34) as well as mesenchymal cells (vimentin, PDGFRα) (Figure 2) (reviewed in [26]). There is now very strong evidence that KSHV infection itself induces these apparently contradictory histological features. Once infected by KSHV, endothelial cells lose the typical cobblestone morphology and assume the characteristic spindle phenotype [25]. The growth of SCs depends on external stimuli, particularly during the early stages [27, 28]. In vitro, SCs do not survive without growth factors, lose the viral genome at early passage and in nude mice do not form tumours [29-31]. SCs are diploid and do not show any genomic abnormality, in contrast to most cancer cells [23, 32]. In addition, latent infection of blood endothelial cells (BECs) skews their differentiation towards This article is protected by copyright. All rights reserved.

lymphatic ECs (LECs) and vice versa [33], thus accounting for the expression of both

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lymphatic and blood vascular endothelial markers on KS spindle cells [34]. This is, at least in part, due to the KSHV-regulated expression of Prox-1, the master regulator of lymphatic endothelial cell differentiation [33]. In addition, KSHV can induce the expression of mesenchymal markers on lymphatic endothelial cells cultured as ‘spheroids’, thereby causing an ‘endothelial to mesenchymal transition’ (EndMT) and explaining the expression of mesenchymal markers on KS spindle cells [26, 35]. Based on currently available experimental data, it is thought that KSHV-induced EndMT is due to the activation of Notch signaling activated by the viral proteins vFLIP and vGPCR [35-37] (Figure 2). The viral FLIP homologue, vFLIP, is also responsible for the spindle cell morphology, since its overexpression in primary endothelial cells induces spindle cell formation and deletion of the vFLIP gene from the viral genome reduces the ability of KSHV to induce spindle cell formation in primary endothelial cells [38, 39]. The transdifferentiation of endothelial cells to mesenchymal cells has also been observed in wound repair experiments [40-44]. Here, the inflammatory stimuli drive the conversion of endothelial cells into spindle shaped fibroblast like cells. EndMT is a key event in wound healing as it allows the cells to acquire invasive properties and to migrate to the damaged tissue, thus contributing to its repair. The main driving forces involved in this process are the inflammatory cytokines IL1β, TNFα and TGFβ [42-44]. EndMT is also involved in solid tumour metastasis formation and fibrosis [42, 44-46]. In contrast to EndMT observed in the context of inflammation or wound healing, KSHV-mediated EndMT appears to be TGFβ independent (see above) [35, 36]. Another unusual feature of KS (compared to other tumours) is the fact that most KS tumours are oligoclonal or polyclonal [47-49]. The oligo-/polyclonal nature of KS, the fact that KS

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cells isolated from KS tumours and implanted into nude mice do not grow into new tumours

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(but induce the growth of murine cells – see below) and the fact that primary human endothelial cells infected with KSHV in tissue culture do not show independent and sustained proliferation, even if grown in 3D culture systems (see evidence summarised above), all suggest that KSHV is not a ‘transforming’ virus, at least for primary human endothelial cells. However, a transforming effect of KSHV has been noted in primary rat mesenchymal precursor cells infected with KSHV [50], in a murine endothelial cell line transfected with a recombinant KSHV genome [51], and in a telomerase-immortalised human endothelial cell line infected with KSHV[52]. It is currently a matter of debate whether the transforming property of KSHV in rodent or immortalised human cells should be interpreted as evidence for its – perhaps weak – transforming potential also for primary human endothelial cells, or if these culture models, based on rodent or immortalised endothelial cells, do not recapitulate faithfully the effects that KSHV has on human endothelial cells in vivo. The recently reported 3D spheroid models of human lymphatic endothelial cells infected with KSHV, which showed the ability of KSHV to induce EndMT and an epigenetic/transcriptome pattern similar to that observed in KS lesions (see above; [35]) may go some way towards a better culture model that recapitulates KSHV-induced effects in endothelial cells in vivo.

Proliferation, inflammation and angiogenesis; important players in KS pathogenesis KS lesions contain very pronounced inflammatory infiltrates of monocytes, plasma cells and T- cells [24]. A leaky neovasculature prone to rupture, a prominent histological feature of KS, gives rise to the accumulation of haemosiderin and red blood cells, thereby accounting for the typical purplish colour of KS lesions [24]. These features of aberrant angiogenesis and inflammation are accompanied by evidence of endothelial cell proliferation. Although most

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(80-90 %) of spindle cells are latently infected, a small fraction, up to 10%, also express lytic

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markers [53] (Figure 1). The importance of lytic reactivation during KS pathogenesis is underlined by epidemiological studies showing that treatment with foscarnet or ganciclovir can prevent the appearance of KS, suggesting that lytic (productive) viral replication, inhibited by these drugs, although occurring in just a small percentage of infected cells (Figure 1), promotes the development of KS tumours [54-56]. Thus, both the latent and lytic programmes (Figure 3) of viral gene expression contribute to KS pathogenesis, the former by virtue of the effect of latent viral proteins and miRNAs on endothelial cell differentiation (see above) and proliferation (see below), the latter in a paracrine manner by inducing the release of cellular or viral cytokines with inflammatory, immune regulatory or angiogenic properties (see below) [57, 58]. PROLIFERATION Strongly expressed in KS spindle cells (Figure 1), during both latent and lytic replication, the major KSHV nuclear latency protein LANA [59] is responsible for replicating the latent circular viral genome during the S-phase of a dividing, latently infected cell and tethering the viral episome to cellular chromosomes during mitosis [60]. This ensures the correct replication of viral genomes and their distribution to daughter cells [60-62]. LANA is thus required for the persistence of the KSHV genome in latently infected, dividing cells. Moreover, LANA can recruit and deregulate the activity of several cellular transcription factors [63-65]. LANA binds to the tumour suppressor p53 and inhibits its ability to induce apoptosis [66]. LANA also targets the G1/S checkpoint proteins pRB and GSK3ß in order to modulate the G1/S transition [67] (Figure 4A). Furthermore, LANA extends the life span of primary endothelial cells in culture and renders them less susceptible to apoptosis [68].

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LANA was also shown to increase the stability of the c-myc oncogene, promoting its

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transcriptional activity [69, 70]. Two other latent KSHV proteins, encoded in the same region of the viral genome, contribute to the atypical characteristics of KSHV-infected spindle cells. A viral homologue of cellular cyclin D [71], vcyc, mediates the phosphorylation, in concert with the cellular cyclindependent kinase cdk6, of a broader range of cellular proteins than cellular cyclins and is not susceptible to the physiological inhibition of cyclins by Cip/Kip or INK4 proteins [71-77]. Vcyc phosphorylates several substrates, among them Cdc6 and Rb, and, when overexpressed experimentally, increases DNA synthesis and triggers progression towards the S phase of the cell cycle (Fig 4A)[74, 76]. The cell responds to the action of vcyc by inducing oncogene induced senescence (OIS) in an autophagy dependent manner [78, 79]. KSHV vcyc is coexpressed together with another latent protein, vFLIP, from a bicistronic mRNA [71, 73, 80]. This concerted expression pattern suggests a functional association between these two proteins. In fact, KSHV succeeds in escaping the vcyc induced authophagy dependent OIS through vFLIP, which prevents LC3 processing by ATG3 and thereby blocks the autophagic flux and vcyc-induced OIS [78] (Figure 4A). Therefore, among the few latent KSHV proteins, several can, when overexpressed in cell lines or primary endothelial cells, enhance cell proliferation. In the context of the whole virus, however, these effects may be muted: Infection of primary endothelial cells with KSHV extends their life span, but does not induce massive proliferation [81]. In KS biopsies, only a small proportion (< 20%) of spindle cells was reported to be labelled with an antibody to PCNA, expressed in proliferating cells [23], while another study found a strong increase of cells staining for p53 and Ki-67 in more advanced KS lesions [23, 82]. While KSHV has the ability to transform rodent endothelial or mesenchymal stem cells in several experimental

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settings [50, 51], KSHV-infected endothelial cells in KS lesions do not appear to be fully

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transformed and do not proliferate autonomously (see also above). Deregulation of the cell cycle and inhibition of apoptosis are not the only changes observed in KSHV-infected endothelial cells. Increase in lactic acid production and in glucose uptake is also a distinctive hallmark of KSHV latency in endothelial cells [83]. Moreover, KSHV latently infected cells are strictly dependent on glycolysis since its inhibition leads to increased cell death as compared to the uninfected control [83]. Metabolic shift toward anerobic glycolysis, decrease of oxygen consumption as well as of oxidative glycolysis, are often observed in cancer cells [84, 85]. In KS cells, the molecular mechanisms behind this process and their role in KS pathogenesis are still not yet fully understood [86]. INFLAMMATION Clinical observations, such as the appearance of KS tumours in surgical scars or sites of trauma (Koebner phenomenon) [87], suggest that cellular mechanisms operating during inflammation and/or wound repair may promote the development of this tumour in KSHVinfected individuals. An epidemiological observation cited in support of this concept is that KS is up to 10,000 fold more common in HIV-infected individuals (who often display signs of immune activation and chronic inflammation) than in the normal population, while the risk in transplant recipients (who have an iatrogenically induced specific T-cell defect) is ‘only’ several hundred fold higher [88]. KS lesions are characterized by the presence of a rich inflammatory infiltrate involving in particular monocytes, eosinophils and plasma cells [89]. Experimental evidence in support of a role for inflammatory cytokines in KS pathogenesis began to accumulate before the discovery of KSHV as the cause of this tumour [90-92]. It is thought that the chronic immune activation seen in AIDS patients may exacerbate the oncogenic effects of KSHV. This article is protected by copyright. All rights reserved.

Experimentally, inflammatory cytokines such as interferon γ, and IL6, have been shown to

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increase the KSHV viral load in peripheral blood B-cells and monocytes [93-96]. Furthermore, increased levels of IL2 and IL10 have been found in the sera of HIV-negative individuals from Uganda, a country known for the higher incidence of endemic (HIVnegative) KS [97], indicating that higher levels of inflammatory cytokines could favour the development of KS. The inoculation of cultured cells from KS biopsies into nude mice induced KS-like lesions of murine origin, suggesting that inflammatory cytokines released from cultured KS cells can have paracrine tumorigenic effects [30, 98]. In addition to cellular inflammatory cytokines [87], KSHV-encoded cytokines may contribute to an inflammatory environment conducive to the development of KS. KSHV encodes three chemokine ligands, vCCL1 (K6), vCLL2 (K4) and vCCL3 (K4.1), homologues, respectively, of MIP1α, MIP1β and CCL2. These viral proteins are able to trigger a Th2 inflammatory response. As a result of their signalling through CCR3 (vCCL2), CCR4 (vCCL3) and CCR8 (vCCL1, vCCL2) receptors, expressed by Th2 cells, these viral proteins are able to chemotactically attract Th2 cells to the site of infection [99-101]. Moreover, vCCL2 acts as an antagonist of CCR1 and 5, which are expressed on the surface of Th1 cells and involved in antiviral responses [99]. Therefore, by encoding factors that stimulate a Th2 inflammatory response, and inhibit the Th1 response, KSHV can down-modulate the Th1 arm of the T-cell response involved in the control of viral infection and avoid host immune surveillance (Figure 4B). Moreover, an inflammatory microenvironment could contribute to the recruitment of target cells that can be newly infected. A known cellular mediator of inflammation involved in many pathological processes is Cox2. Cyclooxygenases (Cox) are enzymes that convert arachidonic acid to prostaglandins (PGs). Unlike the constitutively expressed Cox1, Cox2 expression is induced by mitogenic and pro-

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inflammatory stimuli [102, 103]. It has been shown that Cox2 is overexpressed not only in

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breast, pancreatic and colon cancers [104-106] but also in virus-induced neoplasia such as HPV-induced squamous cell carcinoma [107] and hepatocellular carcinoma caused by HCV [108]. Cox2 is also overexpressed in KS tissue and KSHV de novo infection of primary endothelial cells leads to a significant up-regulation of Cox2 levels [109]. Among KSHV viral proteins, vFLIP and K15 are able to induce Cox2 (Figure 4B), which is, in turn, used by the virus to upregulate LANA expression [110]. In KSHV infected endothelial cells, Cox2 triggers the secretion of chemokines (RANTES, MCP2, TARC, MIP1α, MDC and MIG) that contribute to the recruitment of leukocytes and pro-angiogenic factors (IGF1, PDGF, IL14, MCSF, GM-CSF, VEGF A and C, angiogenin, oncostatin M, TGFβ1) to the site of infection [109, 110]. Moreover, the KSHV-dependent increase in expression of Cox2 augments the secretion of MMPs (Figure 4B), conferring on KSHVinfected cells the ability to digest and invade the extracellular matrix (ECM) [109-111]. Small molecule inhibitors of Cox2 are available and experiments in primary endothelial cells showed that these inhibitors, or silencing of Cox2 by siRNA, reduce both the increased invasiveness of KSHV-infected ECs and the reactivation of the KSHV lytic (productive) replication cycle and/or establishment of latency [109-111]. Thus, KSHV-induced expression of Cox2 may affect the regulation of latent vs lytic infection [111], and contribute to some of the histological features of KS tumours such as (i) the presence of an inflammatory infiltrate (by inducing chemoattractant cytokines) [26, 112], (ii) an increased life span (through the expression of LANA), (iii) increased invasiveness (as a result of the action of metalloproteinases) [81, 111, 112], and (iv) aberrant angiogenesis (due to the increased expression and secretion of pro-angiogenic factors) [26, 81, 98, 113] (Figure 4B).

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If the expression of Cox2 does indeed influence several important aspects of the KSHV life

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cycle and its pathogenesis, it could therefore provide an interesting therapeutic target. ANGIOGENESIS, INVASION AND MIGRATION The term angiogenesis defines the process of new blood vessel formation and vascular remodelling. The histological features of KS (see above) involve elements of aberrant angiogenesis (vascular spaces, leakage, erythrocyte extravasation, abnormal differentiation of endothelial cells including EndMT). KSHV- induced angiogenesis is already microscopically visible at the early stages of KS in the form of leaky and poorly organized newly developed vessels, which result in a red or purple lesion because of the extravasion of blood and haemosiderin [24]. In cell culture systems, KSHV infection of primary endothelial cells induces angiogenic features such as the formation of tubules of endothelial cells plated on matrigel in the absence of externally supplied growth factors, and also enhances their invasiveness and migration [35, 81, 114]. KSHV-infected cells show an increased production and secretion of pro-angiogenic factors such as VEGF, Ang2, IL6 and 8, ephrin B2, MMPs [111, 114, 115]. Multiple KSHV encoded proteins, expressed in both the lytic and latent phase of replication, contribute to KSHV-induced alterations of angiogenesis. The major latency protein, LANA, expressed in KS spindle cells (Figure 1), induces the expression of emmprin, which has previously been implicated in promoting metastasis and angiogenesis in many cancer types and shown to be up-regulated upon KSHV infection of both primary human fibroblast and endothelial cells [110, 111]. KSHV hijacks emmprin expression in order to modify the extracellular microenvironment by, on one hand, inducing the secretion of IL6 and VEGF thus enhancing angiogenesis, and on the other hand increasing the production of MMPs triggering invasiveness [116, 117]. Furthermore, LANA is implicated in the stabilization of This article is protected by copyright. All rights reserved.

the Notch effector Hey1, which represses the expression of Prox-1, the master regulator of

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the differentiation towards lymphatic endothelial cells (see above) (Figure 4C) [118]. Moreover, the Notch mediated activation of PDGFRβ, expressed in KS lesions, contributes to the invasive properties of KSHV infected SCs [35, 119, 120]. Recent studies have also shown that KSHV encodes microRNAs in its cluster of latency genes (Figure 3), and that these microRNAs might contribute to KS angiogenesis, although the mechanism needs to be further clarified [121]. Outside the classical latency locus, which contains LANA, vcyc, vFLIP and the viral miRNAs, three other viral proteins, K1, vIL6 and K15 may be part of a ‘relaxed’ latency programme (Figure 3) and could therefore be expressed in KS tissue [122-124]. Expression of K1 [113] and vIL6 [125, 126] in at least a few KSHV-infected cells in KS tissue has been shown by immunohistochemistry. The non-structural KSHV transmembrane protein K1 is able to increase angiogenic properties in cultured primary endothelial cells [81, 113]. The mechanisms involved include a K1-induced increase in VEGF secretion, which leads to paracrine VEGFR2 activation and downstream signalling. K1 expression also induces several angiogenic and inflammatory cytokines (IL1 α and β, IL8, IL10 and RANTES) [113, 127] (Figure 4C). Furthermore, K1 promotes angiogenesis in vivo, as shown by the more vascularized tumours that were found to develop in mice injected with a HPV negative cervical carcinoma cell line overexpressing K1 [113]. However, these data were obtained in the context of overexpression studies and further investigations are required to elucidate the contribution of K1 to the angiogenic phenotype of endothelial cells infected with KSHV. Moreover, in primary endothelial cells, expression of K1 leads to the activation of the Akt/PI3K pathway driving the inactivation of pro-apoptotic proteins such as PTEN and the activation of mTOR, a major positive regulator This article is protected by copyright. All rights reserved.

of cell survival [113]. It has been reported that, in transplant recipients, use of the mTOR

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inhibitor rapamycin as an immunosuppressive drug promotes regression of KS lesions [128], thus pointing to K1, or other inducers of the mTOR pathway, as possible pivotal players in KS pathogenesis. Another non-structural transmembrane protein, K15, is encoded at the opposite end of the viral genome [124, 129, 130] (Figure 3). This protein has been shown to interact with Src kinase family members and to activate cox2, NFAT and dscr1 promoters [131-134], thus inducing an angiogenic and pro-inflammatory response. Furthermore, in KSHV-infected primary endothelial cells, K15 binds to and activates PLCγ1, thereby leading to VEGFindependent endothelial tubule formation in matrigel-based assays [135] (Figure 4C). Depletion of K15 from the KSHV genome significantly reduces virus-induced angiogenesis in primary endothelial cells [135]. K15 is expressed in latently infected primary endothelial cells [135], but whether this extends to KSHV-infected spindle cells in KS tumours is currently not certain. Known to play a role in KS pathogenesis, as well as in both lymphoproliferative disorders linked to KSHV (see above), the viral homologue of interleukin 6 (vIL6) has been shown to have angiogenic properties. In particular, it has been reported that injection of stably expressing vIL6 cells into nude mice leads to development of highly vascularised tumours [136] and that vIL6 mediated angiogenesis is synergistically enhanced by the HIV 1 nef protein [137]. Moreover, vIL6 seems to have both an anti-apoptotic and mitogenic effect: shRNA depletion of vIL6 resulted in a reduced growth rate in KSHV infected B cell lines [138]. Similarly to cellular IL6 (cIL6), vIL6-mediated signalling occurs through the Jak/STAT, MAPK axis and elicits the expression of VEGFR2, b-FGF, Cyclin D1 and PTX3, a marker of local inflammation [139] (Figure 4C). Interestingly, vIL6 can also induce the

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transcription and secretion of its cellular counterpart [140], thus further supporting the

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inflammatory and pro-angiogenic environment necessary for KS development. Although exhibiting a certain level of homology at the protein level, cellular and viral IL6 display an important difference in receptor requirements. While cIL6 needs to first bind the gp80 αchain of the IL6 receptor in order to recruit and activate the signal transducing component, gp130, vIL6 can directly bind and signal through gp130 independently of gp80 [141, 142] (Figure 4C). As gp130 is shared by other cytokine receptors, the effects of vIL6 also mimic those of other cytokines and vIL6 is therefore much more pleiotropic than cIL6. Cellular and viral IL6 also differ with regard to their secretion rate. Pulse-chase analysis revealed that the half time of vIL6 secretion is about 8 times longer than its cellular counterpart [143]. Furthermore, it has been reported that in KSHV-infected cells vIL6 is mostly retained in the ER where it can trigger autocrine signalling through gp130 [143, 144]. The significance of vIL6 intracellular signalling has been shown in PEL cells, in which vIL6 silencing significantly reduced cell growth [144, 145]. As vIL6 is also expressed in KS lesions [125, 126], intracrine vIL6 signalling could contribute to cell survival and to virus induced tumorigenesis. As already mentioned, a small subset of KSHV-infected cells in KS tumours undergo lytic reactivation (Figure 3). Lytically (productively) infected cells express a much larger complement of viral proteins, which could, by promoting the expression of secreted cellular or viral factors, contribute to the angiogenesis in KS tumours in a paracrine manner. This is thought to be the case for the viral G protein coupled receptor, vGPCR, which is considered an important contributor to KSHV- triggered angiogenesis [146]. vGPCR is a ligandindependent and constitutively active IL8 receptor [147-149]. Expressed during the early phase of the lytic cycle, vGPCR signals through the NF-kB, AP1 and NFAT networks, thereby determining the secretion of many angiogenic factors (VEGF, bFGF, IL1 β, IL2-4-6This article is protected by copyright. All rights reserved.

8 and TNFα), which act in a paracrine manner and could contribute to the modification of the

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extracellular microenvironment towards the development of KS [147-149] (Figure 4C). Experimentally, vGPCR expression in Tie2-TVA transgenic mice leads to the formation of multifocal and aberrantly vascularized tumours with histological similarities to KS [150-152]. Moreover, the viral chemokine homologues (see above) could also make a paracrine contribution to KSHV-mediated angiogenesis. They were first shown to display angiogenic properties in a chorioallantoic membrane assay [153]. Moreover, vCCL2 overexpression in endothelial cells triggers an increase in angiogenic cytokine secretion and has been shown to be pro-angiogenic in vivo [154]. Although this protein increases VEGF secretion also in PEL cells [155], its role in KS angiogenesis needs to be elucidated. Another possible proangiogenic contribution could involve vCCL1- and 2-dependent signalling through CCR8, which has been shown to trigger vascular smooth muscle reorganization and MMP2 activation [156].

Applications to Therapy and Perspectives Twenty years after the discovery of KSHV we have come a long way in understanding the role of this virus in the pathogenesis of KS, but significant challenges remain. Given that KSHV is an indispensable factor in the development of this tumour, and that KS does not appear to result from a classical transformation event, it should be possible to target viral or cellular proteins that contribute to KSHV-induced effects in infected endothelial cells. If successful, such an approach could complement, or replace, current therapy protocols, which essentially rely on anti-retroviral combination therapy for the early stages of AIDS-KS, and the use of cytostatic drugs such as pegylated liposomal doxorubicin for the more advanced

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stages [157, 158]. Inhibitors of other viral DNA polymerases such as cidofovir and foscarnet

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have been shown to inhibit KSHV lytic (productive) replication in vitro [55, 159] and to prevent the development of KS in prophylactically treated patients [55], but their effectiveness in treating established KS lesions is very limited. No other inhibitor of a KSHV protein has so far been described. As a first example of the concept that it may be possible to target cellular factors that play an indispensable role in the viral life cycle, sirolimus, an mTOR inhibitor, has been shown to reduce post-transplant KS lesions that appeared in transplant recipients being treated with calcineurin inhibitors [128]. As a further example, treatment with imatinib, an inhibitor of tyrosine kinases such as Abl, c-kit and PDGFR, some of which have been linked to KS pathogenesis (see above) [160], has resulted in partial regression of KS tumours in about one third of patients with AIDS-KS [161, 162] . Sorafenib, targeting receptors for VEGF and PDGF, has been tried in a single patient [163]. While preliminary, these results are encouraging and support the concept that a better understanding of the role of KSHV in the pathogenesis of this unusual tumour may eventually yield therapeutic benefits.

Acknowledgments & funding sources This study was supported by the German Research Council (DFG) through Collaborative Research Centre 900, project C1, and project grant SCHU-1668/3-1 to TFS. The authors’ work is also supported by the German Centre for Infectious Disease Research (DZIF).

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List of abbreviations

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Abl: Abelson murine leukemia viral oncogene homolog; Ang2: Angiopoietin 2; ATG: Autophagy-related gene; AP1: Activator protein1; b-FGF: Basic fibroblast growth Factor; CCR: CC chemokine receptor; cdc6: Cell division cycle 6; cdk6: Cell division protein kinase 6; cip/kip: CDK interacting protein/Kinase inhibitory protein; COX: Cyclooxigenase; CXCL12: CXC chemokine ligand; CXCR4: CXC chemokine receptor; DLL4: Delta like ligand 4; dscr1: Down Syndrome Critical region 1; Emmprin: Extracellular matrix metalloproteinase inducer; GM-CSF: Granulocyte macrophage-colony stimulating factor; GSK3β: Glycogen synthase kinase β; IGF: Insulin growth factor; IL1β: Interleukin1β; Jak/STAT: Janus kinase/signal transducers and activators of transcription; LC3: Microtubule associated protein 1A/1B light chain 3; MAPK: Mitogenactivated protein kinase; MCP2: Monocyte chemotactic protein 2; MCSF: Macrophage colony stimulating factor; MDC: Macrophage-derived chemokine; MIG: Monokyne induced by IFNγ; MIP: Macrophage inflammatory protein; MMPs: Matrix metalloproteinases; mTOR: Mammalian target of rapamycin; NFAT: Nuclear Factor of Activating T cells; NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells PDGF: Platelet-derived growth factor; PDGFR: Platelet-derived growth factor receptor; PI3K: Phosphatidyl insositol3 kinase; PLCγ1: Phospholipase Cγ; PTEN: Phosphatase and tensin homologue; PTX3: Pentraxin 3; RANTES: Regulated on activation, normal T cell expressed and secreted; Rb: Retinoblastoma protein; TARC: Thymus and activation-regulated chemokine; TGFβ: Transforming growth factor β; TNFα: Tumour necrosis factor α; vCCL: Viral CC chemokine ligand; vcyc: viral cyclin; VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth factor receptor; vFLIP: Viral FLICE-like inhibitory protein; vGPCR: Viral G protein coupled receptor

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Author contributions

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SG and TFS wrote the manuscript together. References 1. 2. 3. 4.

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Figures legend

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Figure 1. Expression of a latent (LANA) and a lytic (ORF 59/PF8) protein in a Kaposi sarcoma biopsy. As discussed in the text, the majority of KSHV-infected spindle cells express latent viral proteins, while a minority of infected cells show evidence of lytic (productive) viral replication, here indicated by the expression of orf 59/PF8, an early lytic gene. Reproduced with kind permission of Carlo Parravicini and Yuan Chang

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Figure 2. Possible mechanism of KSHV-induced endothelial to mesenchymal transition.

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Upon infection of endothelial cells, KSHV induces, through vFLIP and vGPCR, the activation of Notch signaling. This event triggers the expression of mesenchymal markers (see text), here indicated by the yellow star, as well as those of blood and lymphatic endothelium (see text), in purple and orange respectively.

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Figure 3. Map of the KSHV genome. The direction of transcription is shown by the arrows.

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Numbers indicate open reading frames (ORFs), with K designating the ORFs first identified in the KSHV genome; homologues of some of these genes were successively identified in the Old Word primate rhadinoviruses. During the latency phase the virus expresses only a restricted repertoire of ORFs, here depicted in sky blue. In purple are represented the transcripts belonging to the so called ”relaxed latency program” (see text). Upon lytic reactivation, all viral genes are transcribed and particularly those necessary for virus production: in green are the components of the envelope and in orange those of the viral capsid. Genes encoding the proteins discussed in the text are highlighted in bold.

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Figure 4. Molecular mechanisms of KSHV-mediated pathogenesis. A) Proliferation; many

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viral proteins expressed during latency are involved in KSHV-mediated cell proliferation. LANA tethers the viral genome to the host chromatin, and represses p53 and GSK3β proapoptotic functions (see text). Vcyc phosphorylates cdc6 and Rb proteins thus promoting G1/S transition but also inducing autophagy-mediated senescence. VFLIP counteracts vcycinduced senescence by blocking autophagy (see text). B) Inflammation; pro-inflammatory cytokines and MMPs are secreted by KSHV-infected endothelial cells upon K15- and vFLIPmediated COX2 expression. Moreover, KSHV encodes cytokines, namely vCCL1, 2 and 3 able to trigger a Th2 inflammatory response. Viral cytokines can signal through CCR8, 3 and 4 (in blue) expressed by Th2 cells, which are chemotactically attracted to the site of infection, and act as antagonist of CCR1 and 5 (in red) expressed by Th1 cells (see text). C) Angiogenesis; KSHV encodes a variety of proteins exploiting angiogenic properties. K15 recruits and activates PLCγ1 to induce NFAT-mediated transcription of pro-angiogenic genes. vGPCR, K1 and vIL6 trigger the expression of pro-angiogenic cytokines (see text) including VEGF, which, by binding to its cognate receptor, triggers angiogenesis.

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Accepted Article This article is protected by copyright. All rights reserved.