Pone.0019103 1.1

Amplification of the Angiogenic Signal through theActivation of the TSC/mTOR/HIF Axis by the KSHV vGPCRin Kaposi's Sarcoma Bruno C. Jham1, Tao Ma1, Jiadi Hu1, Risa Chaisuparat1, Eitan R. Friedman1, Pier Paolo Pandolfi4, Abraham Schneider1,3, Akrit Sodhi5, Silvia Montaner1,2,3* 1 Department of Oncology and Diagnostic Sciences, School of Dentistry, University of Maryland, Baltimore, Maryland, United States of America, 2 Department of Pathology, School of Medicine, University of Maryland, Baltimore, Maryland, United States of America, 3 Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland, United States of America, 4 Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America, 5 Wilmer Eye Institute, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America Background: Kaposi's sarcoma (KS) is a vascular neoplasm characterized by the dysregulated expression of angiogenic andinflammatory cytokines. The driving force of the KS lesion, the KSHV-infected spindle cell, secretes elevated levels of vascularendothelial 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 inKS through an intricate paracrine mechanism. The cytokines secreted by the few vGPCR-expressing tumor cells activate inneighboring cells multiple pathways (including AKT, ERK, p38 and IKKb) that, in turn, converge on TSC1/2, promoting mTORactivation, HIF upregulation, and VEGF secretion. Conditioned media from vGPCR-expressing cells lead to an mTOR-dependent increase in HIF-1a and HIF-2a protein levels and VEGF upregulation. In a mouse allograft model for KS, specificinhibition of the paracrine activation of mTOR in non-vGPCR-expressing cells was sufficient to inhibit HIF upregulation inthese 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 vGPCRcan contribute to the dramatic amplification of VEGF secretion in KS, and further provide a molecular mechanism for howcytokine dysregulation in KS fuels angiogenesis and tumor development. These data further suggest that activation of HIFby vGPCR may be a vulnerable target for the treatment of patients with KS.
Citation: Jham BC, Ma T, Hu J, Chaisuparat R, Friedman ER, et al. (2011) Amplification of the Angiogenic Signal through the Activation of the TSC/mTOR/HIF Axisby the KSHV vGPCR in Kaposi's Sarcoma. PLoS ONE 6(4): e19103. doi:10.1371/journal.pone.0019103 Editor: Lin Zhang, University of Pennsylvania, United States of America Received February 25, 2011; Accepted March 16, 2011; Published April 29, 2011 Copyright: ß 2011 Jham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant R01CA119911 (National Cancer Institute, National Institutes of Health). The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] VEGF is expressed at elevated levels by KSHV-infectedendothelial cells in vitro, and by KS spindle cells in vivo; a strict Kaposi's sarcoma (KS) is a multifocal vascular neoplasm requirement for VEGF has also been demonstrated for KS spindle invariably associated with infection with the KS-associated human cells grown in vitro [3,4,5,6]. These observations are suggestive of herpesvirus (KSHV/HHV8), which is characterized by cytokine an autocrine mechanism for this angiogenic factor in the growth of dysregulation [1]. The driving force of the KS lesion is the KSHV- this vascular tumor. Indeed, like VEGF, its cognate receptor, infected spindle-shaped tumor (spindle) cell, thought to have a VEGFR2 (KDR), is also upregulated in AIDS-KS primary lesions vascular endothelial or endothelial precursor origin. The promo- as well as in AIDS-KS spindle cell cultures [4,7,8]. However, tion of the angiogenic phenotype in these lesions is supposed to be reasonable disagreement remains as to the precise molecular mediated by elevated levels of pro-inflammatory and pro- mechanism whereby KSHV promotes VEGF secretion.
angiogenic secretions (cytokines, chemokines and growth factors) Of interest, KSHV ORF74 encodes for a viral G protein-coupled from the KS spindle cells [1]. Indeed, KS is often thought to result receptor (vGPCR) with close homology to the mammalian CXCR1 from reactive endothelial hyperproliferation induced by the and CXCR2 and ligand-independent (constitutive) activity [9,10].
chronic release of these molecules and has served as a model for When expressed upon endothelial-specific retroviral infection or as a tumor- and inflammation-induced angiogenesis [1,2].
transgene, vGPCR is sufficient to recapitulate KS-like lesions in mice Among the angiogenic factors elaborated by the KS spindle [11,12,13]. Indeed, emerging data suggest that vGPCR may play a cells, VEGF is unique for its profound impact on KS pathogenesis.
role in KS initiation, progression and maintenance [12,14,15].
PLoS ONE www.plosone.org April 2011 Volume 6 Issue 4 e19103 VEGF Amplification by vGPCR Activation of HIF vGPCR has proven to be a powerful oncogene and a potent vivo (data not shown; [15]). However, these cells (EC-vCYC/ angiogenic activator [1]. Expression of vGPCR in endothelial cells vFLIP) are able to induce allografts in nude mice when co-injected activates intracellular pathways that induce cell survival and with vGPCR-expressing cells (EC-vGPCR) (Fig. 1E). Immunohis- transformation. In addition, vGPCR-expressing cells elaborate tochemical staining of these lesions also shows upregulation of pro-angiogenic factors that are thought to promote the recruit- VEGF in most tumor cells (Fig. 1E). Interestingly, when these ment and transformation of neighboring endothelial cells [16].
mixed-cell allografts are treated with Rapamycin, which is able to Both VEGF and KDR have been previously reported to be block vGPCR tumorigenesis [19], a reduction in VEGF expression upregulated by cells expressing vGPCR [7,8,17,18]. However, the in treated tumors is observed. Collectively, these results suggest limited expression of this viral oncogene in only a few tumor cells that the paracrine upregulation of VEGF by vGPCR requires the in both transgenic KS mouse models and human KS tissues raises activation of the mTOR signaling cascade in vitro and in vivo.
the question as to the relative contribution of vGPCR to VEGFsecretion, suggesting that the relationship between vGPCR and vGPCR paracrine secretions activate mTOR through VEGF is not clearly established. Here we set out to further multiple signaling pathways characterize the contribution of vGPCR to the upregulation of Diverse extracellular stimuli influence mTOR activity through VEGF secretion in KS.
posttranslational modification of TSC1/TSC2 [20]. Indeed,numerous kinases are able to phosphorylate TSC1 or TSC2, integrating extracellular signals and tumorigenesis through thecontrol of TSC1/TSC2/mTOR [21]. Among these kinases, vGPCR activates VEGF expression through a paracrine AKT, ERK, and p38/MK2 have been shown to phosphorylate mechanism dependent on mTOR TSC2 in specific sites, including Thr1462, Ser664 and Ser1254, The KSHV-encoded vGPCR causes angioproliferative lesions respectively [22,23,24,25,26]. Conversely, IKKb induces mTOR remarkably similar to human KS, when expressed upon activation by phosphorylating TSC1 in Ser487 and Ser511 [27].
endothelial-specific retroviral infection in immunocompetent We have previously shown that induction of AKT activity by animals (Fig. 1A–B) [12]. Interestingly, immunohistochemical vGPCR promotes mTOR activation in neighboring cells [19].
staining of these vGPCR tumors using a specific vGPCR antibody However, the contribution of other kinases upstream of TSC/ reveals the presence of this viral protein in only a small percentage mTOR to vGPCR oncogenesis remains unclear. We thus treated of tumor cells, similar to the expression pattern of vGPCR in HMEC1 with media conditioned by control or vGPCR-expressing human KS, suggestive of a paracrine contribution of vGPCR to cells and assessed the activation of TSC kinases by these Kaposi's sarcomagenesis (Fig. 1B) [12,13]. In this regard, vGPCR supernatants. In addition to AKT, we found that ERK, p38 and has been implicated in the induction of the expression of VEGF, a IKKb were also activated by vGPCR paracrine secretions key angiogenic factor highly upregulated in KS [17,18]. However, (Fig. 2A). Activation of these kinases correlated with the staining of vGPCR tumors – and human KS – with a specific phosphorylation of TSC2 in Thr1462, Ser664 and Ser1254, and antibody against VEGF reveals a robust expression of this factor in phosphorylation of TSC1 in Ser511, respectively, and the most tumor cells (Fig. 1B), indicating that vGPCR may also upregulation of mTOR activity, assessed by S6K phosphorylation upregulate VEGF in neighboring cells through an indirect (Fig. 2A). We also evaluated the activation of these TSC1/2 mechanism. To explore this possibility, we used an inducible kinases by individual GPCR angiogenic factors [14,28]. Figure S1 (Tet-on) expression system for vGPCR in immortalized human shows that all the cytokines tested were able to induce microdermal endothelial cells (HMEC1). Using this system, we phosphorylation of TSC1 and/or TSC2. Interestingly, IL-1b, confirmed that induction of vGPCR expression in endothelial cells IL-10, TNFa and VEGF are each able to promote phosphory- leads to the potent upregulation of VEGF, as previously reported lation of TSC1/2 on at least three separate sites (Fig. S1).
(Fig. 1C) [17,18]. Moreover, we observed that exposure of Collectively, these results suggest that the secreted factors HMEC1 to media conditioned by these vGPCR-expressing cells elaborated by vGPCR-expressing cells act together to upregulate (vGPCR CM) is similarly able to promote VEGF expression multiple intracellular signaling pathways that converge on TSC/ We therefore set out to determine the mechanism whereby To determine the relative contribution of these TSC kinases to vGPCR angiogenic factors can induce VEGF upregulation. In this the paracrine mTOR activation by vGPCR, we used specific regard, we have previously reported that vGPCR paracrine inhibitors of PI3K/AKT, MEK/ERK, p38 or IKKb. Surprising- secretions activates TSC/mTOR, a signaling route that has been ly, we observed that either pharmacological inhibition of AKT- shown to regulate the expression of VEGF [19,20]. We thus mediated phosphorylation of TSC2 or IKKb-mediated phosphor- treated HMEC1s with supernatants derived from vGPCR- ylation of TSC1 lead to a complete inhibition of mTOR, expressing cells (vGPCR CM) or control cells (Control CM), in measured by S6K phosphorylation (Fig. 2A). Conversely, the absence or the presence of the mTOR inhibitor, Rapamycin.
pharmacological inhibition of either ERK or p38 leads to only Figure 1D shows that exposure of endothelial cells to vGPCR partial inhibition of ERK-mediated or p38-mediated S6K secreted factors leads to the upregulation of the transcription and phosphorylation, respectively. We then used siRNA to specifically translation of VEGF in a Rapamycin-sensitive manner.
knock-down expression of AKT, ERK1 and 2, p38, or IKKb.
To further evaluate the contribution of the vGPCR-induced Knock-down of these kinases was only sufficient to partially inhibit paracrine activation of mTOR to VEGF upregulation in vivo, we mTOR (Fig. 2B). Collectively, these results suggest that there is a used an allograft model in which (SV-40) immortalized murine redundancy in the pathways leading to the phosphorylation of endothelial cells (SVECs) expressing vGPCR (EC-vGPCR) are TSC1 and 2 and the activation of mTOR by the paracrine mixed with SVECs co-expressing two non-tumorigenic KSHV secretions of vGPCR-expressing cells.
latent genes, vCyclin and vFLIP (EC-vCYC/vFLIP), in a (1:10) We then stained murine vGPCR tumors and human KS tissues ratio that approximates the proportion of expressing cells in with specific antibodies against the phosphorylated (activated) human KS (Fig. 1E) [15]. Cells expressing vCYC and vFLIP do forms of AKT, ERK, p38 or IKKb or against the corresponding not show VEGF upregulation in vitro nor are they tumorigenic in TSC2/TSC1 phosphorylated form, induced by each kinase PLoS ONE www.plosone.org April 2011 Volume 6 Issue 4 e19103

VEGF Amplification by vGPCR Activation of HIF Figure 1. vGPCR activates VEGF expression through an mTOR-dependent paracrine mechanism. (A) KS-like lesion developed uponretroviral transduction of vGPCR in TIE2-tva mice (vGPCR tumor), as described in Materials and Methods. (B) H&E and immunohistochemical stainingsof vGPCR tumor and human KS, using antibodies against vGPCR, VEGF or an isotype-matched control antibody. (C) Upregulation of VEGF in HMEC1stransfected with Tet REV TA and pBIG AU5 vGPCR and treated with doxycycline (vGPCR), respect to untreated cells (Control). VEGF upregulation inHMEC1 exposed for 2 h or 6 h to supernatants collected from vGPCR-expressing cells (vGPCR CM). (D) Supernatants from vGPCR-expressing cells(vGPCR CM) or control cells (Control CM) were used to treat HMEC1 in the presence or absence of Rapamycin (50 nM). RT-PCR and Western blotanalysis were used to determine levels of VEGF mRNA and protein, respectively. (E) EC-vGPCR (10%) were mixed with EC- vCYC/vFLIP (90%), andinjected into athymic nu/nu mice for allograft formation. Tumor weight curves, and immunohistochemical detection in tumor tissue of (AU5-tagged)vGPCR-expressing cells, phosphorylated ribosomal S6 protein or VEGF, upon treatment with (10 mg/kg) Rapamycin of vehicle (Control), are shown.
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VEGF Amplification by vGPCR Activation of HIF Figure 2. vGPCR secretions regulate TSC/mTOR through multiple signaling pathways in vitro and in vivo. (A) HMEC1s were pretreatedwith vehicle or inhibitors of the AKT, ERK, p38 or IKKb pathways, LY294002 (50 mM), U0126 (50 mM), SB203580 (50 mM) or BAY11-7082 (40 mM). Cellswere then exposed to media conditioned by control cells (Control CM) or vGPCR-expressing cells (vGPCR CM). Phosphorylation levels of thecorresponding kinase (AKT, ERK1/2, p38 or IKKb), TSC2/1 targeted phosphorylation site (P-TSC2T1462, P-TSC2S664, P-TSC2S1254 or P-TSC1S511), and S6Kare shown. (B) HMEC1s were transfected with Scrambled siRNA or siRNA for AKT, ERK (ERK1 and 2), p38 or IKKb. Cells were then exposed to mediaconditioned by control or vGPCR-expressing cells. Levels of the corresponding kinase (AKT, ERK1/2, p38 or IKKb) and S6K are shown. (C)Immunohistochemical staining of vGPCR tumors and human KS with antibodies against P-AKT, P-ERK, P-p38 or P-IKKb, and the corresponding TSC2/1targeted phosphorylation site, P-TSC2T1462, P-TSC2S664, P-TSC2S1254 or P-TSC1S511.
doi:10.1371/journal.pone.0019103.g002 (Fig. 2C). In support of our in vitro observations, we found HMEC1 with media conditioned by vGPCR-expressing cells or phosphorylation of these four kinases and the corresponding control cells, in the absence or presence of Rapamycin, and targeted aminoacids in TSC2 or TSC1, in both vGPCR tumors examined the levels of HIF-1a and HIF-2a mRNA and protein.
and human KS (Fig. 2C). These findings suggest a role for these Interestingly, we found that vGPCR angiogenic factors induced an kinases upstream of TSC/mTOR in vGPCR tumorigenesis and upregulation of HIF-1a mRNA (3-fold) and HIF-2a mRNA (18- Kaposi's sarcomagenesis in vivo.
fold); both were blocked by the mTOR inhibitor (Fig. 3A).
Upregulation of HIF-1a, HIF-2a and VEGF protein levels by Paracrine activation of TSC/mTOR by vGPCR angiogenic vGPCR secretions was also blocked by Rapamycin (Fig. 3A).
factors results in the upregulation of HIF-1a/2a Furthermore, the increase in VEGF transcription and translation TSC/mTOR has been shown to regulate Hypoxia Inducible by vGPCR supernatants was blocked by the expression of a Factor (HIF), a family of transcription factors containing an specific siRNA of HIF-1b (Fig. 3B). When we investigated the inducible a subunit and a constitutive b subunit [20,29]. HIF levels of HIF-1a and HIF-2a proteins in vGPCR murine tumors promotes neovascularization and vascular remodeling by control- and KS biopsy specimens by immunohistochemical analysis, we ling the expression of key angiogenic proteins, including VEGF found that both vGPCR murine tumors and human KS showed a [30]. Since vGPCR angiogenic factors activate TSC/mTOR remarkable overexpression of these transcription factors, com- through a variety of intracellular routes, we next investigated pared to normal skin (results not shown) (Fig. 3C). Collectively, whether this activation could lead to the upregulation of VEGF these results suggest that vGPCR may induce paracrine upregula- through a HIF-dependent mechanism. To this end, we treated tion of VEGF through an mTOR/HIF-dependent mechanism.
PLoS ONE www.plosone.org April 2011 Volume 6 Issue 4 e19103

VEGF Amplification by vGPCR Activation of HIF Figure 3. Upregulation of HIF-1a/2a by vGPCR paracrine secretions. (A) HMEC1s were exposed to media conditioned by control (Control CM)or vGPCR-expressing cells (vGPCR CM), in the presence of vehicle or Rapamycin (50 mM). mRNA levels of HIF-1a and HIF-2a and protein levels of HIF-1a, HIF-2a and VEGF are shown. (B) HMEC1s were transfected with increasing doses (20, 40 and 80 nM) of HIF-1b siRNA or Scrambled siRNA. Cellswere then exposed to media conditioned by vGPCR-expressing cells. VEGF mRNA levels are shown. (C) Immunohistochemical analysis of HIF-1a andHIF-2a, and staining using an isotype-matched control antibody, in vGPCR tumor and human KS.
doi:10.1371/journal.pone.0019103.g003 Paracrine activation of mTOR is required for HIF and treated established tumors with Rapamycin or vehicle (Fig. 4).
upregulation in vGPCR sarcomagenesis in vivo Similar to EC-vGPCR+EC-vCYC/vFLIP tumors, growth of vGPCR activates mTOR through both direct and indirect allografts formed upon injection with EC-vGPCR/RR-mTOR + mechanisms and both may thus contribute to endothelial cell EC-vCYC/vFLIP was strongly inhibited by treatment with transformation and angiogenic dysregulation in KS. To assess the Rapamycin, suggesting that protection from the inhibition of relative contribution of vGPCR direct versus paracrine activation direct mTOR activation within vGPCR-expressing cells was not of TSC/mTOR/HIF to vGPCR oncogenesis, we generated cell sufficient to render these tumors sensitive to the drug. Conversely, lines co-expressing vGPCR or vCYC/vFLIP along with a allografts derived from the injection of EC-vGPCR (10%) + EC- Rapamycin-Resistant mTOR mutant (RR-mTOR) that bears a vCYC/vFLIP/RR-mTOR (90%) cells continued growing even Ser2035RIle (SI) substitution in the FKBP12-Rapamycin-binding upon treatment with Rapamycin, suggesting that the sensitivity to domain (EC-vGPCR/RR-mTOR or EC-vCYC/vFLIP/RR- the drug of these allografts is due to the inhibition of the paracrine mTOR) [19]. Expression of RR-mTOR strongly protected activation of mTOR in neighboring cells by the angiogenic factors vGPCR- and vCYC/vFLIP-expressing cells from the ability of elaborated by vGPCR-expressing cells (Fig. 4). Of interest, tissue Rapamycin to inhibit mTOR activation in vitro (data not shown).
staining with a phospho-S6 ribosomal protein specific antibody We then established mixed-cell allografts injecting athymic nu/nu confirmed the inhibition of mTOR activity in most cells of EC- mice with EC-vGPCR (10%) + EC-vCYC/vFLIP (90%) cells, EC- vGPCR (10%) + EC-vCYC/vFLIP (90%) as well as EC-vGPCR/ vGPCR/RR-mTOR (10%) + EC- vCYC/vFLIP (90%) cells or RR-mTOR (10%) + EC- vCYC/vFLIP (90%) tumors, but not EC-vGPCR (10%) + EC- vCYC/vFLIP/RR-mTOR (90%) cells EC-vGPCR (10%) + EC- vCYC/vFLIP/RR-mTOR (90%) PLoS ONE www.plosone.org April 2011 Volume 6 Issue 4 e19103

VEGF Amplification by vGPCR Activation of HIF Figure 4. Paracrine activation of mTOR is required for vGPCR sarcomagenesis in vivo. Tumor allografts were generated upon injection ofathymic nu/nu mice with EC-vGPCR (10%) + EC-vCYC/vFLIP (90%) cells, EC-vGPCR/RR-mTOR (10%) + EC-vCYC/vFLIP (90%) cells or EC-vGPCR (10%) +EC-vCYC/vFLIP/RR-mTOR (90%) cells. Lesions were then treated with (10 mg/kg) Rapamycin or vehicle. Curves of tumor growth andimmunohistochemical staining of tumor tissue with anti-AU1 or anti-AU5 antibodies, revealing expression of RR-mTOR or vGPCR, respectively, areshown.
doi:10.1371/journal.pone.0019103.g004 allografts, upon Rapamycin treatment (Fig. 5). Similarly, HIF average estimated weight of vehicle-treated tumors was 702 mg (a levels were reduced in treated tumors but remained elevated in 4.8 fold increase) vs. an average estimated weight of 234 mg (a most tumor cells of EC-vGPCR (10%) + EC- vCYC/vFLIP/RR- 1.7 fold increase) of Digoxin-treated tumors (Fig. 6). Immuno- mTOR (90%) allografts even after treatment with Rapamycin histochemical analysis of these lesions demonstrated a dramatic (Fig. 5). Collectively, these findings support an essential role of reduction in the levels of HIF as well as VEGF in the Digoxin- vGPCR paracrine secretions in the mTOR-driven promotion of treated animals compared to control mice (Fig. 6). Taken HIF stabilization and VEGF secretion, and in vGPCR tumori- together, our data provide the basis for the early assessment of HIF inhibitors as an anti-KS therapy.
Inhibition of HIF blocks vGPCR tumorigenesis in vivo Of interest, several drugs have been recently described that target HIF expression or activity; these drugs have demonstrated KS is an angioproliferative tumor characterized by the presence anti-angiogenic and anti-cancer effects in vivo [30]. The of angiogenic and inflammatory mediators [1]. The observation identification of HIF as a key factor in vGPCR angiogenic that KS tumors tend to localize to sites of inflammation suggests amplification prompted us to explore inhibition of HIF as a that these lesions thrive in a cytokine-rich environment [1].
potential therapeutic approach for KS treatment. We therefore Indeed, KS spindle cells do not appear to be truly transformed; established tumor allografts by injecting mixed-cell populations of rather, the KS spindle cell elaborates a variety of cytokines, EC-vGPCR and EC-vCYC/vFLIP cells in athymic nu/nu mice chemokines and growth factors that are essential for their growth (Fig. 6). Animals were then treated with either (2 mg/kg) and survival. Indeed, isolated KS spindle cells remain strictly Digoxin, a cardiac glycoside that has been shown to inhibit dependent on cytokines and growth factors to proliferate in vitro HIF-1a synthesis and block tumor formation, or vehicle (Control) and are not tumorigenic when tested in animal models. Among the (Fig. 6) [31]. Drug toxicity, as assessed by weight loss, was numerous angiogenic mediators on which the KS spindle cell is minimal in the treated group (reduction ,5%) during the dependent, VEGF has been shown to be essential for KS spindle treatment period (results not shown). Inhibition of tumor growth cell survival in vitro and KS pathogenesis in vivo.
by the treatment with Digoxin was sustained for the duration of We previously reported a mechanism whereby the KSHV the experiment. At the end of the study, we observed that the vGPCR promotes the upregulation of VEGF transcription in PLoS ONE www.plosone.org April 2011 Volume 6 Issue 4 e19103

VEGF Amplification by vGPCR Activation of HIF Figure 5. Paracrine activation of mTOR is required for HIF upregulation in vGPCR sarcomagenesis. Immunohistochemical staining withspecific antibodies against phospho-S6 ribosomal protein, HIF-1a and HIF-2a of the allografts generated upon injection of EC-vGPCR (10%) + EC-vCYC/vFLIP (90%), EC-vGPCR/RR-mTOR (10%) + EC-vCYC/vFLIP (90%) or EC-vGPCR (10%) + EC-vCYC/vFLIP/RR-mTOR (90%), treated with vehicle or(10 mg/kg) Rapamycin.
doi:10.1371/journal.pone.0019103.g005 vGPCR-expressing cells [17]. We report here that vGPCR also an explanation for how this unusual viral oncogene can play a role upregulates VEGF through a complex indirect (paracrine) in KS despite the observation that its expression is restricted to mechanism. Upregulation of VEGF in neighboring (non- only a few tumor cells. vGPCR angiogenic amplification involves vGPCR-expressing) tumor cells results in a dramatic amplification the secretion of angiogenic and inflammatory cytokines by of the angiogenic signal promoted by vGPCR and helps provide vGPCR-expressing cells which then activate in neighboring cells Figure 6. Inhibition of HIF blocks vGPCR tumorigenesis in vivo. Tumor allografts were generated upon injection of athymic nu/nu mice withEC-vGPCR (10%) + EC-vCYC/vFLIP (90%) cells. Lesions were treated with (2 mg/kg) Digoxin or vehicle. Tumor growth curves andimmunohistochemical staining of tumor tissue with anti-AU5 (revealing vGPCR-expresing cells), anti-HIF-2a or anti-VEGF antibody, are shown.
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VEGF Amplification by vGPCR Activation of HIF multiple signaling pathways that ultimately converge on TSC1 cytokines that promote the activation of IKKb. Ultimately, further and 2, resulting in de-repression of mTOR and the promotion of investigation into the relative contribution of each of these HIF upregulation and VEGF transcription and secretion (Fig. 7).
cytokines to the angiogenic phenotype in KS as well as in other TSC kinases activated by vGPCR paracrine secretions include tumors may be warranted.
AKT, ERK, p38 and IKKb, inducing phosphorylation of TSC2/ Several additional KSHV genes have also been shown to 1 at specific regulatory sites [22,23,24,25,26,27]. Although upregulate cytokine levels. These viral proteins, including pharmacological inhibition of AKT-mediated phosphorylation of vFLIP, kaposin A, kaposin B, K1 and K15, undoubtedly TSC2 or IKKb-mediated phosphorylation of TSC1 lead to a contribute to the inflammatory milieu observed in KS complete inhibition of mTOR activity, siRNA knock down [33,34,35,36,37,38]. As KSHV infection itself has been expression of AKT, ERK1 and 2, p38, or IKKb was only demonstrated to induce cytokine release [1], the relative sufficient to partially inhibit S6K phosphorylation. This suggests contribution of each of the KSHV genes to cytokine dysregu- either that the PI3K/AKT inhibitor, LY294002, and the IKKb lation and the angiogenic phenotype in KS remains to be inhibitor, BAY 11-7082, have non-specific inhibitory effects, or determined. Moreover, various inflammatory cytokines, includ- that the RNAi knockdown of AKT and IKKb were less efficient ing IL-1, TNF-a and interferon-c (IFN—c), are increased upon than their pharmacological inhibition. Although we suspect the HIV infection, an important cofactor in KS development [39].
former to be true, regulation of mTOR through these pathways Collectively, these findings suggest that cytokine dysregulation has proven to be quite complex and the answer may not prove to and TSC/mTOR/HIF/VEGF activation may be a general be straightforward. Nonetheless, our results collectively suggest mechanism linking KS co-factors, inflammation, and dysregu- that there is a redundancy in these pathways and in the lated angiogenesis in KS.
phosphorylation sites for inhibiting TSC activity and further The mTOR signaling pathway is a key modulator of protein provide insight into the complexity of mTOR regulation by translation and has previously been identified as a positive different exogenous stimuli.
regulator of HIF and HIF-dependent responses [20,30].
A number of vGPCR factors promote the phosphorylation of Oncogenes activating this pathway have been implicated in TSC1/2 through several of these signaling pathways. Surprisingly, tumor-induced angiogenesis in other tumors. vGPCR promo- we demonstrate here that phosphorylation of TSC1 in Ser511 by tion of the paracrine activation of mTOR may play a similar IKKb is activated by most of the cytokines tested (Fig. S1), role in the regulation of HIF in KS. Indeed, upregulation of suggesting that regulation of mTOR by IKKb may be quite HIF activity has been observed upon KSHV infection of promiscuous. Moreover, as IKKb activation leads to the activation endothelial cells in culture and HIF stabilization has been of NFkB, this, in turn, may promote a positive feedback loop, previously reported in AIDS-KS lesions [40,41]. Of note, other further enhancing cytokine – and therefore VEGF – secretion in KSHV genes (e.g. LANA-1 and IRF-3) have also been KS. Of note, paracrine secretions from vGPCR-expressing cells suggested to play a role in upregulating HIF activity promote a gene expression profile with an NFkB signature in [42,43,44]. Given the central role of HIF in VEGF regulation, endothelial cells [32]. In light of our results here, this suggests that and the importance of VEGF in KS, it is certainly reasonable to the NFkB signature may be mediated by the specific secreted argue that KSHV may encode a redundancy of mechanisms to Figure 7. vGPCR cytokines activate VEGF secretion through diverse signaling cascades converging in TSC/mTOR/HIF. Schematicshowing the different signaling pathways by which vGPCR cytokines, chemokines and growth factors converge in the phosphorylation of TSC1/2, theactivation of mTOR and the upregulation of HIF levels, leading to VEGF secretion.
doi:10.1371/journal.pone.0019103.g007 PLoS ONE www.plosone.org April 2011 Volume 6 Issue 4 e19103 VEGF Amplification by vGPCR Activation of HIF ensure that this critical endothelial cell growth factor is available thermocycler from eppendorfs (2 min at 94uC; 30 cycles of 94uC in growing KS tumors.
for 30 seconds, 50uC for 30 seconds, 72uC for 1 minute; and Here, we show that pharmacological inhibition of HIF 5 minutes at 72uC). Primers for human VEGF were: 59-GG- upregulation by vGPCR is sufficient to inhibit vGPCR oncogen- GCAGAATCATCACGAAGT-39 (sense) and 59-TGGTGAT- esis. As the master regulator of the hypoxic vascular response, it GTTGGACTCCTCA-39 (anti-sense). Primers for human HIF- should not be surprising that HIF plays a central role in Kaposi's 1a were: 59-GCAAGCCCTGAAAGCGCAAG-39 (sense) and 59- sarcomagenesis. HIF drives transcriptional activation of hundreds GTGAGGCTGTCCGACTTTGA-39 (anti-sense). For HIF-2a, of genes involved in vascular reactivity, angiogenesis, arteriogen- primers employed were: 59-GTCTCTCCACCCCATGTCTC-39 esis, and the recruitment of endothelial precursor cells, all key steps (sense) and 59-GGTTCTTCATCCGTTTCCAC-39 (anti-sense).
toward the development of KS [45]. Indeed, it is tempting to Amplification of GAPDH sequence was used for normalization.
speculate that recent publications describing KS regression inpatients with iatrogenic KS following a switch in their immuno- Western blots and Immunohistochemistry suppressive treatment to the mTOR inhibitor, Sirolimus, may – in Western blots and immunohistochemistry were performed as part – be due to its effect on decreasing HIF activation [46,47].
previously described [12]. Antibodies recognizing AU1, AU5 and Collectively, our data ultimately provide the basis for the early HA epitopes were purchased from Covance. Antibodies against assessment of drugs inhibiting HIF in those patients with the following proteins were employed: S6K, p-S6K (T389), S6, cutaneous and/or systemic KS.
p-S6 (S240/244), AKT, TSC2, p-TSC2 (T1462), ERK1/2,IKKb, p-IKK-a/b (S180/181), mTOR, p-mTOR (S2448), p38, Materials and Methods p-p38 (T180/Y182), HIF-1a and HIF-1b from Cell Signaling; Expression plasmids and reagents HIF-2a and P-TSC2 (S664) (immunohistochemistry) from NovusBiologicals; TSC1 from Invitrogen; p-TSC1 (S511) from Bethyl The expression plasmid encoding for the rapamycin-resistant Laboratories; LANA-1 from Leica Microsystems; p-AKT (S473) mTOR mutant (RR-mTOR) that bears a Ser2035RIle (SI) from R&D Systems; p-ERK1/2 (T202/Y204) from BD Biosci- substitution in the FKBP12-rapamycin-binding domain has been ences; and Actin from Santa Cruz. For p-TSC2 (S1254), described elsewhere [19]. A tetracycline inducible system (Tet-on) antibodies from Enogene and Cell Signaling were used for was used for vGPCR expression. pCEFL Tet REV TA and pBIG Western blot and immunohistochemistry, respectively. For VEGF, AU5 vGPCR were kindly provided by Dr. Silvio Gutkind(NIDCR, NIH). GRO-a was obtained from R&D Systems; IL-8, antibodies from R&D Systems and Abcam were used for Western VEGF, PDGF, IL-1b, IL-10, IL-6, TNFa, IP-10 and SDF-1a blot and immunohistochemistry, respectively. The antibody were obtained from Peprotech. Rapamycin, LY294002, U0126, recognizing vGPCR was kindly provided by Dr. Gary S. Hayward SB203580 and BAY 117082 were purchased from Calbiochem (Johns Hopkins University, Baltimore, MD).
and Digoxin from Sigma. All siRNA oligos were obtained fromQiagen.
Tumorigenesis assays All procedures involving animals were approved by the Cell lines, transfections and supernatant collection Institutional Animal Care and Use Committee. Murine vGPCR Immortalized human dermal microvascular endothelial cells tumors were obtained as described in ref 12. Briefly, TIE2-tva (HMEC1) were obtained from the Centers of Disease Control transgenic mice expressing in vascular endothelium the avian (Atlanta, GA) and grown in Gibco MCDB 131 medium leukosis virus (ALV) receptor, TVA, were injected i.p. with ALV- (Invitrogen), supplemented with 10% FBS, 10 mM/l L-Gluta- derived retrovirus encoding for KSHV vGPCR. Macroscopic mine, 10 ng/ml epidermal growth factor, 1 mg/ml hydrocortisone vGPCR tumors developed in 4 months predominantly in ear, tail, and 1% antibiotic antimycotic. (SV-40) immortalized murine paw and GI tract. For tumor allograft formation, cells expressing endothelial cells (SVEC) and SVEC-derived stable cell lines vGPCR (EC-vGPCR or EC-vGPCR/RR-mTOR) were mixed expressing KSHV vGPCR or KSHV vCyclin and vFLIP (EC- with cells expressing vCyclin and vFLIP (EC-vCYC/vFLIP or EC- vGPCR, EC-vCYC/vFLIP) were described previously [15]. The vCYC/vFLIP/RR-mTOR), in a (1:10) ratio (105:106 cells). These Rapamycin-Resistant mTOR (RR-mTOR) was transfected along mixed cell populations were then injected into the right flank of 8- with the pTracer-EF/Bsd plasmid (Invitrogen) into EC-vGPCR or wk-old athymic (nu/nu) nude female mice as previously described EC-vCYC/vFLIP. Cells were then stably selected with Blasticidin [15]. For these in vivo studies, Digoxin stock solution (Sigma- (Invitrogen). siRNA delivery into cultured cells was performed Aldrich) was dissolved in DMSO and Rapamycin (LC Laborato- using Hiperfect (Qiagen). For conditioned media preparation, ries) was dissolved as previously described [19]. Drug treatment HMEC1 were transfected with Tet REV TA and pBIG AU5 was initiated when estimated tumor weight reached around 150– vGPCR, serum starved and treated with doxycycline (Dox; 1 200 mg. For this procedure, tumor-bearing animals were mg/ml). 24 h later, supernatants from untreated (Control CM) and randomly grouped (control, n = 5; drug-treated group, n = 5) and Dox treated (vGPCR CM) cells were collected, centrifuged at treated with Rapamycin (10 mg/kg) or Digoxin (2 mg/kg) or an 1000 g for 10 min, and concentrated 10 times with centrifugal equal volume of vehicle. For Rapamycin, treatment schedule was a single injection per animal given i.p. for 5 consecutive days [19].
For Digoxin, treatment schedule was a single injection per animal Reverse transcriptase PCR given i.p. for 10 consecutive days [31]. The animals were Total RNA was isolated using the GenElute Mammalian Total monitored twice weekly for tumor formation. The longest length RNA Miniprep kit (Sigma-Aldrich) according to manufacturer's (L) and shortest width (W) of the tumor were measured using a protocol and reverse transcription was performed using Super- caliper at different time points throughout the experiment. Tumor Script III First-Strand Synthesis System (Invitrogen). Upon volume was then converted into tumor weight using the formula extraction of mRNA, cDNA was obtained using the SuperScript LW2/2, as described previously [19]. Results of animal experi- III First-Strand Synthesis System from Invitrogen. Subsequently, ments were expressed as mean estimated tumor weight 6 SD.
the PCR reaction was carried out using the Mastercycler When appropriate, animals were euthanized, and tissue was fixed PLoS ONE www.plosone.org April 2011 Volume 6 Issue 4 e19103 VEGF Amplification by vGPCR Activation of HIF in 4% paraformaldehyde and embedded in paraffin for further S6K and S6 upon treatment of HMEC1 with different vGPCR (recombinant) proteins: IL-8, GROa, VEGF, PDGF, IL-1b, IL-10, IL-6, TNFa, IP-10, or SDF1a. Cells were pretreated with (50 nM) Rapamycin, where corresponding. (B) Phosphorylation of Four patients diagnosed with KS (multiple lesions) at University TSC2/1 (TSC2T1462, TSC2S664, TSC2S1254 and TSC1S511) or of Maryland Dental School or School of Medicine, between 1990 S6K upon treatment of HMEC1 with different recombinant and 2010, were identified. Formalin-fixed paraffin-embedded cytokines, chemokines, and growth factors, as described in (A). (C) tissue samples were obtained from the pathology archives for Analysis of the levels of phosphorylation of TSC2/1 (TSC2T1462, immunohistochemical studies. Representative hematoxylin and TSC2S664, TSC2S1254 and TSC1S511) shown in (B).
eosin sections of each tumor were reviewed and the diagnosis was confirmed by immunohistochemistry using a specific antibodyrecognizing (LANA-1). The Institutional Review Board (IRB) protocol wasexempt.
We thank Dr. Gary S. Hayward for kindly providing the specific antibodyrecognizing KSHV vGPCR. BCJ is a recipient of a predoctoral fellowshipfrom the CNPq-Brazil. We thank Xin Wang for her help with the Statistical Analysis In all cases, results are shown as a mean value 6 SD from at least three independent experiments. Western blot scans are Author Contributions representative of at least three independent experiments. Statisticalanalysis was performed with Prism 4.2 software (GraphPad).
Conceived and designed the experiments: A. Schneider A. Sodhi SM.
ANOVA test: ***, p,0.001; **, p,0.01; *, p,0.05.
Performed the experiments: BCJ TM JH RC ERF. Analyzed the data: BCJTM JH RC ERF SM. Contributed reagents/materials/analysis tools: PPP.
Wrote the paper: SM.
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