Molecular mechanisms, therapeutic targets, and clinically tested drugs in AIDS-KS paracrine viral oncogenesis.

<p>In KS spindle cells lytically infected with KSHV cells or latently infected spindle cells expressing early lytic genes, KSHV genes such vGPCR, K1, and ORF45 constitutively trigger signaling cascades, leading to mTOR and ROS activation, which induce transcription and translation of PDGF and VEGF. These secreted growth factors can act in a paracrine manner to activate the same signaling cascades in latently infected cells expressing VEGF and PDGF receptors to drive KS cell proliferation and angiogenesis. Rapamycin (RAPA), which inhibits mTOR, and Imatinib (IMA), which inhibits PDGFR, can interrupt this paracrine loop to target KSHV tumorigenesis. Both drugs have shown efficacy in AIDS-KS clinical trials.</p

KSHV pathobiology in healthy and HIV/AIDS patients.

<p>(A) In a healthy host, KSHV infection of a KS progenitor is not oncogenic, since it leads to latent infection or to cytopathic lytic replication. Reactivation leading to oncogenic lytic gene expression is under immunological control. (B) Scenario for AIDS-KS pathogenesis according to the Paracrine Oncogenesis and Abortive Lytic Hypotheses. In HIV/AIDS, decreased immunosurveilance, inflammatory cytokines and HIV Tat lead to KSHV reactivation and reinfection. This leads to increased, uncontrolled, early lytic oncogenic gene expression, with concomitant risk of cell transformation by somatic host cell oncogenic alterations. Upon transformation, cells shut down early lytic oncogenes. Latently infected transformed cells are stimulated in a paracrine manner by angiogenic and proliferative factors released from lytically infected or abortive lytic cells (paracrine oncogenesis, see details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004154#ppat-1004154-g002" target="_blank">Figure 2</a>). In addition, lytically infected cells provide a constant source of virions for reinfection, while angiogenesis and inflammation recruit target KS progenitors. ART inhibits KSHV reactivation and lytic replication through immune reconstitution and decreased levels of HIV viral loads. Gancyclovir inhibits viral replication and lytic gene expression.</p

rKSHV.219 lytic replication <i>in vivo</i> results in productively infected tumors culminating in the formation of virus-like particles.

<p>(A) rKSHV.219 lytic gene expression increases <i>in vivo</i> relative to mECK^null.rK133 cells in culture during tumorigenesis. RNA was isolated from tumors and cells in culture for analysis of rKSHV.219 gene expression by qRT-PCR. A representative comparative analysis is shown; error bars represent the SD of experimental duplicates. (B) RT-PCR analysis of rKSHV.219 transcripts was performed as a preliminary confirmation that the virus was able to express genes representative of the entire viral replicative cycle. RNA was isolated from tumors, reverse transcribed and run on a 3% agarose gel. Gene expression <i>in vivo</i> reveals the presence of transcripts that span the entire KSHV genome and replicative potential. Reverse transcriptase negative and non-template controls were run to confirm the absence of contamination. (C) Transmission electron microscopy (TEM) analysis of tumors: Tumors were excised and fixed in gluteraldehyde. TEM revealed the presence of herpesvirus-like particles (100 nm–200 nm) <i>in vivo</i>.</p

Replacing BACK36 in mECK36 cells for rKSHV.219 generates the tumorigenic cells, mECK^null.rK.

<p>(A) Graph depicting mECK36 tumor kinetics in athymic nu/nu mice. 3×10⁶ mECK36 cells were subcutaneously injected into the hind flanks of 3 athymic nu/nu mice. Concurrently, 3×10⁶ mECK^null cells were subcutaneously injected into another group of 10 athymic nu/nu mice. Within 4–6 weeks solid mECK36 tumors were palpable and growth was monitored by caliper measurement (open squares). Error bars represent the standard deviation between 3 different tumors. mECK^null cells did not form tumors (black dots). (B) Fluorescence microscopy of mECK^null.rK. <i>In vitro</i>, mECK^null.rK express GFP constitutively, indicating rKSHV.219 infection, and maintain tight latency as determined by the absence of RFP expression. (C) Cell cultures of mECK^null.rK were prepared for immunofluorescence for the KSHV LANA which exhibited the classic punctate nuclear pattern of the protein.</p

mECK^null. rK133 cells consistently form KS-like tumors in immunocompromised mice.

<p>(A) Graph depicting tumor kinetics in athymic nu/nu mice. 3×10⁶ mECK^null. rK133 cells were subcutaneously injected into the hind flanks of 5 athymic nu/nu mice. Concurrently, 3×10⁶ mECK^null cells were subcutaneously injected into another group of 10 athymic nu/nu mice. Within 4–6 weeks solid mECK^null. rK133 tumors were palpable and growth was monitored by caliper measurements. mECK^null cells did not form tumors (red line, x-axis). (B) Dissection site showing a GFP+ tumor indicating the presence of rKSHV.219. The subcutaneous tumor was visualized under UV. (C) Tumor morphology was analyzed by H&E staining of paraffin embedded sections. Pathologically, they are composed of spindle cells arranged in bundles with RBCs in slit-like vasculature (top panels, black arrows). Frozen sections were prepared for immunofluorescence for KSHV LANA which showed that the spindle cells of the tumor express the viral latent nuclear antigen, LANA (bottom panels). (D) Immunofluorescence analysis of angiogenic protein expression <i>in vivo</i> reveals that the tumor cells express VEGF-R2 and podoplanin. A representative tumor is shown.</p

CD133/Prominin-1 enrichment of the mECKnull.rK results in a lytically-inducible population that harbors KSHV as an episome.

<p>(A) mECK^null.rK were harvested from cell culture and CD133-expressing cells were positively selected using immunomagnetic beads. The histogram shows the parental population (green), the CD133 enriched (mECK^null.rK133) (blue) and the CD133-depleted populations (red) were analyzed by flow cytometry to confirm CD133 enrichment. A representative histogram is shown. (B) Graph depicting the percentage of CD133 expression in the mECK^null.rK, prior to CD133-enrichment, the percentage of CD133 in the enriched population of mECK^null.rK133 immediately post-enrichment, and the mECK^null.rK133 one month after CD133 enrichment. Error bars represent standard deviation between triplicate wells. (C) <i>In vitro</i>, mECK^null.rK133 express GFP, indicating rKSHV.219 infection, and maintain tight latency as determined by the absence of RFP expression (top panels). When treated with TSA lytic replication is induced as determined by the expression of RFP (bottom panels). (D) RFP induction after lytic reactivation is concurrent with the upregulation of KSHV lytic gene expression as determined by qRT-PCR for RTA, K8, vIRF-1 and K8.1. Error bars represent the SD of duplicate wells. Data are representative of three independent experiments. (E) mECK^null.rK133 cells are episomally-infected with KSHV. mECK^null.rK133 cells were serially passaged in the absence of hygromycin and GFP was measured by flow cytometry. While almost 100% of cells are GFP positive at day 0, by day 70, they are 100% GFP negative, suggesting that the virus exists as an episome in the murine cells.</p

Newly isolated murine bone marrow-derived cells infected with rKSHV.219 generate productively infected tumors in nude mice.

<p>(A) Graph depicting growth kinetics in athymic nu/nu mice. 3×10⁶ mECrK cells were subcutaneously injected into the hind flanks of 4 athymic nu/nu mice. Within 4–6 weeks solid tumors were palpable and growth was monitored by caliper measurements. (B) mECrK tumor cells express CD31. Cells from a dissociated tumor were immunostained for CD31, an angiogenic marker and analyzed by flow cytometry for GFP/CD31 co-expression. About half of the CD31+ cells are also rKSHV.219 infected as determined by GFP expression. The vast majority of rKSHV.219 infected cells also expressed CD31. (C) Tumors were excised and fixed in gluteraldehyde for TEM imaging. TEM revealed the presence of herpesvirus-like particles <i>in vivo</i>. Bar = 300 nm.</p

Microarray analysis rKSHV.219-infected murine cells confirms that the primary bone marrow-derived cells and mECK^null. rK133, are closely related cells of the endothelial lineage.

<p>Dendrogram and heatmap of microarray data comparing the host transcriptome of mECK^null. rK133 cells (denoted as mEC36nullrK.219 in the figure) and mECrK.219 (denoted as mECrK.219 in the figure) to other murine cells of various lineages: ESC, embryonic stem cells; BM, total bone marrow; FLE, fetal liver erythroblasts; EC_NCadh, N-cadherin expressing endothelial cells; EC_VE+NCadh, VE+N-cadherin expressing endothelial cells; EC_VECadh, VE-cadherin expressing endothelial cells; BM_MSC, bone marrow derived mesenchymal stem cells; AEC, aortic endothelial cells; mECrK.219, primary bone marrow-derived mEC infected with rKSHV.219; mECK^null.rK133, mECK^null cells infected with rKSHV.219 and then enriched for Prominin-1; BM_Macro, bone marrow-derived macrophages; Macro, peripheral macrophages; HSC, hematopoietic stem cells; LSC, leukemia stem cells; GEC, glomerular endothelial cells; SubQ, subcutaneous tissue. Similar to the mECK36 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087324#pone.0087324-Cancian1" target="_blank">[9]</a>, the mECK^null.rK133 are of the endothelial lineage and cluster with murine aortic endothelial cells.</p

rKSHV.219 infected cells and viral DNA can be detected throughout the murine host.

<p>(A) Graph depicts viral DNA copy number per 500 ng total DNA in 10×10⁶ cells purified from bone marrow and in 500 µl whole blood of tumor bearing mice. (B) rKSHV.219 infected cells were detected in murine lymph nodes. Cells from murine lymph nodes were dissociated into single cell suspensions and plated in chamber slides and fixed for fluorescence microscopy for GFP expression. The top panels depict IgG control antibody and native GFP expression, which is quite dim. GFP expression was enhanced with an antibody directed against GFP in the middle and bottom panels. (C) rKSHV.219 infected cells are present in murine spleen. Spleen from a tumor bearing mouse was excised, dissociated with collagenase IV and cultured in puromycin containing selective medium. GFP expressing LANA positive cells grew from the splenic cell culture.</p

Image_1_A major role for Nrf2 transcription factors in cell transformation by KSHV encoded oncogenes.tif

Kaposi’s sarcoma (KS) is the most common tumor in AIDS patients. The highly vascularized patient’s skin lesions are composed of cells derived from the endothelial tissue transformed by the KSHV virus. Heme oxygenase-1 (HO-1) is an enzyme upregulated by the Kaposi´s sarcoma-associated herpesvirus (KSHV) and highly expressed in human Kaposi Sarcoma (KS) lesions. The oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR) is expressed by the viral genome in infected cells. It is involved in KS development, HO-1 expression, and vascular endothelial growth factor (VEGF) expression. vGPCR induces HO-1 expression and HO-1 dependent transformation through the Ga13 subunit of heterotrimeric G proteins and the small GTPase RhoA. We have found several lines of evidence supporting a role for Nrf2 transcription factors and family members in the vGPCR-Ga13-RhoA signaling pathway that converges on the HO-1 gene promoter. Our current information assigns a major role to ERK1/2MAPK pathways as intermediates in signaling from vGPCR to Nrf2, influencing Nrf2 translocation to the cell nucleus, Nrf2 transactivation activity, and consequently HO-1 expression. Experiments in nude mice show that the tumorigenic effect of vGPCR is dependent on Nrf2. In the context of a complete KSHV genome, we show that the lack of vGPCR increased cytoplasmic localization of Nrf2 correlated with a downregulation of HO-1 expression. Moreover, we also found an increase in phospho-Nrf2 nuclear localization in mouse KS-like KSHV (positive) tumors compared to KSHV (negative) mouse KS-like tumors. Our data highlights the fundamental role of Nrf2 linking vGPCR signaling to the HO-1 promoter, acting upon not only HO-1 gene expression regulation but also in the tumorigenesis induced by vGPCR. Overall, these data pinpoint this transcription factor or its associated proteins as putative pharmacological or therapeutic targets in KS.</p