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KSHV-induced ligand mediated activation of PDGF receptor-alpha drives Kaposi's sarcomagenesis.

ABSTRACT: Kaposi's sarcoma (KS) herpesvirus (KSHV) causes KS, an angiogenic AIDS-associated spindle-cell neoplasm, by activating host oncogenic signaling cascades through autocrine and paracrine mechanisms. Tyrosine kinase receptor (RTK) proteomic arrays, identified PDGF receptor-alpha (PDGFRA) as the predominantly-activated RTK in KSHV-induced mouse KS-tumors. We show that: 1) KSHV lytic replication and the vGPCR can activate PDGFRA through upregulation of its ligands PDGFA/B, which increase c-myc, VEGF and KSHV gene expression in infected cells 2) KSHV infected spindle cells of most AIDS-KS lesions display robust phospho-PDGFRA staining 3) blocking PDGFRA-signaling with N-acetyl-cysteine, RTK-inhibitors Imatinib and Sunitinib, or dominant-negative PDGFRA inhibits tumorigenesis 4) PDGFRA D842V activating-mutation confers resistance to Imatinib in mouse-KS tumorigenesis. Our data show that KSHV usurps sarcomagenic PDGFRA signaling to drive KS. This and the fact that PDGFRA drives non-viral sarcomas highlights the importance for KSHV-induced ligand-mediated activation of PDGFRA in KS sarcomagenesis and shows that this oncogenic axis could be successfully blocked to impede KS tumor growth.

SUBMITTER: Cavallin LE

PROVIDER: S-EPMC6053240 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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KSHV-induced ligand mediated activation of PDGF receptor-alpha drives Kaposi's sarcomagenesis.

Cavallin Lucas E LE Ma Qi Q Naipauer Julian J Naipauer Julian J Gupta Sachin S Kurian Mani M Locatelli Paola P Romanelli Paolo P Nadji Mehrdad M Goldschmidt-Clermont Pascal J PJ Mesri Enrique A EA

PLoS pathogens 20180709 7

Kaposi's sarcoma (KS) herpesvirus (KSHV) causes KS, an angiogenic AIDS-associated spindle-cell neoplasm, by activating host oncogenic signaling cascades through autocrine and paracrine mechanisms. Tyrosine kinase receptor (RTK) proteomic arrays, identified PDGF receptor-alpha (PDGFRA) as the predominantly-activated RTK in KSHV-induced mouse KS-tumors. We show that: 1) KSHV lytic replication and the vGPCR can activate PDGFRA through upregulation of its ligands PDGFA/B, which increase c-myc, VEGF ...[more]

PMID: 29985958

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Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT/enhancer binding protein alpha (C/EBPalpha) and p21(CIP-1) expression, resulting in G(0)/G(1) cell cycle arrest during the lytic cycle. RAP and C/EBPalpha are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C/EBPalpha; this analysis showed that one of these sites has a very high affinity for C/EBPalpha. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPalpha can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C/EBPalpha together resulted in a threefold increase in activity over that obtained with C/EBPalpha alone. Importantly, the introduction of exogenous Flag-tagged C/EBPalpha triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C/EBPalpha and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C/EBPalpha and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPalpha, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPalpha or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPalpha-mediated transactivation of the RAP promoter. However, the C/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C/EBPalpha and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C/EBPalpha in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C/EBPalpha was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C/EBPalpha and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPalpha occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C/EBPalpha and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPalpha. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPalpha but that full activity requires more than just the core C/EBP binding site.

Dataset Information

KSHV-induced ligand mediated activation of PDGF receptor-alpha drives Kaposi's sarcomagenesis.

Publications

KSHV-induced ligand mediated activation of PDGF receptor-alpha drives Kaposi's sarcomagenesis.

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