KSHV-induced Angiogenesis Research Articles

An oncogenic viral interferon regulatory factor upregulates CUB domain-containing protein 1 to promote angiogenesis by hijacking transcription factor lymphoid enhancer-binding factor 1 and metastasis suppressor CD82.

Kaposi's sarcoma (KS), a highly angiogenic and invasive vascular tumor, is the most common AIDS-associated cancer caused by KS-associated herpesvirus (KSHV) infection. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) contributes to KSHV-induced cell motility (PLoS Pathog. 15:e1007578, 2019). However, the role of vIRF1 in KSHV-induced angiogenesis remains unknown. Here, using two in vivo angiogenesis models including the chick chorioallantoic membrane assay (CAM) and the matrigel plug angiogenesis assay in mice, we show that vIRF1 promotes angiogenesis by upregulating CUB domain (for complement C1r/C1s, Uegf, Bmp1) containing protein 1 (CDCP1). Mechanistically, vIRF1 enhances the expression of transcription factor lymphoid enhancer-binding factor 1 (Lef1) and binds to Lef1 to promote CDCP1 transcription. Meanwhile, vIRF1 degrades metastasis suppressor CD82 through an ubiquitin-proteasome pathway by recruiting E3 ubiquitin ligase AMFR to CD82, which protects CDCP1 from CD82-mediated, palmitoylation-dependent degradation. CDCP1 activates AKT signaling, which is required for vIRF1-induced cell motility but not angiogenesis. Our results illustrate that, by hijacking Lef1 and CD82, vIRF1 upregulates CDCP1 to promote angiogenesis and cell invasion. These novel findings demonstrate the vIRF1 targets multiple cellular proteins and pathways to promote the pathogenesis of KS, which could be attractive therapeutic targets for KSHV-induced malignancies.

An APE1 inhibitor reveals critical roles of the redox function of APE1 in KSHV replication and pathogenic phenotypes.

APE1 is a multifunctional protein with a DNA base excision repair function in its C-terminal domain and a redox activity in its N-terminal domain. The redox function of APE1 converts certain transcription factors from inactive oxidized to active reduced forms. Given that among the APE1-regulated transcription factors many are critical for KSHV replication and pathogenesis, we investigated whether inhibition of APE1 redox function blocks KSHV replication and Kaposi’s sarcoma (KS) phenotypes. With an shRNA-mediated silencing approach and a known APE-1 redox inhibitor, we demonstrated that APE1 redox function is indeed required for KSHV replication as well as KSHV-induced angiogenesis, validating APE1 as a therapeutic target for KSHV-associated diseases. A ligand-based virtual screening yielded a small molecular compound, C10, which is proven to bind to APE1. C10 exhibits low cytotoxicity but efficiently inhibits KSHV lytic replication (EC50 of 0.16 μM and selective index of 165) and KSHV-mediated pathogenic phenotypes including cytokine production, angiogenesis and cell invasion, demonstrating its potential to become an effective drug for treatment of KS.

A KSHV microRNA enhances viral latency and induces angiogenesis by targeting GRK2 to activate the CXCR2/AKT pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Most tumor cells in these malignancies are latently infected by KSHV. Thus, viral latency is critical for the development of tumor and induction of tumor-associated angiogenesis. KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced angiogenesis remains unknown. We have recently shown that miR-K12-3 (miR-K3) promoted cell migration and invasion by targeting GRK2/CXCR2/AKT signaling (PLoS Pathog, 2015;11(9):e1005171). Here, we further demonstrated a role of miR-K3 and its induced signal pathway in KSHV latency and KSHV-induced angiogenesis. We found that overexpression of miR-K3 not only promoted viral latency by inhibiting viral lytic replication, but also induced angiogenesis. Further, knockdown of GRK2 inhibited KSHV replication and enhanced KSHV-induced angiogenesis by enhancing the CXCR2/AKT signals. As a result, blockage of CXCR2 or AKT increased KSHV replication and decreased angiogenesis induced by PEL cells in vivo. Finally, deletion of miR-K3 from viral genome reduced KSHV-induced angiogenesis and increased KSHV replication. These findings indicate that the miR-K3/GRK2/CXCR2/AKT axis plays an essential role in KSHV-induced angiogenesis and promotes KSHV latency, and thus may be a potential therapeutic target of KSHV-associated malignancies.

Inhibiting the Recruitment of PLCγ1 to Kaposi's Sarcoma Herpesvirus K15 Protein Reduces the Invasiveness and Angiogenesis of Infected Endothelial Cells.

Kaposi’s sarcoma (KS), caused by Kaposi’s sarcoma herpesvirus (KSHV), is a highly vascularised tumour of endothelial origin. KSHV infected endothelial cells show increased invasiveness and angiogenesis. Here, we report that the KSHV K15 protein, which we showed previously to contribute to KSHV-induced angiogenesis, is also involved in KSHV-mediated invasiveness in a PLCγ1-dependent manner. We identified βPIX, GIT1 and cdc42, downstream effectors of PLCγ1 in cell migration, as K15 interacting partners and as contributors to KSHV-triggered invasiveness. We mapped the interaction between PLCγ1, PLCγ2 and their individual domains with two K15 alleles, P and M. We found that the PLCγ2 cSH2 domain, by binding to K15P, can be used as dominant negative inhibitor of the K15P-PLCγ1 interaction, K15P-dependent PLCγ1 phosphorylation, NFAT-dependent promoter activation and the increased invasiveness and angiogenic properties of KSHV infected endothelial cells. We increased the binding of the PLCγ2 cSH2 domain for K15P by substituting two amino acids, thereby creating an improved dominant negative inhibitor of the K15P-dependent PLCγ1 activation. Taken together, these results demonstrate a necessary role of K15 in the increased invasiveness and angiogenesis of KSHV infected endothelial cells and suggest the K15-PLCγ1 interaction as a possible new target for inhibiting the angiogenic and invasive properties of KSHV.

Activation of NF-κB by the Kaposi's sarcoma-associated herpesvirus K15 protein involves recruitment of the NF-κB-inducing kinase, IκB kinases, and phosphorylation of p65.

Kaposi's sarcoma herpesvirus (KSHV) (or human herpesvirus 8) is the cause of Kaposi's sarcoma, primary effusion lymphoma (PEL), and the plasma cell variant of multicentric Castleman's disease (MCD). The transmembrane K15 protein, encoded by KSHV, has been shown to activate NF-κB and the mitogen-activated protein kinases (MAPKs) c-jun-N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk) as well as phospholipase C gamma (PLCγ) and to contribute to KSHV-induced angiogenesis. Here we investigate how the K15 protein activates the NF-κB pathway. We show that activation of NF-κB involves the recruitment of NF-κB-inducing kinase (NIK) and IKK α/β to result in the phosphorylation of p65/RelA on Ser536. A K15 mutant devoid in NIK/IKK recruitment fails to activate NF-κB but remains proficient in the stimulation of both NFAT- and AP1-dependent promoters, showing that the structural integrity of the mutant K15 protein has not been altered dramatically. Direct recruitment of NIK represents a novel way for a viral protein to activate and manipulate the NF-κB pathway. KSHV K15 is a viral protein involved in the activation of proinflammatory and angiogenic pathways. Previous studies reported that K15 can activate the NF-κB pathway. Here we show the molecular mechanism underlying the activation of this signaling pathway by K15, which involves direct recruitment of the NF-κB-inducing kinase NIK to K15 as well as NIK-mediated NF-κB p65 phosphorylation on Ser536. K15 is the first viral protein shown to activate NF-κB through direct recruitment of NIK. These results indicate a new mechanism whereby a viral protein can manipulate the NF-κB pathway.

Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

Kaposi's Sarcoma (KS), caused by Kaposi's Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.

Cancer Angiogenesis Induced by Kaposi Sarcoma–Associated Herpesvirus Is Mediated by EZH2

EZH2 is a component of the epigenetic regulator PRC2 that suppresses gene expression. Elevated expression of EZH2 is common in human cancers and is associated with tumor progression and poor prognosis. In this study, we show that EZH2 elevation is associated with epigenetic modifications of Kaposi sarcoma-associated herpesvirus (KSHV), an oncogenic virus that promotes the development of Kaposi sarcoma and other malignancies that occur in patients with chronic HIV infections. KSHV induction of EZH2 expression was essential for KSHV-induced angiogenesis. High expression of EZH2 was observed in Kaposi sarcoma tumors. In cell culture, latent KSHV infection upregulated the expression of EZH2 in human endothelial cells through the expression of vFLIP and LANA, two KSHV-latent genes that activate the NF-κB pathway. KSHV-mediated upregulation of EZH2 was required for the induction of Ephrin-B2, an essential proangiogenic factor that drives endothelial cell tubule formation. Taken together, our findings indicate that KSHV regulates the host epigenetic modifier EZH2 to promote angiogenesis.

Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is associated with the angioproliferative KS lesions characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth factors, and angiogenic factors. De novo KSHV infection of human microvascular dermal endothelial cells results in increased secretion of several growth factors, cytokines, chemokines, and angiogenic factors, and the multifunctional angiogenic protein angiogenin is one of them. KS tissue sections were positive for angiogenin, highlighting the importance of angiogenin in KS pathogenesis. Examination of KSHV-mediated angiogenin upregulation and secretion and potential outcomes revealed that during infection of primary endothelial cells, KSHV induced a time- and dose-dependent increase in angiogenin gene expression and protein secretion beginning as early as 8 h postinfection and lasting until the fifth day of our observation period. TIVE latently transformed cells (TIVE-LTC) latently infected with KSHV secreted high levels of angiogenin. Angiogenin was also detected in BCBL-1 cells (human B cells) carrying KSHV in a latent state. Significant induction of angiogenin was observed in cells expressing KSHV ORF73 (LANA-1; latent) and ORF74 (lytic) genes alone, and moderate induction was seen with the lytic KSHV ORF50 gene. Angiogenin bound to surface actin, internalized in a microtubule-independent manner, and translocated into the nucleus and nucleolus of infected cells. In addition, it increased 45S rRNA gene transcription, antiapoptosis, and proliferation of infected cells, thus demonstrating the multifunctional nature of KSHV-induced angiogenin. These activities were dependent on angiogenin nuclear translocation, which was inhibited by neomycin. Upregulation of angiogenin led to increased activation of urokinase plasminogen activator and generation of active plasmin, which facilitated the migration of endothelial cells toward chemoattractants, including angiogenin, and chemotaxis was prevented by the inhibition of angiogenin nuclear translocation. Treatment of KSHV-infected cell supernatants with antiangiogenin antibodies significantly inhibited endothelial tube formation, and inhibition of nuclear translocation of angiogenin also blocked the expression of KSHV-induced vascular endothelial growth factor C. Collectively, these results strongly suggest that by increasing infected endothelial cell 45S rRNA synthesis, proliferation, migration, and angiogenesis, KSHV-induced angiogenin could be playing a pivotal role in the pathogenesis of KSHV infection, including a contribution to the angioproliferative nature of KS lesions. Our studies suggested that LANA-1 and vGPCR play roles in KSHV-induced angiogenesis and that the angiogenic potential of vGPCR might also be due to its ability to induce angiogenin.

Angiogenesis, Kaposi’s sarcoma and Kaposi’s sarcoma-associated herpesvirus

Tumor angiogenesis is the uncontrolled growth of blood vessels in tumors, serving to supply nutrients and oxygen, and remove metabolic wastes. Kaposi's sarcoma (KS), a multifocal angioproliferative disorder characterized by spindle cell proliferation, neo-angiogenesis, inflammation, and edema, is associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV). Recent studies indicate that KSHV infection directly promotes angiogenesis and inflammation through an autocrine and paracrine mechanism by inducing pro-angiogenic and pro-inflammatory cytokines. Many of these cytokines are also expressed in KS lesions, implicating a direct role of KSHV in the pathogenesis of this malignancy. Several KSHV genes are involved in KSHV-induced angiogenesis. These studies have provided insights into the pathogenesis of KS, and identified potential therapeutic targets for this malignancy.

RBP-J (CSL) is essential for activation of the K14/vGPCR promoter of Kaposi's sarcoma-associated herpesvirus by the lytic switch protein RTA.

The Kaposi's sarcoma-associated herpesvirus (KSHV) gene product virally encoded G protein-coupled receptor (vGPCR) is a homolog of cellular GPCRs and has been proposed to play important roles in KSHV-induced angiogenesis. The most abundant vGPCR-containing transcripts are K14/vGPCR bicistronic RNAs that are strongly induced during lytic reactivation. Here we show that the promoter governing this transcript is strongly responsive to activation by the viral lytic switch protein RTA. By deletion mapping and scanning mutation analyses, we have identified three putative RTA response elements (A, B, and C) in this promoter. However, none of these sites appear to directly bind RTA in electrophoretic mobility shift assays (EMSA). Site C corresponds to a canonical binding site for RBP-J, a sequence-specific transcriptional repressor that is normally the target of Notch signaling. RBP-J can bind RTA and recruit it to its cognate recognition site; when this happens, the activation function of RTA can relieve RBP-J-mediated repression and upregulate expression of the targeted gene. EMSA studies reveal that both sites A and C can bind to RBP-J; sequence inspection reveals that site A is a novel functional variant of known RBP-J recognition sites. (Site B corresponds to an as-yet-unknown host DNA-binding protein.) The importance of sites A and C in vivo is underscored by the observation that K14/vGPCR promoter function is dramatically inhibited in cells genetically deficient in RBP-J. The regulation of K14/vGPCR transcripts by RBP-J raises the possibility that other modulators of Notch signaling might be able to induce expression of this RNA outside the context of lytic KSHV replication.