• Activation of signal transduction pathways by HHV-8 chemokine receptor homologue ORF74: Evidence for a paracrine mechanism of Kaposi's sarcoma pathogenesis

      Pati, Sibani; Reitz, Marvin (2001)
      Infection with Human Herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), is necessary for the development of KS. The HHV-8 lytic phase gene ORF74 is related to G protein-coupled receptors, particularly CXCR2. ORF74 has been shown to activate inositol phosphate/phospholipase C and the downstream MAP kinases. We show here that ORF74 activates the transcription factor NF-kappaB, independent of ligand, when expressed in KS-derived HHV-8 negative endothelial cells, primary vascular endothelial cells, or T-lymphoid cells. We found that activation of NF-kappaB by ORF74 occurs primarily through the PI-3 Kinase/Akt pathway. ORF74 also results in the activation of a number of factors downstream of Akt, other than NF-kappaB, which are relevant to KS pathogenesis. Activation of NF-kappaB and the Akt pathway by ORF74 was enhanced by the addition of HIV-1 Tat protein, suggesting a collaborative role between the two in the promotion of AIDS KS. Mutation of Val to Asp in the ORF74 second cytoplasmic loop did not affect ligand independent signaling activity, but greatly increased the response to GROalpha. In KS cells, endothelial cells, and T-lymphoid cells, ORF74 upregulated the expression of NF-kappaB-dependent inflammatory cytokines and adhesion molecules. KS cells and T cells expressing ORF74 showed increased levels of adhesion to one another. Supernatants from transfected KS cells activated NF-kappaB signaling in untransfected cells and elicited the chemotaxis of monocytoid and T lymphoid cells. Expression of ORF74 conferred a morphology on primary endothelial cells that was strikingly similar to that of spindle cells present in KS lesions. Taken together, these data, demonstrating that ORF74 activates signaling pathways and induces the expression of pro-angiogenic and pro-inflammatory factors, suggest that expression of ORF74 in a minority of cells in KS lesions could influence uninfected cells or latently infected cells via autocrine and paracrine mechanisms, thereby contributing to KS pathogenesis.
    • Human herpesvirus K1 open reading frame activates NFkappaB and contributes to the inflammatory phenotype

      Bose, Debashish; Reitz, Marvin (2002)
      Human herpesvirus 8 (HHV8) has been epidemiologically linked to the development of Kaposi's sarcoma (KS) and primary effusion lymphoma in individuals infected with human immunodeficiency virus. The HHV8 genome contains a large number of open reading frames that may encode viral genes that play a role in the disease process. A unique open reading frame designated K1 has been demonstrated to encode a transmembrane protein involved in signal transduction that may be a viral oncogene. In order to assess the activities of the K1 open reading frame, we cloned K1 and developed cellular expression systems and a transgenic mouse model. K1-specific RNA was detected in RNA from BC-3 HHV8-infected primary effusion lymphoma cells upon treatment with phorbol ester, indicating that K1 is a mainly expressed during the lytic cycle of the virus. K1-specific RNA was detected in RNA extracted from KS tumor tissue by reverse transcriptase polymerase chain reaction. We found that expression of K1 in NIH3T3 cells conferred upon them anchorage-independence, as indicated by growth in methylcellulose, and the ability to form tumors in nude mice, while control cells demonstrated neither characteristic of transformed cells. We found that expression of K1 in ECV304 endothelial cells resulted in the loss of contact inhibition. The predicted amino acid sequence of K1, derived from the nucleotide sequence of K1, suggested that several domains involved in signal transduction pathways were present, particularly in the spleen tyrosine kinase (Syk)/nuclear factor of activated T cells (NFAT) pathway, and the nuclear factor kappaB (NFkappaB) pathway. We used reporter and electrophoretic mobility shift assays to further characterize the way in which K1 may activate NFkappaB. Our results suggest that K1 engages tumor necrosis factor receptor activated factors (TRAFs) to drive NFkappaB activation by enlisting the inhibitor of kappaB kinase complex (IKK) to phosphorylate inhibitor of kappaB (IkappaBalpha) and allow NFkappaB to translocate to the nucleus. Our final results show that K1 induces an inflammatory phenotype that would be expected of an activator of NFkappaB. We conclude that K1 has a role to play in disease by contributing to the inflammatory phenotype and transformation of HHV8-infected cells.
    • Molecular Basis of Kaposi's Sarcoma Herpesvirus vGPCR-induced Paracrine Neoplasia

      Jham, Bruno; Montaner, Silvia (2010)
      Kaposi's sarcoma (KS), one of the most common AIDS-associated neoplasms, is a multifocal vascular tumor invariably associated with infection with the KS-associated herpesvirus (KSHV or HHV8). KS has a complex histopathology with respect to its cellular composition, origin and pathogenesis. The driving force of the KS lesion is the KSHV-infected spindle-shaped tumor cell, thought to have a vascular endothelial or endothelial precursor origin. KS tumors are also characterized by infiltrating inflammatory cells, slit-like blood vessels, and extravasated erythrocytes. The recruitment of these cells and the promotion of the angiogenic phenotype in these lesions are thought to be mediated by elevated levels of pro-inflammatory and pro-angiogenic secretions (cytokines, chemokines and growth factors) from the KS tumor cells. We have previously found that expression of a single KSHV gene, the viral G protein-coupled receptor (vGPCR) is able to recapitulate KS-like lesions in mice. Indeed, our results suggest that vGPCR may be responsible for KS initiation, progression and tumor maintenance, underscoring the key role of this viral oncogene in Kaposi's sarcomagenesis. vGPCR has proven to be a powerful oncogene and a potent angiogenic activator by inducing intracellular signaling pathways that promote the survival and transformation of expressing cells and by releasing secreted factors (cytokines, chemokines and growth factors), that may promote the recruitment and subsequent paracrine transformation of neighboring endothelial cells. However, the role of these vGPCR angiogenic factors, and their relative contribution to KS development, remains unclear. Here we describe 1) the molecular mechanism by which vGPCR paracrine secretions upregulate vascular endothelial growth factor (VEGF) in KS lesions; 2) the upregulation by vGPCR of a novel angiopoietin-related factor, angiopoietin-like 4 (ANGPTL4), that plays a critical role in promoting vGPCR-induced angiogenesis and vascular permeability; and 3) the essential role of the transcription factor hypoxia inducible factor (HIF) in vGPCR sarcomagenesis, highlighting the therapeutic potential of HIF inhibitors as an alternative treatment for KS.
    • The Role of the Akt/TSC/mTOR Signaling Pathway in Kaposi's Sarcoma- Associated Virus G Protein-Coupled Receptor Neoplasia

      Chaisuparat, Risa; Montaner, Silvia (2009)
      Kaposi's sarcoma (KS), a multifocal vascular neoplasm, is the most frequent cancer arising in HIV-infected individuals and in immunosuppressed patients. KS is caused by the KS-associated herpesvirus (KSHV). A single lytic gene of KSHV genes, G protein-coupled receptor (vGPCR), is sufficient to induce Kaposi-like sarcomas in mice. This vGPCR has been shown to promote the activation of PI3K/Akt, involved in transformation of endothelial cells. However, the Akt downstream effectors required for vGPCR to promote Kaposi's sarcomagenesis are still unknown. Here, we have found that vGPCR induces the phosphorylation and inactivation of tuberin (TSC2), promoting the activation of mTOR. Moreover, over-activation of TSC/mTOR is sufficient to render endothelial cells oncogenic. Treatment with rapamycin (mTOR inhibitor) efficiently prevented the growth of vGPCR tumors in vivo. Collectively, these results implicate the mTOR signaling route in Kaposi's sarcomagenesis and provide experimental evidence demonstrating that drugs targeting mTOR may represent an effective mechanism-based therapy for the treatment of KS. We have observed that, although rapamycin has been shown to be an efficient therapy for patients with iatrogenic or classic KS, the activity of Akt was transiently increased in vGPCR-expressing endothelial cells (EC-vGPCR). We therefore investigated the efficacy of PI-103, a novel dual PI3Kα/mTOR inhibitor, in preventing vGPCR transformation. PI-103 treatment effectively and independently blocked the activation of both PI3K and mTOR in EC-vGPCR. This resulted in the effective inhibition of endothelial cell proliferation and survival in vitro, and tumor growth in vivo, suggesting that PI-103 may be an effective therapeutic option for the treatment of KS. We also found that the angiogenic growth factors secreted by EC-vGPCR can induce the activity of mTOR in endothelial cells, suggesting that vGPCR regulates mTOR through both direct and indirect (paracrine) mechanisms. In addition, we found that the effect of rapamycin on vGPCR sarcomagenesis is not dependent on the direct activation of mTOR in vGPCR-expressing cells. Rather, the profound sensitivity of these tumors to rapamycin treatment may be due, in part, to the inhibition of the paracrine activation of mTOR in neighboring (bystander) cells by the angiogenic factors elaborated by EC-vGPCR. Collectively, these results support the key role of paracrine transforming mechanisms in vGPCR sarcomagenesis.