An NF-κB gene expression signature contributes to Kaposi's sarcoma virus vGPCR-induced direct and paracrine neoplasia

Abstract

Kaposi's sarcoma (KS) is the most frequent AIDS-associated malignancy, etiologically linked to the infection with the human herpesvirus 8 (HHV-8/KSHV). This member of the gamma-herpesviridae family encodes 81 open reading frames, several bearing oncogenic potential. A constitutively active virally encoded G protein-coupled receptor (vGPCR) readily induces KS-like lesions when expressed in endothelial cells in vivo, and unmasks the oncogenic potential of other HHV-8 genes in a paracrine fashion. How vGPCR causes endothelial cell transformation is still not fully understood. Using full-genome microarray analysis we show here that the expression of nuclear factor-kappaB (NF-kappaB)-regulated genes is a prominent feature triggered by vGPCR in cells expressing this viral oncogene and in cells exposed to vGPCR-induced secretions, thus mimicking its paracrine effect. Indeed, vGPCR activates the NF-kappaB pathway potently, and NF-kappaB activation is a hallmark of both human and experimental KS. Of interest, whereas constitutive NF-kappaB signaling is not sufficient to promote endothelial cells transformation, NF-kappaB function is strictly required for vGPCR-induced direct and paracrine neoplasia. Taken together, these results strongly support the role of NF-kappaB regulated genes in KS pathogenesis, thus providing the rationale for the development of novel mechanism-based therapies for this angioproliferative disease.

Remodeling of VE-cadherin junctions by the human herpes virus 8 G-protein coupled receptor

“…Furthermore, these few vGPCR-expressing cells might promote the secretion of multiple cytokines and chemokines, which act in a paracrine manner thereby activating signaling pathways, which trigger proliferation and vascular leakiness within the tumor microenvironment. Of interest, high levels of inflammatory cytokines and local release of VEGF has been measured in vGPCRinduced KS tumors in mice (Ascherl et al, 1999;Montaner et al, 2004;Martin et al, 2008). Hence, the dramatic loss of vascular integrity observed in human KS might be controlled both directly by vGPCR signaling to VE-cadherin and indirectly by the vGPCR secretome, which might also converge on VE-cadherin function.…”

“…Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and Src kinase is also detected, albeit to a reduced degree compared with VEGF stimulation. This can likely be attributed to autocrine effects including VEGF-A secretion (Martin et al, 2008). Using a permeability assay measuring fluorescently-conjugated dextran transendothelial passage, we then tested whether vGPCR impacts on paracellular permeability in vitro (Gavard and Gutkind, 2006).…”

“…This was Figure 1 HHV8 vGPCR-driven tumors display a leaky blood vessel network. (a) SVEC were transfected with pCEFL-empty (control) or pCEFL-AU5-HHV8-vGPCR (vGPCR) and stable cell pools were selected by G418 for 3 weeks, as described in (Martin et al, 2008). Confocal analysis of AU5 staining (green) in stable cells, together with actin staining by phalloidin (red) and nucleus counterstaining (DAPI, blue).…”

“…Cell culture and siRNA transfection SVEC were purchased from the ATCC (Manassas, VA, USA) and vGPCR-expressing SVECs were described previously (Martin et al, 2008). siRNA transfections of non-silencing control (Dharmacon, Chicago, IL, USA), mouse Rac and RhoA silencing (Invitrogen (Gavard et al, 2008)) and mouse PI(3)Kg (Clinisciences) were performed using the Hiperfect reagent (Qiagen, Courtaboeuf, France).…”

“…G418-selected stable cell lines were obtained by transfection of the corresponding pCEFLderived plasmids as described previously (Martin et al, 2008).…”