Novel Cellular Genes Essential for Transformation of Endothelial Cells by Kaposi's Sarcoma–Associated Herpesvirus

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is involved in the development of lymphoproliferative diseases and Kaposi's sarcoma. The oncogenicity of this virus is reflected in vitro by its ability to transform B cells and endothelial cells. Infection of dermal microvascular endothelial cells (DMVEC) transforms the cells from a cobblestone-like monolayer to foci-forming spindle cells. This transformation is accompanied by dramatic changes in the cellular transcriptome. Known oncogenes, such as c-Kit, are among the KSHV-induced host genes. We previously showed that c-Kit is an essential cellular component of the KSHV-mediated transformation of DMVEC. Here, we test the hypothesis that the transformation process can be used to discover novel oncogenes. When expression of a panel of KSHV-induced cellular transcripts was inhibited with antisense oligomers, we observed inhibition of DMVEC proliferation and foci formation using antisense molecules to RDC1 and Neuritin. We further showed that transformation of KSHV-infected DMVEC was inhibited by small interfering RNA directed at RDC1 or Neuritin. Ectopic expression of Neuritin in NIH 3T3 cells resulted in changes in cell morphology and anchorage-independent growth, whereas RDC1 ectopic expression significantly increased cell proliferation. In addition, both RDC1-and Neuritin-expressing cells formed tumors in nude mice. RDC1 is an orphan G proteincoupled receptor, whereas Neuritin is a growth-promoting protein known to mediate neurite outgrowth. Neither gene has been previously implicated in tumorigenesis. Our data suggest that KSHV-mediated transformation involves exploitation of the hitherto unrealized oncogenic properties of RDC1 and Neuritin. (Cancer Res 2005; 65(12): 5084-95)

The insulin receptor is essential for virus-induced tumorigenesis of Kaposi's sarcoma

“…The IR joins a growing list of host cell proteins essential for the transformed phenotype in ECs infected by KSHV in vitro (Moses et al, 2002;McAllister et al, 2004;Raggo et al, 2005). It seems that multiple mechanisms are required to support the postconfluent growth of DMVEC.…”

“…It seems that multiple mechanisms are required to support the postconfluent growth of DMVEC. Yet, not every growth factor receptor or oncogene that is expressed or upregulated in KSHV-infected cells is necessary for KSHV-mediated transformation, exemplified by the IGF-IR, as well as by a number of other KSHV-induced transcripts previously shown to be dispensable for this process (Raggo et al, 2005).…”

“…Total RNA was purified from E-DMVEC, primary DMVEC and TIME cells using RNeasy spin columns (Qiagen Inc., Valencia, CA, USA). Human tissue was a generous gift from H Koon and BJ Dezube and prepared as previously described (Raggo et al, 2005). Total RNA was treated with DNase I (DNase Free: Ambion, Austin, TX, USA) before synthesis of cDNA by random hexamers and Superscript III (Invitrogen, Carlsbad, CA, USA).…”

“…Upon infection with KSHV, E-DMVECs develop a spindle cell phenotype, lose contact inhibition, form foci when cultured postconfluence and acquire the ability to form colonies in soft agar. The E-DMVEC system has thus been extremely useful for evaluating changes in the cellular transcriptome and proteome, ultimately leading to the identification of both known as well as novel oncogenes that are likely to be involved in KS tumorigenesis (Moses et al, 2002;McAllister et al, 2004;Raggo et al, 2005).…”

Induction of spindle cell morphology in human vascular endothelial cells by human herpesvirus 8-encoded viral FLICE inhibitory protein K13

“…As shown in Figure 3a, treatment of K13-ER TAM HUVEC cells with 4-OHT was accompanied by strong induction of IL-6 and IL-8 production in the cellular supernatant, thereby demonstrating that K13-induced spindle cell transformation of HUVEC cells is accompanied by production of proinflammatory cytokines. RDC1 is one of the most upregulated genes in HHV8-transformed vascular endothelial cells and was recently shown to play a key role in the HHV8-induced transformation of dermal vascular endothelial cells (Raggo et al, 2005). We used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to investigate whether K13-induced spindle cell transformation is Figure 1 Development of an inducible system to control K13 activity at the post-translational level.…”