A normal progenitor cell-derivative containing the whole KSHV genome, allowed for the reproduction of the tumorigenic and angiogenic characteristics of an infected KS spindle cell, and led to the generation of a cell and animal model of KSHV-induced KS. This new model not only illustrates several features that makes it useful for biological and preclinical studies, but it also challenges our current thinking on key issues of KSHV biology and KS pathogenesis.
We found that the KSHV genome is angiogenic and tumorigenic in endothelial lineage cells of adherent bone marrow cell preparations. These findings suggest that an endothelial lineage cell type is a natural target of KSHV infection and a progenitor of tumorigenic KS spindle cells. It should be added that although our approach allows us to “target” putative progenitors in the transfected population and may have been critical for inclusion of the right cell type(s) in the right microenvironment, it does not allow us to unequivocally identify the KSHV targets. Nevertheless, phenotypic marker expression and transcriptome profile clustering results are consistent with the KSHVBac36 being transfected to endothelial lineage cells that are among the bone marrow adherent cell population, including endothelial cells and endothelial cell progenitors (Rafii and Lyden, 2003
We showed that the KSHV genome can independently induce the KS phenotype when its genome is expressed within the context of an appropriate normal cell progenitor and when the KSHV-bearing cells are grown in vivo. mECK36 lead to the growth of subcutaneous tumors or multifocal KS pulmonary lesions consisting of vascular spindle cell sarcomas, that expressed angiogenic and KS phenotypic markers such as podoplanin and the VEGF and angiopoietin receptors and ligands. Moreover, genome wide transcriptome analysis of mECK36 tumors revealed that 81% of human KS signature genes behaved as mECK36 tumor signature genes, and were similarly up-or down-regulated in mouse and human tumors. These findings suggest that the KSHV genome encodes the ability to recreate the full KS pathophysiology including visceral invasiveness and disease localization.
The pattern of KSHV expression in tumors, as evidenced by RT-PCR array analysis and immunodetection of K8.1, was consistent with an increase in lytic transcripts. Although it was reported that most spindle cells in KS lesions are latently infected (Staskus et al., 1997
), we found that a subpopulation of KS lesions (Dittmer, 2003
) exhibits a pattern of KSHV expression similar to mECK36 spindle sarcomas. Thus, the KSHV expression profile of mECK36 in tumors can be biologically relevant. In future studies, it will be of importance to establish possible correlations between the mECK36-like KSHV expression profile and KS clinical presentation. Interestingly enough, the mECK36 pattern of KSHV gene expression was not accompanied by virus production indicating that mECK36 were in an abortive lytic replication state. Others have shown that in permissive human cells KSHVBac36 transfection leads to virion production (Zhou et al., 2002
). In contrast, both in vitro
and in vivo
grown mECK36 showed an absence of viral replication. Since transcritptome analysis shows that the viral genome is intact, it is unlikely that the defects in viral replication were due to Bac36 alterations. Instead, viral replication and maturation events are most likely being blocked. A recent report (Parsons et al., 2006
), points to productive KSHV infection in SCID/NOD mice upon injection of KSHV virions suggesting that the use of KSHVBac36 transfection may relate to the abortive lytic status of mECK36. Interestingly, KS lesions contain lytic replication-defective KSHV genomes that may play a role in KS tumorigenesis (Deng et al., 2004
). Thus, the replication-defective infection achieved by Bac36 transfection may be relevant to actual occurrences of the disease and might have been critical to capture a KSHV-induced KS phenotype.
We found that KSHV episome-loss led to loss of tumorigenicity. This indicates that the mECK36 tumorigenic phenotype is strictly dependent on KSHV and reversible, since mECK36 reverted to a non-tumorigenic phenotype upon KSHV loss in vitro
. This resembles explanted KS spindle cells that lose KSHV in culture becoming non-tumorigenic (Aluigi et al., 1996
; Dictor et al., 1996
; Ganem, 2006
). It also suggests that KSHV-induced oncogenesis, at least in its initial stages, does not lead to the accumulation of further oncogenic hits which could contribute to a malignant phenotype independent of KSHV. This also resembles human KS lesions in which cellular oncogenic alterations such as p53 and ras mutations, or Bcl-2 overexpression together with increased clonality tend to be found only in advanced KS (Gill et al., 1998
; Nicolaides et al., 1994
; Pillay et al., 1999
; Rabkin et al., 1997
). It can also be concluded that the maintenance of the KSHV episome during tumor formation—which is carried out in the absence of hygromycin—indicates that KSHV does provide a selective advantage for in vivo
growth that leads to tumorigenesis. This selective advantage of KSHV+ versus non-infected cells provides an alternative explanation to re-infection (Grundhoff and Ganem, 2004
) for maintenance of the KSHV episome in infected spindle cells in KS lesions.
KSHVBac36-mediated tumorigenesis correlated with the upregulation of several KSHV lytic transcripts and the induction of a KS-like angiogenic phenotype upon implantation into nude mice. This further supports the notion, evidenced by recent publications, that in KS as well as in PEL (An et al., 2006
; Staudt et al., 2004
) the host microenvironment influences KSHV gene expression. However, the regulatory effects are indeed dialectic. We found that KSHV together with in vivo
growth conditions trigger in the infected cell the expression of genes related to angiogenesis and malignancy which provides a selective advantage leading to tumor formation. We found that mECK36 grown in vitro
did not display signs of cell transformation. When subjected to in vivo
growth conditions, however, the same cells induced angiogenic spindle cell sarcomas with upregulation of key angiogenic markers ( and ). In concordance, analysis of the mouse-KS-like signature of mECK36 tumors showed that almost 50% of it derives from genes that were only expressed in mECK36 tumors. The concomitant upregulation of KSHV lytic genes in vivo
and the induction of an angiogenic phenotype in KS-like lesions pointed to a series of potentially pathogenic KSHV genes as critical determinants for KS-like tumorigenesis. We selectively inhibited one of these genes to prove its involvement in mECK36 tumorigenesis and to test the suitability of our model for genetic analysis of KSHV pathogenic function. siRNA-mediated suppression of the early lytic angiogenic oncogene vGPCR (Bais et al., 1998
; Bais et al., 2003
; Yang et al., 2000
), blocked VEGF secretion and tumorigenesis, leading to significant retardation in tumor growth. It is unlikely that siRNA was also targeting the expression of orf K14, which is co-expressed with vGPCR in bi-cistronic messages (Kirshner et al., 1999), as orf K14 mRNA levels were not affected by the vGPCR shRNA (). These results with vGPCR silencing were verified by preliminary studies with vGPCR-null Bac36 mutants (LC, EM unpublished). Our results confirm single gene experiments with vGPCR (Bais et al., 1998
; Bais et al., 2003
; Grisotto et al., 2006
; Guo et al., 2003
; Montaner et al., 2003
; Montaner et al., 2006
; Yang et al., 2000
), indicating that it plays a non-redundant role in angiogenesis and tumorigenesis within the context of full KSHV genome expression, and further supports the notion that this KSHV gene is a good therapeutic target. Our results with vGPCR suppression show that our model is sensitive to genetic manipulation and can be used to analyze the contribution of other KSHV genes in tumorigenesis. Furthermore, they point to mECK36 in vivo
malignant phenotype as a consequence of upregulation of KSHV genes, such as vGPCR, that provide a selective advantage to the cell—in this case angiogenicity- leading to positive selection of the KSHV episome and KS-like tumor formation.
The suitability of mECK36 for genetic studies of KS pathogenesis could help to reveal the role of both KSHV and host genes implicated in the KS phenotype. A recent report showed that long-term latent KSHV infection of immortalized human endothelial cells leads to the outgrowth of transformed and tumorigenic clones (An et al., 2006
). However, the use of hTERT-immortalized cells as substrate, and the sporadic occurrence of the in vitro-transformed outgrowths points to the accumulation of oncogenic alterations in the host genome; thus, making it difficult to strictly link the malignant phenotype to specific KSHV genes. In our case, the fact that Bac36-transfection of mEC leads to a non-productive infection also imposes limitations to the mECK36 model, in particular for studying viral entry and viral replication. However, several key characteristics make the mECK36 model appropriate for genetic studies of pathobiology and experimental therapeutics: 1) The progenitor target population(s) are normal mouse bone marrow cells, making it possible to use available knockout (K/O) or conditional K/O mice to generate different Bac36-transfected cells that could be used to identify genetic determinants of KS pathogenesis. 2) The KSHV dependence of the KS phenotype makes the mECK36 system highly sensitive for studies of KSHV genes critical for tumor growth, and makes it suitable for testing therapeutic strategies targeting KSHV pathogenic genes or host angiogenic machinery. 3) The mECK36 population expresses the full KSHV genetic complement, allowing for manipulation of latent and lytic KSHV genes using Bac or siRNA in functional studies. 4) mECK36 viral expression was stable through multiple passages (DD and EAM unpublished) and had reproducible in vivo
In summary, our results show that the mEC Bac36 system and the mECK36 KSHV-harboring spindle sarcomas are good phenotypic, physiologic, molecular and viral surrogates of human KS that could unlock novel research avenues on KSHV pathobiology. They define mECK36 as a biologically sensitive cell and animal model of viral Kaposi’s sarcoma suitable for analyzing the role of KSHV and host cell genes in KS pathogenesis, as well as for preclinical testing of anti-KS drugs.