Our results reveal the presence of a novel xenotropic MLV that, like XMRV, was likely generated through the recombination of endogenous murine proviral sequences via xenograft experiments in nude mice. We postulate that such recombination events may be extremely rare and yet the generation of similar viruses may be enhanced as a consequence of extraordinary selection pressures inherent in typical xenograft and culture experiments. We further show that defective blood vessel formation is a functional consequence of infection by these xenotropic MLVs, resulting in larger tumors that possess greater numbers of blood vessels that are immature and hemorrhagic. Remarkably, our in vitro results indicate that exposure to the xenotropic MLV envelope proteins is sufficient to induce tumor cells to produce factors that alter vascular SMCs to an immature phenotype, suggesting a novel mechanism by which xenotropic viral infection of tumor cells leads to the formation of immature blood vessels.
Disrupted tumor vascular maturation has long been equated with high rates of tumor cell shedding and metastasis [16, 28]. Highly hemorrhagic tumors that contain an abundance of dilated, immature blood vessels poorly invested with vascular SMCs and pericytes exhibit increased metastatic potential [18, 29]. Consistent with this possibility, Ramaswamy et al.[20, 30] used microarray analysis of over 800 human solid tumors and metastases and demonstrated that 4 of the 9 genes whose down-regulation is highly predictive of tumor metastasis encode markers of differentiated SMC/pericytes, suggesting that a lack of differentiated SMC/pericytes within tumor biopsy samples is highly predictive of tumor metastatic potential. Immature vascular networks are also highly inefficient for blood delivery and exhibit impaired delivery of chemotherapeutic agents, resulting in higher levels of agents in normal versus tumor tissues , which is also thought to contribute to the poor overall efficacy of anti-angiogenic therapies due to counteracting effects of combined therapies. The mechanisms that contribute to incomplete maturation of tumor vessels are poorly understood, although it may result in part from a hyper-VEGF state, as supported by studies in both animal models and human clinical trials showing at least partial normalization of vessel structure with anti-VEGF therapies [22, 23].
Although neither XMRV nor B4rv are likely to infect humans and contribute to tumor pathogenesis, our observations that xenotropic MLV envelope proteins induce signalling that results in an immature vascular phenotype provide a novel mechanism by which viral gene products might promote tumor pathogenesis. However, there are a number of key unresolved questions: First, what are the mechanisms by which the XMRV envelope proteins interact with tumor cells? Although the known viral entry receptor XPR1 has been shown to mediate entry of XMRV into some cells [24, 25], there is also recent evidence that XMRV can infect cells that do not express XPR1 . Indeed, we have been unable to inhibit the effects of XMRV envelope protein in suppressing SMC differentiation by siRNA-induced inhibition of XPR1 (Additional file 8: Figure S8e). As such, the effects of XMRV envelope protein observed in the present studies appear to be mediated by a receptor or membrane protein or moiety other than XPR1. Based on our data showing an increase in VEGF-dependent endothelial tube formation in the presence of conditional media from B4rv- or XMRV-infected tumor cells (Figure 3C, Additional file 8: Figure S8d), this receptor might be one of a wide range of cell surface molecules that, when activated, may result in the release of VEGFs. Of interest, our observation that production of soluble factors that repress vascular SMC differentiation persisted in LNCaP cells chronically infected with B4rv or XMRV suggests that the envelope protein receptor may not be down-regulated or de-sensitized despite chronic exposure to ligand, and/or that there are positive feedback mechanisms that result in continual regeneration of receptor. Second, what are the rate-limiting mechanisms that prevent xenotropic and/or polytrophic MLVs from infecting humans? Of significance, productive infection by XMRV or B4rv of human cells in vitro appears to be limited to cells that contain defective defences to retroviruses. Indeed, the prostate cancer LNCaP cell line used herein lacks XMRV-restricting APOBECs  and contains a deletion mutation of one allele of RNaseL , a gene critical in the innate immune response to retroviruses. Inactivating polymorphisms of RNaseL are highly associated with familial prostate cancer in young males, and the hypothesis that such deficiencies in innate immunity leaves one vulnerable to a pathogenic retrovirus was the basis for the study that lead to the discovery of XMRV. Since then, any association of XMRV and human infection has been invalidated. However, a critical question is whether utilization of RNaseL defective tumor cells to generate xenograft derived cell lines might allow for the evolution of retroviruses that ordinarily cannot escape cellular immune defences. Third, how does binding of envelope protein to XPR1 or alternative receptor on tumor cells induce production, and/or release of soluble factors that disrupt tumor vascular maturation?
Signalling through retroviral envelope proteins has been shown in other disease models, notably by the Jaagsiekte sheep retrovirus (JSRV) whose envelope proteins cause ovine pulmonary adenocarcinoma through the activation of multiple signaling pathways including the phosphoinositide-3-OH kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) signaling cascades, binding/degradation of hyaluronidase 2 (Hyal2), and activation of the RON receptor . Degradation products of Hyal2 in particular have been associated with an increase in angiogenesis in the context of xenograft prostate cancer models, outside of the context of JSRV infection. A recent study reports an increase in pro-angiogenic factors within tumors infected with JSRV, particularly VEGF-C and PDGFR-alpha. However the role of the envelope protein in factor production has yet to be elucidated. Other examples of retroviral envelope induced cellular signaling include the Friend spleen focus-forming virus (SFFV), mouse mammary tumor virus (MMTV), enzootic nasal tumor virus (ENTV), and avian hemangioma retrovirus (AHV), all in which envelope proteins contribute to, or induce, transformation . Although previous work by other labs demonstrates that XMRV does not induce transformation [1–3], no work to date has shown whether xenotropic MLVs as a class may activate signaling pathways that promote a pro-tumorigenic environment through indirect mechanisms, including through inducing the secretion of pro-angiogenic factors. This is especially important to examine given that unlike other MLVs that arise in a more organic fashion, all xenotropic MLVs identified to date have been found in the context of xenograft experiments that establish cell lines in the laboratory setting [2, 31], providing a unique and specific selection on this class of MLV. Indeed, given that XMRV and B4rv evolved independently but elicit very similar functional consequences in tumor cells, it is interesting to speculate that there is a unique aspect to xenograft experiments that selects for retroviruses that induce larger, more hemorrhagic tumors.