The development of oncolytic viral therapy has recognized the importance of host cell and virus interactions in optimizing therapeutic efficacy. The mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. In particular, the mechanism of VV cell entry is opaque. It has been suggested that VV cell entry may occur by passive fusion with the cell membrane, or via uncharacterized receptors, or by macropinocytosis, followed by internalization requiring the coordination of complex actin dynamics (32
). VVs, including vaccine strains, have been shown to inherently target tumors (10
). This tumor selectivity is partially attributed to the activation of the epidermal growth factor/Ras-MEK-extracellular signal-regulated kinase (ERK) pathways and deregulation of interferon (IFN) pathways in cancer cells (36
). A greater understanding of the cellular factors essential for efficient VV infection would offer further insights into poxvirus biology and help to improve the development of VV as a novel anticancer therapeutic.
We have previously shown that the cytotoxicity of oncolytic VV is augmented by hypoxia in some PDAC cell lines (14
). In this study, we show that this is only in cell lines where there is hypoxic induction of VEGF-A. We used PDAC cell models with both VEGF-A overexpression and siRNA-mediated gene silencing to demonstrate that VEGF-A increases viral reporter gene expression, replication, and cytotoxicity. Quantitative PCR and direct fluorescent confocal microscopy of a fluorophore-labeled virus showed that VEGF-A increases the rate of virion internalization. The use of the clinically relevant inhibitor of the VEFGR (pazopanib) in cells stably expressing VEGF-A also demonstrated that VEGF-A can act in an autocrine manner to increase viral internalization, gene expression, and replication. We show here that VEGF-A can increase Akt phosphorylation, which is prevented by inhibition of the VEGFR, and that selective Akt inhibition reduces VV internalization. Using systemic administration of VVL15, a recombinant VV expressing fLuc, we were able to show that this finding is consistent in vivo
. We also demonstrated that this is replicated in NHBE cells, which are a natural host cell for VV.
Although other growth factors such as EGF and its receptor have recently been reported to augment the uptake of adeno-associated virus 6 (AAV6) and influenza virus A (38
), we have shown the role of VEGF-A in the VV life cycle. It is interesting that orf virus, from the genus Parapoxvirus
, produces a viral virulence factor, VEGF-E, which is the VEGF homologue most closely related to VEGF-A and contains all eight cysteine residues of the central cysteine knot motif characteristic of the VEGF family (40
). In addition, myxoma virus, a rabbit-specific poxvirus, has been shown to infect and kill human cancer cells only where there is endogenous Akt phosphorylation and activation (41
). Neuropilin-1, a coreceptor of VEGF, is a component of receptor complexes involved in the entry of human T-cell leukemia virus (42
This is the first study to our knowledge to demonstrate that VEGF-A signaling facilitates VV entry. In our study, silencing VEGF-A or inhibiting Akt activation did not prevent viral infection completely, supporting the concept that there are likely to be multiple pathways by which VV may enter cells, which may differ across cell types (43
). Integrin β1-mediated phosphatidylinositol 3-kinase (PI3K)/Akt activation has very recently been reported to be essential for vaccinia virus entry (44
). Integrin β1 is important for both vaccinia virus attachment and penetration. It has been reported that VEGFs and their receptors interact with integrins, including β1, on endothelial and tumor cells (45
). Further investigations are warranted to dissect whether VEGF interacts with integrin to affect the life cycle of VV and which VEGFR(s) is involved in such effects. However, our present study at least suggests that VEGF-A is one of the cellular factors that are responsible for the tumor tropism of VV.
In addition, an increase in vascularity induced by overexpression of VEGF-A may account for some of the effects in vivo
, although it does not give a conclusive indication as to the permeability of these vessels, which may also be important for VV spread (47
). Consequently, VEGF-A results in increased tropism of VV for malignant cells by increasing internalization and may also improve virus delivery in vivo
due to its role in tumor angiogenesis. The effect of the angiogenic state on tumor uptake of VV, cross traveling of VV from blood to tumor cells, and their relationship with VEGF expression are very interesting topics that warrant further investigations. Of note, our findings have implications for regimens that combine anti-VEGF/Akt agents with oncolytic VV for cancer treatment. It might be more reasonable to use anti-VEGF/Akt agents after treatment with oncolytic VV based on our findings in this study.
VV was widely used in the mass vaccination programs that eradicated smallpox in 1972. Although the immunity of vaccinated individuals lasts for decades (48
), a large proportion of the general population is still recognized to be extremely susceptible to variola virus and other poxviruses in the event of an intentional or unintentional release (49
). Therefore, it is important to look into more approaches to prevent and or interrupt poxvirus infection, especially in normal human cells. The identification of VEGF-A and Akt signaling as influences in VV infection in normal human epithelial cells () has important implications for treating poxvirus infection through targeting host cellular genes.