Hypoxia is a well characterized feature of solid tumors, and the hypoxic environment has a detrimental effect on the response of tumors to radiation3
Oncolytic viruses that selectively replicate in tumor cells have made the transition from bench to bedside over the past decade. Data from recent studies, showing reduced adenoviral replication in hypoxic cells,14,15
raise concerns that, much like the effects of chemotherapy and radiation, hypoxic conditions in cells could also diminish the efficacy of oncolytic viruses.
We found that the oncolytic HSV G207 exhibited increased replication in hypoxic cells, while wild-type HSV strain F exhibited a smaller increase in replication in such cells. The reproducible half-log enhancements in G207 replication that we describe after 48 hours of viral replication under hypoxic conditions (as compared to normoxic culture conditions) in culture, and after 5 days under hypoxic conditions (as compared to normoxic conditions) in vivo
may seem small, but are actually significant in the light of several studies showing that relatively small increases in viral yield in vivo
, comparable to the increase we observed under hypoxic conditions, can have large impacts on inhibition of tumor growth. For example, ionizing radiation was shown to increase oncolytic HSV yield in vivo
by approximately threefold, and yet it increased the tumor cure rate from 14 to 56%.23
We have shown that temozolomide increases G207 yield in vivo
by two- to sixfold, and yet the combination increases long-term survival from 10 to 100%.13
In addition, our in vivo
observations held true not only in “glucose sensitive” glycolytic-dependent U87 cells, which are less tolerant of low glucose concentrations because of cellular expression of the LDH-B isoform alone,24,25
but also in “glucose resistant” T98 cells, which can tolerate low glucose concentrations26
and which rely on oxidative phosphorylation (aerobic respiration) for survival, probably as a result of expression of both the LDH-A and the B isoforms. The in vivo
data presented here, therefore, suggest that hypoxia can enhance oncolytic HSV replication in glioma cell lines, whether these are glycolytic-dependent or independent.
The enhanced replication in wild-type strain F in hypoxic cells relative to normoxic cells, which was much smaller than the hypoxia-related enhancement of oncolytic HSV G207 replication but still statistically significant, might reflect the natural tropism of HSV for cells with reduced oxygen tension and the stimulation of HSV replication by DNA damage induced by oxygen-derived free radicals. Indeed, if hypoxia naturally enhances HSV replication, this could reflect HIF-1α-mediated transcription of cellular factors that enhance HSV replication or direct HIF-1α effects on the HSV genome, given the recent demonstration that the genome of Kaposi's sarcoma-associated herpesvirus contains hypoxia-responsive elements that can be specifically activated by HIF-1α.27
However, the larger hypoxia-mediated enhancement of G207 replication shows that the viral gene deletions in G207 dramatically enhance the slight hypoxia-related enhancement of HSV replication that is intrinsic to strain F. A portion of the hypoxia-mediated enhancement of G207 replication may reflect our finding of hypoxia-mediated upregulation of GADD34 and our further finding that blocking of the GADD34 expression impairs one-third of the hypoxia-mediated upregulation of viral replication. GADD34 is a mammalian gene whose product complements the replication of HSVs such as G207 that are deficient in the viral gene γ34.5.22
This finding is consistent with previous demonstrations that GADD34 is expressed in response to cellular stressors such as hypoxia,28
and suggests that, in addition to HSV's natural tropism for hypoxic environments, the specific viral gene deletion found in G207 may cause an even greater augmentation of viral replication in hypoxic environments. In addition to the explanation relating to hypoxia-mediated GADD34 upregulation, other potential explanations for the finding that hypoxia upregulates G207 replication far more than strain F does could be: (i) hypoxia, which upregulates protein phosphatase 1 expression,29
causes protein phosphatase 1 levels to rise high enough that protein phosphatase 1 causes some dephosphorylation and activation of eIF-2α in a γ34.5-independent fashion, thereby promoting viral protein synthesis; and (ii) hypoxia upregulates the expression of other GADD proteins to sufficient levels as to make them achieve a degree of complexing with proliferating-cell nuclear antigen (PCNA), similar to the action of GADD34 in cells infected with γ34.5-deficient HSVs.
A review of the literature shows that oncolytic HSV, which was investigated in this study, is the fourth oncolytic virus to be studied by investigators to characterize how hypoxia affects viral replication. Also, this is the first study to report hypoxic upregulation of oncolytic viral replication. An earlier study found that hypoxia impairs replication of oncolytic adenovirus,14
a DNA virus. Another study found that hypoxia has no effect on adenoviral uptake or exogenous gene expression, and therefore does not affect gene delivery by replication-incompetent adenoviruses; however, hypoxia impairs adenoviral replication by reducing E1A levels and thereby affects the propagation of oncolytic adenoviruses.15
While one study showed that GADD34 expression (induced by hypoxia in our study) impairs replication of vesicular stomatitis virus,30
an RNA virus, another study argued that hypoxia does not affect vesicular stomatitis virus replication, using indirect evidence in the form of viral replication in hypoxic areas of a tumor.31
Hypoxia has also been shown to inhibit the replication of the oncolytic DNA virus, Minute virus of mice.32
Further, hypoxia has been demonstrated to inhibit the replication of other DNA viruses that have not yet been used as oncolytic viruses, such as simian virus 40 (ref. 33
). These reports from earlier studies underscore the fact that the hypoxia-mediated upregulation of oncolytic HSV replication, as identified by us in this study, is a unique feature among viruses in general.
Importantly, our study is the first to find an oncolytic virus (oncolytic HSV) that demonstrates an increase in replication in hypoxic environments. Other researchers have attempted to use HIF-1α-driven promoters to regulate the expression of essential viral genes in oncolytic adenovirus34
or oncolytic HSV.35
However, before embarking on these viral engineering approaches that are designed to increase the safety of these viruses by limiting their replication to hypoxic areas, it is important to keep in mind the direct effects of hypoxia on the oncolytic virus itself. In other words, hypoxia-driven promoters in the virus might not overcome the ability of hypoxia to limit the translation of proteins from adenoviruses14
and might not improve upon the intrinsic hypoxia-mediated upregulation of viral replication that we found with the HSV G207.
Further, in this study we not only showed replication in hypoxic areas in vivo
but also used manipulations of tumor oxygenation in vivo
to demonstrate that there is greater viral replication in tumors that are more hypoxic. Such preclinical data, along with other considerations such as the ability to generate large titers and the rate of viral replication relative to the rate of tumor migration and growth, will likely need to be considered when choosing which oncolytic viruses warrant further clinical study. Given the high levels of hypoxia found in most human solid tumors,16
and the recent demonstration that oncolytic viruses such as vaccinia virus and vesicular stomatitis virus eventually cause reduced blood supply to the tumor36
thereby potentially worsening tumoral hypoxia, the ability of oncolytic HSVs to not only tolerate hypoxia but to benefit from it may provide these viruses with a key intrinsic therapeutic advantage.