VSV is an oncolytic virus, which selectively grows and kills a variety of cancer cells and shows an attenuated growth phenotype in normal cells. VSV selectivity is achieved by exploiting molecular defects in cancer cells, which compromise the innate antiviral defense or, on the other hand, create advantages for malignant growth and survival (38
). The potent cytolytic properties of VSV in conjunction with its rapid replication cycle have made VSV an extremely promising candidate for oncolytic virotherapy (1
). Interestingly, many of the signaling pathways that viruses use are the same ones deregulated during malignant evolution. Due to their relevance in oncogenesis, these same pathways have become targets for anticancer drug development. Now that oncolytic viruses are finally entering the clinic, the time has come to take a further step forward and explore possible new application strategies involving the most up-to-date and refined anticancer agents. Therefore, it is reasonable to foresee the great potential of synergistic combinations of oncolytic viruses and small cell-permeable inhibitors of protein kinases to enhance tumor killing. For example, the various MAPK pathways (ERK1/2, JNK, and p38 MAPK) appear to be some of the most significant cellular signaling cascades in the development of several malignancies, including HCC (36
). Therefore, the inhibition of ERK can be useful to control the growths of several human cancers (40
), while JNK modulators are able to induce cancer cell death or sensitize tumor cells to apoptotic stimuli (12
). The JNK pathway has also been linked to cell cycle progression and antiviral responses (3
). In addition, the MAPK pathways play an important role in the life cycles of certain viruses (10
). In view of a combinatorial approach with new drugs based on the specific targeting of MAPK and oncolytic viruses, we investigated the use of such combinations using VSV in the context of HCC.
In this work, we have studied the influence of three of the major MAPK inhibitors on VSV oncolysis in vitro
, comparing HCC cell lines with primary human hepatocytes. It was previously shown that ERK facilitates VSV-mediated oncolysis by the negative regulation of the IFN-α response (43
). In many HCC cell lines, the innate immunity response to pathogens is compromised, especially due to multiple defects in the type I IFN system (38
). The reestablishment of a functional type I IFN response in HCC would be seriously detrimental to the therapeutic efficacy of VSV. Our studies demonstrated that in HCC cell lines, the activation of the ERK signaling pathway does not enhance VSV oncolysis, since protection from lytic infection was not improved by the coadministration of the ERK inhibitor U0126 and IFN-α/β. Therefore, the disruption of ERK signaling by anticancer drugs seems to be compatible with VSV therapy in HCC, at least in vitro
. The discrepancy of our results compared with those of previous reports (43
) emphasizes the need to consider each cancer type as a unique environment. For this reason, preliminary in vitro
studies assume a great significance in view of subsequent clinical investigations.
Since their discovery in the early 1990s, JNKs have attracted intense interest due to the increasing evidence of the involvement of JNK-dependent signaling events in the development of several pathological conditions. The potential therapeutic application of JNK-specific inhibitors for the treatment of different human diseases, from ischemia, diabetes, and cancer to viral infectious diseases, has been explored (25
). Notably, JNK1 has an essential oncogenic role in HCC development, and direct evidence comes from in vivo
studies with JNK1 knockout mice. In mice lacking JNK1, diethylnitrosamine-triggered liver tumorigenesis was remarkably reduced, and treatment with chemical JNK inhibitors resulted in the reduced growth of xenografted human HCC cells (22
). Besides SP600125 (3
), several small-molecule compounds inhibiting JNK kinase activity with higher selectivity and efficacy have been developed (35
), and the combination of these new inhibitors with VSV virotherapy could potentially be beneficial for HCC treatment.
Increasingly, it has been shown that viral infection can lead to stress-activating protein kinase (SAPK)/JNK and p38 MAPK activation, which is needed for viral replication and release (21
). In this report, we observed a strong activation of JNK upon the infection of HCC cell lines with VSV, while the levels of activation of ERK and p38 MAPK were very weak. Inhibitors of p38 MAPK (SB203580) and of ERK (U0126) did not reduce the viral yield in HCC cells. On the other hand, the JNK inhibitor SP600125 dramatically decreased viral titers in all cell types tested, consistent with previous studies with dengue virus, rotavirus, and circovirus (7
). SP600125 and other similar anthrapyrazoles are considered to be valuable therapeutic agents; their usefulness against cancer (3
), liver injury, and fibrosis (20
) has been associated with minimal toxicity in vivo
. Unfortunately, in light of our results, we excluded the possibility of a conjunctive application of SP600125 and VSV therapy. However, our results indicate that the attenuation of VSV by SP600125 is due to a nonspecific mechanism that does not involve the inhibition of JNK, and therefore, the combination of VSV and other specific JNK inhibitors still represents a viable treatment option.
Most interestingly, despite the fact that the numbers of copies of the viral genome in the supernatants of SP600125-treated cells did not differ substantially from those in the untreated controls, the infectious viral titers were significantly lower, up to 10,000- to 100,000-fold. These results led us to conclude that SP600125 affects VSV infectivity such that only a fraction of the new viral progeny released into the culture supernatant retains the ability to reinfect cells. As a cause of a lack of infectivity of newly formed virions, we have found that the viral particles incorporate at least two different forms of the G protein in the presence of SP600125: one that comigrates with the normal G protein and the other that is significantly higher in molecular mass than the normal G protein (VSV-G*). The same result was observed when a VSV-G protein-expressing plasmid was transfected in the presence of the JNK inhibitor.
At this point, we speculated that VSV-G* could represent either a hyperglycosylated form of VSV-G or a VSV-G dimer, since the size was roughly twice the size of the monomeric G protein. An alteration of glycosylation can have a dramatic impact on the infectivity of viruses; as observed previously by Whitt and colleagues, virions incorporating a glycoprotein with an additional N-linked oligosaccharide in the extracellular domain were not infectious, apparently due to the formation of heterodimers that lacked fusion activity (58
). In our experiments, digestion with N
- or O
-glycosidases did not completely abolish VSV-G* expression, leading us to the conclusion that this form was not assignable to a hyperglycosylated status (C). Intriguingly, VSV-G* may represent a protein complex or an as-yet-unknown modification of the G protein that is thermostable and resistant to SDS; additionally, it does not dissociate under reducing conditions (DTT) and is not denatured by urea. Moreover, exposure to an acidic pH at an elevated temperature did not to affect the detection of VSV-G* (data not shown).
The incubation of extracellular VSV with SP600125 did not lead to VSV-G* formation, nor did it hamper viral infectivity, ruling out a possible cross-linking activity of SP600125. Pretreatment alone with infection carried out in the absence of SP600125 resulted in reduced levels of VSV-G* and a partial recovery of viral titers.
Mass spectrometry analysis revealed that VSV-G* contains only peptides from the VSV G protein. In repeated experiments, we did not identify any cellular protein consistently in VSV-G* preparations, including the ones related to major posttranslational modifications, such as ubiquitination, sumoylation, neddylation, and ISGylation, etc. (23
). Despite the fact that we were able to exclude several mechanisms involved in protein modifications, the nature of VSV-G* still remains enigmatic, yet it is possible that unidentified modifications through covalent linkages are responsible for the formation of VSV-G*. Given the difficulty in identifying these processes, further studies will be required to address this important aspect.
The presence of VSV-G* species compromised the fusion activity of the VSV glycoprotein; in the presence of SP600125, the expression of VSV-G* led to a reduction in levels of syncytium formation. Since increased levels of VSV-G* expression depend on the SP600125 concentration and correlate inversely with viral titers, we postulate that SP600115 attenuates VSV by hampering the VSV glycoprotein fusogenic activity.
In conclusion, our in vitro
results support the concept that combination therapy using oncolytic VSV and MAPK inhibitors might result in synergistic antitumor activity against HCC, and we plan to test this hypothesis in future in vivo
studies. Furthermore, at the molecular level, we have provided new insights into the antiviral properties of the inhibitor SP600125. SP600125 also attenuates the growths of several viruses of different families, suggesting a possible common mechanism of action that could be exploited for the development of antiviral treatment. A very intriguing application of SP600125 could be as a treatment of viral infections that are accompanied by malignant transformation. Both antiviral and antitumor effects of the drug could have significant benefits, for example, in the treatment of hepatitis C virus or human papillomavirus infection (16
). The elucidation of viral posttranslational control and viral mechanisms of infectivity can also be investigated by means of the effect of SP600125 on VSV-G maturation, leading to the development of new and targeted antiviral strategies.