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original article

The RAS/Raf1/MEK/ERK Signaling Pathway Facilitates VSV-mediated Oncolysis: Implication for the Defective Interferon Response in Cancer Cells Josh A Noser1, Amber A Mael1, Ryuta Sakuma1, Seiga Ohmine1, Paola Marcato2, Patrick WK Lee2 and Yasuhiro Ikeda1 Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, Minnesota, USA; 2Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada 1

Vesicular stomatitis virus (VSV) can replicate in malignant cells more efficiently than in normal cells. Although the selective replication appears to be caused by defects in the interferon (IFN) system in malignant cells, the mechanisms which render these cells less responsive to IFN remain poorly understood. Here we present evidence that an activated RAS/Raf1/MEK/ERK pathway plays a critical role in the defects. NIH 3T3 or human primary cells stably expressing active RAS or Raf1 were rapidly killed by VSV. Although IFNα treatment no longer protected the RAS- or Raf1-overexpressing cells from VSV infection, responsiveness to IFNα was restored following treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Similarly, human cancer-derived cell lines became more responsive to IFNα in conjunction with U0126 treatment. Intriguingly, dual treatment with both IFNα and U0126 severely reduced the levels of viral RNAs in the infected cells. Moreover, cancer cells showed defects in inducing an IFNα-responsive factor, MxA, which is known to block VSV RNA synthesis, and U0126 restored the MxA expression. Our observations suggest that activation of the extracellular signal-regulated protein kinase (ERK) signaling leads to the defect in IFNα-mediated upregulation of MxA protein, which facilitates VSV oncolysis. In view of the fact that 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway, VSV would be an ideal oncolytic virus for targeting such cancers. Received 10 October 2006; accepted 2 April 2007; published online 15 May 2007. doi:10.1038/sj.mt.6300193

Introduction In developed countries cancer is a major public health problem.1 Mutated RAS signaling is found in many forms of cancer, and the oncogenic RAS promotes metastasis, angiogenesis, and loss of growth control. In addition to mutated activation, RAS signaling

can also be constitutively activated by members of the epidermal growth factor family of receptor tyrosine kinases as well as other tyrosine kinases which are commonly over-expressed in cancers. Consequently RAS has been shown to be the modulator of tumor cell invasion and metastasis caused by tyrosine kinases.2 Within the RAS signaling pathway, RAS/Raf/MEK/ERK signaling is critically important for RAS-mediated transformation, as shown in rodent models.3–5 Although RAS activation alone may not be enough to transform human cells,6 it is significant that 30% of all cancers have constitutive activation of the RAS/Raf/MEK/ERK pathway.7 Importantly, these are most often cancers that offer limited therapeutic options.8 Many viruses have oncolytic activity, and are currently being assessed for use in cancer therapy.9–17 More than 50 phase I, phase II, or phase III clinical trials based on oncolytic viruses are currently open. These trials include adenoviruses (phase III), reovirus, herpes simplex virus, and measles virus (for a review see ref. 18). Vesicular stomatitis virus (VSV) has many favorable features for cancer virotherapy, in that it is able to replicate in malignant cells more efficiently than in normal cells and shows potent oncolytic properties.19 The primary immune defense against VSV infection is through the interferon (IFN) response.20,21 Phosphorylation of the doublestranded RNA-dependent protein kinase PKR, in combination with the IFN response, is necessary for the IFN-mediated blockage of VSV in normal cells.19,22 Dysfunction of the IFN response occurs during the evolution of many cancers.23–28 VSV is highly sensitive to the antiviral state induced by IFN signaling in normal cells; therefore the virus replicates selectively in cancer cells, with defects in the IFN system.19 A better understanding of the mechanism that enables VSV’s to replicate preferentially in cancer cells will open new possibilities for utilizing VSV as an oncolytic vector. In this study, we examined the effects on the oncolytic properties of VSV caused by signaling downstream of activated RAS. By using several mutants of RAS and downstream elements, as well as signaling inhibitors, we showed that an activated RAS/ Raf1/MEK/ERK pathway plays a major role in VSV-mediated oncolysis.

The first two authors contributed equally to this work. Correspondence: Yasuhiro Ikeda, Molecular Medicine Program, Mayo Clinic College of Medicine, Guggenheim 18-11c, 200 First Street SW, Rochester, Minnesota 55905, USA. E-mail: [email protected] Molecular Therapy vol. 15 no. 8, 1531–1536 aug. 2007

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Ras Enhances the VSV Oncolysis

Results Involvement of RAS effector pathways in VSV-mediated oncolysis We first asked whether activation of RAS signaling pathways could alter the oncolytic properties of VSV in NIH 3T3 cells. By retroviral transduction we established 3T3 cells stably expressing a constitutively active form of H-RAS (12V) or effector binding domain mutants of RAS, 12V35S and 12V40C. RAS12V40C is an active form of RAS that has lost the ability to bind Raf kinase and RalGEFs but retains signaling capability to PI3-kinase, whereas the RAS12VS35 mutant cannot activate RalGEFs or PI3-kinase but does stimulate Raf activity.29 3T3 cells infected by a retroviral vector carrying the empty vector pBabe-puro were used as controls. Following confirmation of H-RAS expression in the cells (Figure 1a), the 3T3 cell lines were infected with a green fluorescent protein (GFP)-expressing VSV at a multiplicity of infection (MOI) of 0.05. Sixteen hours after infection, the infected cells were examined under a UV microscope. The RAS12V- and RAS12V35Spositive cells expressed very high levels of GFP and showed remarkable fusion formation, while RAS12V40C-expressing and control cells showed no apparent cytotoxic effects (Figure 1b). Although the RAS12V40C and control cells were eventually killed

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Figure 1 Involvement of RAS effector pathways in vesicular stomatitis virus (VSV)-mediated oncolysis. (a) 3T3 cells were infected with retroviral vectors coding for the active form of RAS (RAS12V) and two RAS mutants (12V35S and 12V40C), and an empty vector control (Ctrl). Western blotting was performed to verify RAS expression in the 3T3 cell lines. (b) The cells were infected with VSV expressing green fluorescent protein (GFP) at a multiplicity of infection (MOI) 0.05 and the cytopathic effects were observed 18 hours after infection. Following VSV infection, efficient oncolysis was evident in the RAS12V35S (nonRalGEF or PI3 kinase signaling)-expressing but not in the RASV12C40 (non-Raf/MEK/ERK signaling)-expressing cell lines. (c) Detection of elevated levels of phosphorylated ERK (p-ERK1 and p-ERK2) in the 3T3 cells over-expressing the active-form of Raf1 by Western blotting. (d) Control, active-forms of RAS- or Raf1-expressing 3T3 cells were infected with the GFP-expressing VSV at an MOI of 0.05. Twelve hours after infection, GFP-positive cells were analyzed by fluorescence-activated cell sorting. The numbers indicate the percentage of GFP-positive cells. The mean fluorescent intensity (MFI) of the GFP-positive population is also shown. UV, ultraviolet; TM, transmission.

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within 3 days after infection, it was clear that the active RAS12V effector pathways did enhance VSV-mediated oncolysis. Because VSV glycoprotein (VSV G protein) generally requires acidification in order to mediate fusion between the membrane of the virus and the membrane of the endosome, it is possible that the VSV G protein acquires a fusion-competent form in RAS12V-expressing cells during exocytotic transport, through intracellular exposure to low pH, as previously reported in HEC-1A cells.30 Our observation that constitutive expression of RAS12V35S, but not RAS12V40C, enhances the oncolytic properties of VSV suggested that Raf kinase activation was involved in the RASmediated enhancement of VSV replication. In order to examine whether the Raf1/MEK/ERK pathway was involved in the RASmediated increase in permissiveness to VSV, we made a 3T3 cell line stably expressing an active form of Raf1. We confirmed that the 3T3 cells with active Raf1 had elevated levels of phosphorylated extracellular signal-regulated protein kinase1/2 (ERK1/2) (Figure 1c). As in the case of RAS12V, active Raf1 expression also made 3T3 cells more permissive to VSV (Figure 1d and Figure 2a), thereby indicating that the Raf1/MEK/ERK signaling pathway is responsible for the enhanced oncolysis. Interestingly, the sizes of syncytia formed in the Raf1-positive cells were smaller than those observed in the RAS12V- and RAS12V35S-expressing cells (Figure 1b and Figure 2a). This suggests the involvement of another RAS effector pathway(s), such as RalGEF or PI3 kinase signaling pathway, in the large fusion formation by VSV.

The role of the RAS/Raf1/MEK/ERK signaling pathway in creating a defective IFNα system Approximately 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway.7 It has been suggested that many cancer cells do not fully respond to IFNα.19 We therefore hypothesized that the RAS/Raf1/MEK/ERK pathway might play a role in causing defects in the IFN system. We pre-treated RAS12V-positive, Raf1-positive, and control 3T3 cells with IFNα and/or with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, for 6 hours. The cells were then challenged with VSV at an MOI of 0.05. We used the p38 inhibitor SB203580 as a control. The infected cells were examined 24 hours after infection under a microscope (Figure 2a), and the culture supernatants were harvested for viral titration assay (Figure 2b). IFNα almost completely blocked VSV replication in the control 3T3 cells (Figure 2a). Indeed, the IFNα-treated control cells were apparently free from GFP-positive cells for more than 5 days (data not shown). In sharp contrast, the RAS12V- and Raf1positive 3T3 cells did not respond efficiently to the IFNα treatment (Figure 2a and b) and were killed within 2 days. These observations strongly indicate that activation of RAS or Raf1 weakens the IFNα-dependent anti-VSV effects. In order to demonstrate the involvement of down stream MEK/ERK signaling in causing the defect, we treated the cells with U0126, which inhibits both active and inactive MEK1/2. As we expected, pre-treatment with U0126 rescued the IFNα response in the Raf1- and RAS12V-expressing 3T3 cells (Figure 2a and b), while U0126 alone did not strongly reduce the permissivity of the RAS 3T3 cells. The U0126-treated Raf1-positive 3T3 cells showed a permissivity to VSV very similar to that of the control 3T3 cells www.moleculartherapy.org vol. 15 no. 8 aug. 2007


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Figure 2 Involvement of the RAS/Raf1/MEK/ERK signaling pathway in the defects of interferon α (IFNα) system. (a) 3T3 RAS, Raf1, and empty vector control cells were tested for responsiveness to INFα and/or U0126 treatment. A p38 inhibitor, SB203580, was used as a control. Expression of RAS 12V or Raf1 rendered cells less responsive to IFNα treatment. (b) Progeny viral production from the IFNα and/or U0126treated cells. The IFNα- or U0126-treated cells were infected at a multiplicity of infection of 0.05 for 24 hours. The culture supernatants were harvested and their viral titers were measured on 293T cells as described in Materials and Methods. (c) Negative regulation of the IFNαmediated antiviral response by RAS in primary human cardiac fibroblast cells. Western blot analysis was performed to ensure RAS expression. The cells were infected as before and the viral titers were measured at 24 hours after infection on 293T cells.

(Figure 2a). Given that U0126 blocks phosphorylation of ERK by MEK, our results indicate that the RAS/Raf1/MEK/ERK pathway causes the deficient IFNα response. We also demonstrated this by using primary human cardiac fibroblast (HCF) cells. As with NIH 3T3 cells, HCF cells stably expressing RAS12V were less responsive to IFNα treatment (Figure 2c), and additional U0126 treatment potentiated the response to IFNα in the RAS12V-positive cells.

Restoration of the deficient IFNα response in cancer cells by the MEK inhibitor We then examined whether defects in the IFNα response in actual cancer-derived cells are caused by activation of the RAS/Raf1/MEK/ ERK pathway. We picked nine commonly used cancer-derived cell lines and examined their responsiveness to IFNα treatment. We also examined 293T cells, which stably express adenoviral proteins and simian virus 40 large T antigen. The 10 cell lines were pre-treated with IFNα and/or U0126 for 6 hours and then infected by VSV at an MOI of 0.05. At day 2 after infection, almost all the cancer Molecular Therapy vol. 15 no. 8 aug. 2007

Figure 3 Restoration by U0126 of defects in the interferon α (IFNα) response of cancer cells. (a) Cancer-derived cell lines were pre-treated with IFNα and U0126 for 12 hours then infected at a multiplicity of infection (MOI) of 0.05. Two days after infection, the infected cells were observed under the microscope. (b) Culture supernatants were harvested 18 hours after infection at an MOI 0.05. The viral titers were determined on 293T cells.

cells were killed by VSV (Figure 3a). As reported previously,19 in all cancer cell lines, except MDA468 and CFPAC1, IFNα treatment alone could not efficiently block VSV replication and failed to protect the cells from lytic infection (Figure 3a). However, the protection from lytic infection was enhanced by IFNα treatment in conjunction with U0126 treatment. Figure 3b shows the results of the multiple cancer cell lines screened for VSV production. The cancer cells were infected by VSV at an MOI of 0.05 for 24 hours and their supernatants were examined for progeny virus production. The data shows that the IFNα response was consistently enhanced, to varying degrees, with U0126 treatment (Figure 3b), thereby suggesting that the impaired IFNα-mediated blockage of VSV replication is caused by elevated levels of active MEK/ERK signaling. Exceptions were CFPAC1 and MDA468, which strongly responded to IFNα treatment alone, and 293T cells, which were highly permissive to VSV even under dual treatment with both IFNα and U0126. Because 293T cells stably express adenoviral proteins as well as simian virus 40 large T antigen, we speculate that some of the viral proteins were efficient in abrogating the IFNα-mediated antiviral response.

Dual treatment with IFNα and U0126 drastically r educes the levels of VSV RNAs in cancer cells In order to understand further how U0126 enhances the IFNαmediated VSV block, we examined VSV protein synthesis in cancer cells. Four tumor cell lines were selected: HT1080, A375, and 1533



MDA231 for their enhanced IFNα-mediated antiviral activity upon U0126 treatment, and CFPAC1 for its very strong antiviral response to IFNα alone. The cell lines were incubated with IFNα and/or U0126 for 3 hours and subsequently challenged with VSV at an MOI of 2. Total cellular proteins were harvested at 12 hours after infection and probed for VSV-specific antigens. As reported previously,31 IFNα-treated cells showed significantly reduced levels of viral proteins (Figure 4a). Although U0126 treatment alone had little effects on VSV protein synthesis, VSV antigens were under detectable levels in the cells dual-treated in both IFNα and U0126. In order to determine how U0126 enhances the IFNα-mediated antiviral effects—whether it is by blocking viral protein synthesis or at an earlier step in the viral life cycle—we examined the effect of IFNα and/or U0126 treatment on the levels of viral RNAs in cancer cells. HT1080 and A375 cells were incubated with IFNα and/or U0126 overnight and, then challenged with VSV at an MOI of 20. Two hours after infection, total RNA was extracted and used in reverse transcriptase polymerase chain reaction (RT-PCR). The antisense primer for L gene was utilized to synthesize complementary DNA (cDNA) from the viral positive strand RNA (viral messenger RNAs and the genome-sense RNA), while the sense primer for L gene was utilized to synthesize cDNA from the negative strand RNA (viral genomic RNA). Semi-quantitative PCR was performed using the cDNAs to detect viral transcripts as well as viral genomic RNA. As shown in Figure 4b, IFNα treatment alone decreased the levels of positive strand RNA but showed little effect on the levels of negative strand RNA. Although U0126 treatment alone had no remarkable effect on the viral RNAs, dual treatment with U0126 and IFNα strongly reduced the levels of positive strand RNA and, to a lesser degree, the negative strand RNA. We also examined

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Figure 4 Dual treatment with interferon α (IFNα) and U0126 reduced the levels of viral RNAs in cancer cells. (a) In order to examine the effects of IFNα and/or U0126 treatment on viral protein synthesis, Western blotting was performed to detect vesicular stomatitis virus (VSV)-specific protein expression in the infected cells. (b) VSV-specific negative- and positive-stranded RNAs were detected by reverse transcriptase polymerase chain reaction (RT-PCR). As a control, α-tubulin complementary DNA (cDNA) was amplified by RT-PCR. (c) Primary human cardiac fibroblast (HCF) cells and cancer cell lines were treated with IFNα and/or U0126 overnight. Induction of MxA-specific RNA was examined by RT-PCR. As a control, the same RNA samples were utilized to amplify α-tubulin cDNA.

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the levels of viral messenger RNA of the N gene, as well as a control α-tubulin transcript, by using the cDNA synthesized with an oligo(dT) primer. Again, IFNα treatment alone decreased the levels of positive strand viral RNA, while dual treatment with U0126 and IFNα strongly reduced the levels of positive strand RNA. In contrast, treatment with either U0126 or IFNα did not strongly affect the levels of α-tubulin transcript in the treated cells. Our RT-PCR results (Figure 4b) suggested that U0126 enhances the IFNα-mediated antiviral effects by affecting the levels of viral RNA, rather than by blocking viral protein synthesis. Since an IFNα-inducible factor, MxA, is known to restrict VSV replication by inhibiting messenger RNA synthesis,32 we examined the possible link between U0126 treatment and induction of MxA expression. HT1080, A375, and primary HCF cells were incubated with IFNα and/or U0126 overnight. Total RNA was extracted and 2.0 μg of RNA was utilized to synthesize the first strand DNA with an oligo(dT) primer. One-tenth of the reverse transcription products were used for amplifying MxA-specific cDNA with primers, 5'-CTGATCTCCAGGGTGATTAGCTCAT-3' and 5'-ATGGTTGTTTCCGAAGTGGACA-3'. As expected, MxA induction was evident after IFNα treatment in primary HCF cells (Figure 4c). However, to our surprise, high levels of MxA messenger RNA were detectable only after U0126 treatment both in HT1080 and A375. In contrast, U0126 did not affect induction of another IFNα-inducible factor, RNase-L, which degrades both cellular and viral RNA species (data not shown). These data strongly suggest that elevated levels of RAS/Raf1/MEK/ERK signaling pathway negatively regulate the induction of MxA.

Discussion The rapid replication cycle and potent oncolytic properties of VSV have made it a promising candidate for cancer virotherapy. The aim of this study was to investigate the molecular signaling events that determine host cell permissiveness to VSV. We used model systems of NIH 3T3 and human fibroblast cells to examine whether activation of the RAS signaling pathway could render the cells more susceptible to VSV. We found that RAS-overexpressing cells were rapidly killed by VSV infection (Figure 1). Following VSV infection, efficient oncolysis was evident in the RAS12V35S (non-RalGEF nor PI3 kinase signaling) mutant-expressing cell lines, but not in the RASV12C40 (non-Raf/MEK/ERK signaling) mutant-expressing ones, thereby suggesting that the RAS/Raf1/ MEK/ERK pathway is important for VSV-mediated oncolysis. The use of Raf1-expressing 3T3 cells and the MEK inhibitor U0126 further confirmed that the oncolytic properties of VSV are dependent on Raf/MEK/ERK signaling. The ERK signaling pathway plays an essential role in the replication life cycle of certain viruses, including Coxsackievirus, influenza A, Vaccinia and Borna virus.33–36 For example, Coxsackievirus B3 infection activates ERK1/2, and inhibition of this activation decreases progeny virus release.34 Similarly, infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ ERK cascade; inhibition of Raf signaling results in nuclear retention of the viral ribonucleoprotein complexes and impaired function of the nuclear-export protein and concomitant inhibition of virus production.36 Our study clearly shows that constitutive activation of RAS/Raf1/MEK pathway positively affects the oncolytic www.moleculartherapy.org vol. 15 no. 8 aug. 2007


roperties of VSV. This can be partly explained by the enhanced p VSV replication/production upon activation of this pathway. The other notable effect of RAS or Raf1 over-expression was the negative regulation of the IFNα-induced antiviral responses (Figure 2). Intriguingly, the MEK inhibitor U0126 enhanced the antiviral activity of IFNα in tumor cells (Figure 3), thereby indicating that constitutive activation of the MEK/ERK pathway in malignant cells also plays an important role in the defective IFNα-mediated antiviral response found in many cancer cell lines. ERK activation has been shown to mediate innate antiviral responses including induction of type 1 IFN through interferon regulatory factor 3 (IRF3) activation.37 In contrast, our results showed negative regulation of the antiviral responses by MEK activation. These apparently opposing concepts can be explained by the dysregulation of ERK signaling. In the NIH 3T3 model, we found very high levels of phosphorylated IRF3 upon stable Raf1 expression (Supplementary Figure S1), which confirms the results from the previous study.37 However, such constitutive activation of IRF3 failed to induce notable antiviral responses in those cells. One possible explanation is that prolonged phosphorylation of IRF3 results in the dysfunction of its downstream antiviral pathway. It is likely that well-regulated ERK signaling is required for the optimal antiviral response through IRF3. IFNα induces antiviral responses through the Jak/STAT pathway.38 PKR is also required for an efficient IFNα-mediated antiviral response against VSV.19,22 We were not successful in our attempts to identify the possible link between ERK signaling and activation of these pathways (Supplementary Figures S2–4). Our observations are at variance with some of the previous studies using murine cell models,39,40 which showed the inhibition of PKR phosphorylation by RAS signaling through ERK activation. However, considering that IFNα could induce a certain level of antiviral response in all tumor-derived cell lines tested (Figure 3b), our finding that there is no apparent involvement of active MEK/ERK in Jak/STAT or PKR pathways (Supplementary Figure S4) may not be surprising. We were not alone in finding this, because in STAT1 or PKR knockout murine embryonic fibroblast cells, with or without RAS over-expression, the RAS-expressing cells were shown to have a marked increase in VSV replication kinetics.31 This suggests that the RAS-mediated enhancement in VSV replication/oncolysis is independent of the PKR- or STAT1-mediated antiviral signaling pathways. Recently, Dr. Barber and his colleague have reported frequent abnormal eIF2Bε expression in transformed and malignant cells, and have implied that the elevated levels of eIF2Bε are a likely reason for the increased permissiveness of transformed cells to VSV replication.31 We examined whether constitutive activation of ERK signaling controls the abnormal expression of eIF2Bε. Although constitutive eIF2Bε expression is readily detected in A375, HT1080, CFPAC1 (Supplementary Figure S4), and MDA231 (data not shown), U0126 treatment had no significant effects on the eIF2Bε expression in these cells (Supplementary Figure S4), thereby suggesting that there is no link between the ERK activation and the eIF2Bε expression. Unexpectedly, the most remarkable effect of IFNα and U0126 dual treatment on VSV replication was on the levels of viral RNAs, but not on the viral translation step (Figure 4b). The inhibition of MEK activity in the presence of IFNα severely reduced Molecular Therapy vol. 15 no. 8 aug. 2007


the positive- and negative-strand viral RNAs, while U0126 treatment alone did not strongly affect them. Intriguingly, we also found that, in cancer cells, IFNα treatment failed to induce an IFNα-responsive factor MxA, which is known to inhibit VSV RNA synthesis32 (Figure 4c). Moreover, MxA expression in cancer cells was restored by U0126 treatment. It is therefore conceivable that the negative regulation of IFN systems in cancer cells was in part due to the defect in inducing MxA protein. It remains to be determined why MxA upregulation alone, following U0126 treatment, did not affect the levels of viral RNAs in HT1080 and A375 cells. We assume that VSV protein(s) may counteract the MxA-mediated antiviral effects. If this is the case, viral replication can be severely affected by MxA when infected cells express significantly reduced levels of viral proteins following IFNα treatment. In the course of this study, using a NIH 3T3 and VSV model, Battcock et al.41 reported a negative regulation of IFNα by the RAS/Raf/MEK pathway. By using small interfering RNA specific to MEK1 and MEK2, the authors demonstrated that MEK2 is responsible for this negative regulation. It may therefore be interesting to see the effects of MEK2 knockout on the MxA protein induction in human cancer cells. Collectively, our study has demonstrated that RAS/Raf/MEK/ ERK signaling enhances VSV-mediated oncolytic properties. Activation of this pathway appears to enhance VSV replication/ oncolysis in cancer cells through negative regulation of IFNαmediated response. Given that 30% of all cancers have constitutive activation of the RAS/Raf/MEK/ERK pathway, and that most of these are cancers offering only limited therapeutic options,7,8 VSV should serve as an ideal oncolytic agent to treat such cancers.

MATERIALS AND METHODS Cells. NIH 3T3 cells were obtained from American Type Culture Collection

(Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium with 4 mmol/l L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l glucose (American Type Culture Collection, Manassas, VA) supplemented with 10% calf serum and antibiotics. In order to minimize spontaneous transformation, cells were used by no more than 10 passages. HCF cells were grown in fibroblast media (Sciencell, San Diego, CA) and used for no more than 6 passages. The following cell lines were from American Type Culture Collection (Manassas, VA): CFPAC-1 (CRL-1918), HT-1080 (CCL121), MIA PaCa-2 (CRL-1420), A375 (CRL-1619), A549 (CCL-185), Hep 3B (HB-8064), and HT 29 (HTB-38). MDA-231 is from ECACC (92020424, Salisbury, UK) and MDA-468 is from ICLC (HTL99024, Genova, Italy). All cancer cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum and antibiotics. Universal type I IFN, constructed from the recombinant human IFNs alpha A and alpha D, was obtained from PBL Biomedical Laboratories (Piscataway, NJ) and used at 100 U/ml. U0126 (Promega, Madison, WI) was dissolved in dimethyl sulfoxide and used at 2.5 µmol/l. Viruses. H-RAS-expressing plasmids, pBabe-puro-RAS(12V), pBabe-

puro-RAS(12V35S), pBabe-puro-RAS(12V40C), and their control, pBabepuro, were kindly provided by Dr. Channing Der. pBabe-puro-Raf1 was made by cloning the active form of Raf1 fragment into the BamHI-EcoRI site of pBabe-puro. The active form of Raf1 has a deletion in the CR1 region of human Raf1. The infectious retroviral vectors were made as previously described.42 The NIH 3T3 cells expressing H-RAS mutants, Raf1 and control, as well as HCF RAS cells were all made by retroviral transduction and selection in the presence of puromycin (2 μg/ml). VSV-expressing GFP was a kind gift from Dr. Glen Barber. VSV titrations were performed in 96-well

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flat bottom plates. Fivefold serially diluted viruses were added to the wells with 2 × 104 293T cells. Virus infection was monitored through the detection of GFP-positive cells and the appearance of cytopathic effects. Western blotting analysis. All Western blots were run on SDS-PAGE gels and transferred to polyvinylidene fluoride membranes using the semi-dry method. Membranes were then blocked overnight. The antiH-RAS, p-STAT1 (Tyr701) antibodies were purchased from Upstate (Chicago, IL), and p-IRF3, p-PKR, p-eIF2α, and eIF2Bε were purchased from Cell Signaling (Danvers, MA). The p-ERK antibody was purchased from R&D systems. Anti-VSV sera from infected mice were kindly provided by Dr. T. Mikami. Immunostaining of cells. Approximately 5 × 104 cells were grown on

12 mm cover slips in 24-well plates overnight. The cells were treated with 100 U/ml of IFNα and/or 2.5 µmol/l U0126 for 2 hours, then washed with cold phosphate-buffered saline (PBS) and incubated with 3.7% formalin in PBS for 1 minute. Following the removal of the formalin the cells were treated with 1 ml of cold methanol for 1 minute. The cells were then treated with blocking solution (1% calf serum in PBS) for 30 minutes and incubated with anti- p-STAT1 (Tyr701) antibody (R&D Systems, Minneapolis, MN). After one hour of incubation, the cells were washed three times with PBS. The cells were then incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen, Carlsbad, CA) for 1 hour. The cells were again washed three times with cold PBS. The cover slips were then mounted on a slide. The images were obtained with a Zeiss LSM 510 confocal laser scanning microscope and processed with Zeiss imaging software.

Acknowledgments We express our appreciation and gratitude to Glen N. Barber (University of Miami School of Medicine) for providing VSV-expressing GFP and to Channing J. Der (University of North Carolina at Chapel Hill) for providing the mutant RAS constructs. We thank Stephen J. Russell and Roberto Cattaneo (Mayo Clinic) for helpful discussions. Funding for this work was provided by the Mayo Foundation for Research and Education.

Supplementary Material Figure S1. Raf1 overexpression resulted in constitutive IRF3 phosphorylation in NIH 3T3 cells. Figure S2. The influence of INFα and/or U0126 treatment on STAT1 phosphorylation at Y701 and subsequent nuclear translocation of STAT1(p-Y701). Figure S3. The influence of INFα and/or U0126 treatment on STAT1 phosphorylation at Y701. Figure S4. The effects of IFNα and/or U0126 treatment on phosphorylation of PKR, eIF2α, IRF3, and ERK, as well as eIF2Bε expression.

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