Kensuke Hirasawa

Reovirus Oncolysis of Human Breast Cancer

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.

Oncolytic Viral Therapy for Prostate Cancer: Efficacy of Reovirus as a Biological Therapeutic

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor.

Negative Regulation of the Alpha Interferon-Induced Antiviral Response by the Ras/Raf/MEK Pathway

ABSTRACT Interferon (IFN) is one of the molecules released by virus-infected cells, resulting in the establishment of an antiviral state within infected and neighboring cells. IFN-induced antiviral response may be subject to modulation by the cellular signaling environment of host cells which impact the effectiveness of viral replication. Here, we show that cells with an activated Ras/Raf/MEK signaling cascade allow propagation of viruses in the presence of IFN. Ras-transformed (RasV12) and vector control NIH 3T3 cells were infected with vesicular stomatitis virus (VSV) or an IFN-sensitive vaccinia virus (delE3L) in the presence of alpha interferon. While IFN protected vector control cells from infection by both viruses, RasV12 cells were susceptible to viral infection regardless of the presence of IFN. IFN sensitivity was restored in RasV12 cells upon RNA interference (RNAi) knockdown of Ras. We further investigated which elements downstream of Ras are responsible for counteracting IFN-induced antiviral responses. A Ras effector domain mutant that can only stimulate the Raf kinase family of effectors was able to suppress the IFN response and allow VSV replication. IFN-induced antiviral mechanisms were also restored in RasV12 cells by treatment with a MEK inhibitor (U0126 or PD98059). Moreover, by using RNAi to MEK1 and MEK2, we determined that MEK2, rather than MEK1, is responsible for suppression of the IFN response. In conclusion, our results suggest that activation of the Ras/Raf/MEK pathway downregulates IFN-induced antiviral response.

Activated Ras/MEK Inhibits the Antiviral Response of Alpha Interferon by Reducing STAT2 Levels

ABSTRACT The ability of interferon (IFN) to induce the expression of antiviral genes, and therefore suppress viral infection, is dependent on the activity of cellular suppressors. The Ras/MEK pathway is one of these cellular suppressors, since the activation of Ras/MEK permits viral replication in the presence of alpha IFN (IFN-α). Here, we have investigated the mechanism by which activated Ras/MEK inhibits the IFN-α response. We found that the induction of antiviral proteins in response to IFN-α was impaired in Ras-transformed NIH 3T3 (RasV12) cells. The inhibition of the Ras/MEK pathway restored the IFN-mediated induction of antiviral genes, indicating that activated Ras interrupts the IFN pathway upstream of antiviral gene transcription. Indeed, the IFN-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2 was inhibited in RasV12 cells compared to that of vector control cells. In addition, we found that the total amount of STAT2 was reduced in RasV12 cells. To determine if the impaired IFN-α response can be rescued by restoring the overall level of STAT2, we overexpressed STAT2 in RasV12 cells. The IFN-α-induced phosphorylation of STAT1 and STAT2, as well as the expression of antiviral protein, were restored, and IFN-induced antiviral protection was partially restored. Moreover, we demonstrated that the downregulation of STAT2 levels by Ras/MEK was mediated at the transcriptional level. Thus, the activation of the Ras/MEK pathway reduces the amount of STAT2 available for propagating the IFN signal, resulting in the impairment of the IFN-α-induced antiviral response.

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

Bicistronic vectors are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. Internal ribosome entry site (IRES) elements have been utilized to initiate expression of an additional protein from a bicistronic vector. The IRES element commonly used in current bicistronic vectors originates from the encephalomyocarditis virus (EMCV). As IRES-mediated translation is dependent on availability of IRES trans-acting factors, which vary between cell types and species, adequate gene expression from the EMCV IRES element is not always achieved. To identify a novel IRES element that mediates gene expression consistently with a higher efficiency than the EMCV IRES, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the vascular endothelial growth factor and type 1 collagen-inducible protein (VCIP) IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results suggest that VCIP-IRES containing vectors improve gene expression compared with those harboring an EMCV-IRES. This could increase the potential benefits of bicistronic vectors for experimental and therapeutic purposes.

Suppression of IFN-Induced Transcription Underlies IFN Defects Generated by Activated Ras/MEK in Human Cancer Cells

Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes.

Oncogenic Ras inhibits IRF1 to promote viral oncolysis

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. Previously, we demonstrated that oncogenic RAS/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. Here, we investigated how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, namely guanylate-binding protein 2 and IFN gamma inducible protein 47 (Ifi47), we identified the IFN regulatory factor 1 (IRF1) binding site as the promoter region responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild type mouse embryonic fibroblasts (MEFs), but not in IRF1−/− MEFs, showing that IRF1 is involved in MEK-mediated downregulation of IFN-inducible genes. Furthermore, IRF1 protein expression was lower in RasV12 cells compared with vector control NIH3T3 cells, but was restored to equivalent levels by inhibition of MEK. Similarly, the restoration of IRF1 expression by MEK inhibition was observed in human cancer cells. IRF1 re-expression in human cancer cells caused cells to become resistant to infection by the oncolytic vesicular stomatitis virus strain. Together, this work demonstrates that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis.

Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E2) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER− and ER+ breast cancer cell lines, we showed that E2 attenuated HLA-DR in two ER+ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER− lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα+ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα− VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E2 and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E2 treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E2 inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E2 in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα− breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα+ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.

Promotion of viral internal ribosomal entry site- mediated translation under amino acid starvation

Cap-dependent and internal ribosomal entry site (IRES)-mediated translation are regulated differently within cells. Viral IRES-mediated translation often remains active when cellular cap-dependent translation is severely impaired under cellular stresses induced by virus infection. To investigate how cellular stresses influence the efficiency of viral IRES-mediated translation, we used a bicistronic luciferase reporter construct harbouring IRES elements from the following viruses: encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV) or human rhinovirus (HRV). NIH3T3 cells transfected with these bicistronic reporter constructs were subjected to different cellular stresses. Increased translation initiation was only observed under amino acid starvation when EMCV or FMDV IRES elements were present. To identify cellular mechanisms that promoted viral IRES-mediated translation, we tested the involvement of eukaryotic initiation factor 4E-binding protein (4E-BP), general control non-depressed 2 (GCN2) and eukaryotic initiation factor 2B (eIF2B), as these are known to be modulated under amino acid starvation. Knockdown of 4E-BP1 impaired the promotion of EMCV and FMDV IRES-mediated translation under amino acid starvation, whereas GCN2 and eIF2B were not involved. To further investigate how 4E-BP1 regulates translation initiated by EMCV and FMDV IRES elements, we used a phosphoinositide kinase-3 inhibitor (LY294002), an mTOR inhibitor (Torin1) or leucine starvation to mimic 4E-BP1 dephosphorylation induced by amino acid starvation. 4E-BP1 dephosphorylation induced by the treatments was not sufficient to promote viral IRES-mediated translation. These results suggest that 4E-BP1 regulates EMCV and FMDV IRES-mediated translation under amino acid starvation, but not via its dephosphorylation.

Restoration of IRF1-dependent anticancer effects by MEK inhibition in human cancer cells

Interferon regulatory factor (IRF1) is a potent antiviral, antitumor and immune regulatory protein. Recently, we found that activated Ras/MEK inhibits antiviral response by downregulating IRF1 expression and renders cancer cells susceptible to oncolytic viruses. In this study, we sought to determine whether IRF1 downregulation underlies oncogenesis induced by Ras/MEK activation in human cancer cells. Treatment of the MEK inhibitor U0126 promoted IRF1 expression in 7 of 11 cancer cell lines we tested. IRF1 promotion was also observed in human cancer cell lines treated with different MEK inhibitors or with RNAi oligonucleotides against extracellular signal-regulated kinases (ERKs). Restoration of the expression of antitumor genes, p27 and p53 upregulated modulator of apoptosis (PUMA), by MEK inhibition was less in IRF1 shRNA knockdown cancer cells than in vector control cancer cells, suggesting that Ras/MEK targets IRF1 for the downregulation of the antitumor genes. Moreover, apoptosis induction by U0126 was significantly reduced in IRF1 shRNA knockdown cells than vector control cells. This study demonstrates that IRF1 expression is suppressed by activated Ras/MEK in human cancer cells and that IRF1 plays essential roles in apoptosis induced by Ras/MEK inhibition.