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author:("Kaur, baleen")
1.  Indirubins Decrease Glioma Invasion by Blocking Migratory Phenotypes in Both the Tumor and Stromal Endothelial Cell Compartments 
Cancer research  2011;71(16):5374-5380.
Invasion and proliferation in neoplasia require the cooperation of tumor cell and endothelial compartments. Glycogen synthase kinase-3 (GSK-3) is increasingly recognized as a major contributor to signaling pathways that modulate invasion and proliferation. Here we show that GSK-3 inhibitors of the indirubin family reduce invasion of glioma cells and glioma-initiating cell-enriched neurospheres both in vitro and in vivo, and we show that β-catenin signaling plays an important role in mediating these effects. Indirubins improved survival in glioma-bearing mice in which a substantial decrease in blood vessel density was seen in treated animals. In addition, indirubins blocked migration of endothelial cells, suggesting that anti-invasive glioma therapy with GSK-3 inhibitors in vivo not only inhibits invasion of tumor cells, but blocks migration of endothelial cells, which is also required for tumor angiogenesis. Overall, our findings suggest that indirubin inhibition of GSK-3 offers a novel treatment paradigm to target 2 of the most important interacting cellular compartments in heterotypic models of cancer.
doi:10.1158/0008-5472.CAN-10-3026
PMCID: PMC4288480  PMID: 21697283
2.  Oncolytic HSV-1 Virotherapy: Clinical Experience and Opportunities for Progress 
Current pharmaceutical biotechnology  2012;13(9):1842-1851.
Oncolytic virotherapy with mutants derived from Herpes simplex virus (HSV) type 1 exhibit significant antitumor effects in preclinical models. Several mutants have now been tested in clinical trials for a variety of cancer types, and all have been found to be safe. While there have been hints of antitumor efficacy with prolonged survival in some cases compared with historical controls, dramatic responses have been elusive. We review the clinical experience published to date and discuss some of the biologic factors that may be limiting for virus infection and spread, as well as new strategies currently under development to enhance antitumor efficacy.
PMCID: PMC4233127  PMID: 21740359
Herpes simplex virus; oncolytic viruses; tumor microenvironment; angiogenesis; extracellular matrix; cytokines; immune response
3.  DNA demethylating agents synergize with oncolytic HSV1 against malignant gliomas 
Purpose
Oncolytic viruses (OV) based on herpes simplex virus type 1 (HSV1) are being utilized in clinical trials for a variety of cancers. The OV, rQNestin34.5, utilizes a nestin promoter/enhancer to selectively drive robust viral replication in malignant glioma cells. We have discovered that this promoter becomes extensively methylated in infected glioma cells, reducing OV efficacy.
Experimental Design
We utilized demethylating drugs (5-azacytidine), Decitabine or Valproic Acid (VPA) in both in vitro and in vivo malignant glioma models to determine if they improved the efficacy of rQNestin34.5 therapy.
Results
Utilization of demethylating agents, such as 5-azacytidine (5-Aza), improved OV replication and tumor cell lysis in vitro and, in fact, synergized pharmacologically by Chou-Talalay analysis. In vivo the combination of the demethylating agents, 5-Aza or Decitabine, with rQNestin34.5 significantly prolonged the survivorship of athymic mice harboring intracranial human glioma xenografts over single agent alone.
Conclusion
These results thus provide further justification for the exploration of demethylating agents when combined with the OV, rQNestin34.5, in preclinical therapeutics and possibly clinical trials for malignant glioma.
doi:10.1158/1078-0432.CCR-12-3588
PMCID: PMC3860592  PMID: 24056786
Glioblastoma multiforme; experimental therapeutics; virotherapy; decitabine; rQNestin34.5
4.  FHIT Suppresses Epithelial-Mesenchymal Transition (EMT) and Metastasis in Lung Cancer through Modulation of MicroRNAs 
PLoS Genetics  2014;10(10):e1004652.
Metastasis is the principal cause of cancer death and occurs through multiple, complex processes that involve the concerted action of many genes. A number of studies have indicated that the Fragile Histidine Triad (FHIT) gene product, FHIT, functions as a tumor suppressor in a variety of common human cancers. Although there are suggestions of a role for FHIT loss in progression of various cancers, a role for such loss in metastasis has not been defined. Here, via in vivo and in vitro assays, we reveal that the enforced expression of FHIT significantly suppresses metastasis, accompanied by inhibition of the epithelial-mesenchymal transition (EMT), a process involved in metastasis through coordinate modulation of EMT-related genes. Specifically, miR-30c, a FHIT-upregulated microRNA, contributes to FHIT function in suppression of EMT and metastasis by directly targeting metastasis genes Metadherin (MTDH), High-mobility group AT—hook 2 (HMGA2), and the mesenchymal markers, Vimentin (VIM) and Fibronectin (FN1), in human lung cancer. Finally, we demonstrate that the expression pattern of FHIT and miR-30c is inversely correlated with that of MTDH and HMGA2 in normal tissue, non-metastatic and metastatic tumors, serving as a potential biomarker for metastasis in lung cancer.
Author Summary
Although Fragile Histidine Triad (FHIT) is known as a potential tumor suppressor gene in terms of tumor initiation and progression, the role of FHIT in the metastatic process is not well characterized. Here it is shown that FHIT reduces the motility and invasiveness of lung cancer cells in vitro and ability to metastasize in vivo, at least partially through the miR-30c-mediated suppression of EMT, a critical process during tumor metastasis. This study provides new insights into the role of FHIT and a FHIT-activated miRNA, miR-30c, as crucial modulators in lung metastasis.
doi:10.1371/journal.pgen.1004652
PMCID: PMC4207614  PMID: 25340791
5.  SapC-DOPS-induced lysosomal cell death synergizes with TMZ in glioblastoma 
Oncotarget  2014;5(20):9703-9709.
SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.
PMCID: PMC4259431  PMID: 25210852
SapC-DOPS; glioblastoma; lysosomal dysfunction; TMZ; synergy
6.  Cytomegalovirus Contributes to Glioblastoma in the Context of Tumor Suppressor Mutations 
Cancer research  2013;73(11):3441-3450.
To study the controversial role of cytomegalovirus (CMV) in glioblastoma, we assessed the effects of murine CMV (MCMV) perinatal infection in a GFAP-cre; Nf1loxP/+; Trp53−/+ genetic mouse model of glioma (Mut3 mice). Early on after infection, MCMV antigen was predominantly localized in CD45+ lymphocytes in the brain with active viral replication and local areas of inflammation, but, by 7 weeks, there was a generalized loss of MCMV in brain, confirmed by bioluminescent imaging. MCMV-infected Mut3 mice exhibited a shorter survival time from their gliomas than control Mut3 mice perinatally infected with mock or with a different neurotropic virus. Animal survival was also significantly shortened when orthotopic gliomas were implanted in mice perinatally infected with MCMV versus controls. MCMV infection increased phosphorylated STAT3 (p-STAT3) levels in neural stem cells (NSC) harvested from Mut3 mice subventricular zone, and, in vivo, there was increased p-STAT3 in NSCs in MCMV-infected compared with control mice. Of relevance, human CMV (HCMV) also increased p-STAT3 and proliferation of patient-derived glioblastoma neurospheres, whereas a STAT3 inhibitor reversed this effect in vitro and in vivo. These findings thus associate CMV infection to a STAT3-dependent modulatory role in glioma formation/progression in the context of tumor suppressor mutations in mice and possibly in humans.
doi:10.1158/0008-5472.CAN-12-3846
PMCID: PMC4136413  PMID: 23729642
7.  In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles 
We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.
doi:10.3791/51187
PMCID: PMC4120271  PMID: 24837630
Medicine; Issue 87; Saposin C (SapC); Dioleoylphosphatidylserine (DOPS); Brain tumor; Arthritis; Fluorophore; Fluorescence; Optical imaging; Multi-angle rotational optical imaging (MAROI)
8.  Choindroitinase ABC I-Mediated Enhancement of Oncolytic Virus Spread and Anti Tumor Efficacy: A Mathematical Model 
PLoS ONE  2014;9(7):e102499.
Oncolytic viruses are genetically engineered viruses that are designed to kill cancer cells while doing minimal damage to normal healthy tissue. After being injected into a tumor, they infect cancer cells, multiply inside them, and when a cancer cell is killed they move on to spread and infect other cancer cells. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove a major glioma ECM component, chondroitin sulfate glycosoamino glycans from proteoglycans without any deleterious effects in vivo. It has been shown that Chase-ABC treatment is able to promote the spread of the viruses, increasing the efficacy of the viral treatment. In this paper we develop a mathematical model to investigate the effect of the Chase-ABC on the treatment of glioma by oncolytic viruses (OV). We show that the model's predictions agree with experimental results for a spherical glioma. We then use the model to test various treatment options in the heterogeneous microenvironment of the brain. The model predicts that separate injections of OV, one into the center of the tumor and another outside the tumor will result in better outcome than if the total injection is outside the tumor. In particular, the injection of the ECM-degrading enzyme (Chase-ABC) on the periphery of the main tumor core need to be administered in an optimal strategy in order to infect and eradicate the infiltrating glioma cells outside the tumor core in addition to proliferative cells in the bulk of tumor core. The model also predicts that the size of tumor satellites and distance between the primary tumor and multifocal/satellite lesions may be an important factor for the efficacy of the viral therapy with Chase treatment.
doi:10.1371/journal.pone.0102499
PMCID: PMC4105445  PMID: 25047810
9.  In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles 
We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.
doi:10.3791/51187
PMCID: PMC4120271  PMID: 24837630
Medicine; Issue 87; Saposin C (SapC); Dioleoylphosphatidylserine (DOPS); Brain tumor; Arthritis; Fluorophore; Fluorescence; Optical imaging; Multi-angle rotational optical imaging (MAROI)
10.  NK cells impede glioblastoma virotherapy via NKp30 and NKp46 natural cytotoxicity receptors 
Nature medicine  2012;18(12):1827-1834.
The role of the immune response to oncolytic Herpes Simplex viral (oHSV) therapy for glioblastoma is controversial. Within hours of oHSV infection of human or syngeneic glioblastoma in mice, activated natural killer (NK) cells are recruited to the site of infection. This response significantly diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on NK cell natural cytotoxicity receptors (NCR) NKp30 and NKp46, whose ligands were up-regulated in oHSV-infected glioblastoma cells. HSV titers and oHSV efficacy were increased in Ncr1−/− mice and in a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These in vitro and in vivo (mouse) results demonstrate that glioblastoma virotherapy is partly limited by an antiviral NK cell response involving specific NCRs, uncovering novel potential targets to enhance cancer virotherapy.
doi:10.1038/nm.3013
PMCID: PMC3668784  PMID: 23178246
Herpes simplex virus; gene therapy; oncolytic virus; brain tumor; microglia; macrophages
11.  Anti-angiogenic Gene Therapy in the Treatment of Malignant Gliomas 
Neuroscience letters  2012;527(2):62-70.
More than four decades ago, Dr. Judah Folkman hypothesized that angiogenesis was a critical process in tumor growth. Since that time, there have been significant advances in understanding tumor biology and groundbreaking research in cancer therapy that have validated his hypothesis. However, in spite of extensive research, glioblastoma multiforme (GBM), the most common and malignant primary brain tumor, has gained little in the way of improved median survival. There have been several angiogenesis targets that have resulted in drugs that are in clinical trials or FDA approved for clinical use in several cancers. GBM is a highly angiogenic tumor and several drugs are showing promise in clinical trials with one (bevacizumab), clinically approved for use. We will review several possible angiogenic targets in GBM as well as the vector methodologies used for delivery. In addition, GBMs present several therapeutic challenges related to structure, tumor immune microenvironment and resistance to angiogenesis. To overcome these challenges will require novel approaches to improve therapeutic gene expression and vector biodistribution in the glioma.
doi:10.1016/j.neulet.2012.08.001
PMCID: PMC3471371  PMID: 22906922
VEGF; Vasculostatin; oncolytic virus; Astrocytoma; glioma; brain tumor
12.  Copper chelation enhances anti-tumor efficacy and systemic delivery of oncolytic HSV 
Purpose
Copper in serum supports angiogenesis and inhibits replication of wild type HSV-1. Copper chelation is currently being investigated as an anti-angiogenic and anti-neoplastic agent in patients diagnosed with cancer. Herpes simplex virus derived oncolytic viruses (oHSVs) are being evaluated for safety and efficacy in patients, but several host barriers limit their efficacy. Here, we tested if copper inhibits oHSV infection and replication and if copper chelation would augment therapeutic efficacy of oHSV.
Experimental Design
Subcutaneous and intracranial tumor bearing mice were treated with oHSV ± ATN-224 to evaluate tumor burden and survival. Virus replication and cell killing was measured in the presence or absence of the copper chelating agent ATN-224 and in the presence or absence of copper in vitro. Micro-vessel density and changes in perfusion were evaluated by immuno-histochemistry and DCE-MRI. Serum stability of oHSV was measured in mice fed with ATN-224. Tumor bearing mice were injected intravenously with oHSV; tumor burden and amount of virus in tumor tissue was evaluated.
Results
Combination of systemic ATN-224 and oHSV significantly reduced tumor growth and prolonged animal survival. Immunohistochemistry and DCE-MRI imaging confirmed that ATN-224 reduced oHSV-induced blood vessel density and vascular leakage. Copper at physiologically relevant concentrations inhibited oHSV replication and glioma cell killing and this effect was rescued by ATN-224. ATN-224 increased serum stability of oHSV and enhanced the efficacy of systemic delivery.
Conclusion
This study shows that combining ATN-224 with oHSV, significantly increased serum stability of oHSV and greatly enhanced its replication and antitumor efficacy.
doi:10.1158/1078-0432.CCR-12-0697
PMCID: PMC3784008  PMID: 22753591
Glioblastoma (GBM); Oncolytic Virus (OV); Herpes Simplex Virus-1 (HSV-1); ATN-224; Copper ions (Copper)
13.  Thymoquinone Inhibits Autophagy and Induces Cathepsin-Mediated, Caspase-Independent Cell Death in Glioblastoma Cells 
PLoS ONE  2013;8(9):e72882.
Glioblastoma is the most aggressive and common type of malignant brain tumor in humans, with a median survival of 15 months. There is a great need for more therapies for the treatment of glioblastoma. Naturally occurring phytochemicals have received much scientific attention because many exhibit potent tumor killing action. Thymoquinone (TQ) is the bioactive compound of the Nigella sativa seed oil. TQ has anti-oxidant, anti-inflammatory and anti-neoplastic actions with selective cytotoxicity for human cancer cells compared to normal cells. Here, we show that TQ selectively inhibits the clonogenicity of glioblastoma cells as compared to normal human astrocytes. Also, glioblastoma cell proliferation could be impaired by chloroquine, an autophagy inhibitor, suggesting that glioblastoma cells may be dependent on the autophagic pathway for survival. Exposure to TQ caused an increase in the recruitment and accumulation of the microtubule-associated protein light chain 3-II (LC3-II). TQ also caused an accumulation of the LC3-associated protein p62, confirming the inhibition of autophagy. Furthermore, the levels of Beclin-1 protein expression were unchanged, indicating that TQ interferes with a later stage of autophagy. Finally, treatment with TQ induces lysosome membrane permeabilization, as determined by a specific loss of red acridine orange staining. Lysosome membrane permeabilization resulted in a leakage of cathepsin B into the cytosol, which mediates caspase-independent cell death that can be prevented by pre-treatment with a cathepsin B inhibitor. TQ induced apoptosis, as determined by an increase in PI and Annexin V positive cells. However, apoptosis appears to be caspase-independent due to failure of the caspase inhibitor z-VAD-FMK to prevent cell death and absence of the typical apoptosis related signature DNA fragmentation. Inhibition of autophagy is an exciting and emerging strategy in cancer therapy. In this vein, our results describe a novel mechanism of action for TQ as an autophagy inhibitor selectively targeting glioblastoma cells.
doi:10.1371/journal.pone.0072882
PMCID: PMC3767730  PMID: 24039814
14.  STAT3 Activation Promotes Oncolytic HSV1 Replication in Glioma Cells 
PLoS ONE  2013;8(8):e71932.
Recent studies report that STAT3 signaling is a master regulator of mesenchymal transformation of gliomas and that STAT3 modulated genes are highly expressed in the mesenchymal transcriptome of gliomas. A currently studied experimental treatment for gliomas consists of intratumoral injection of oncolytic viruses (OV), such as oncolytic herpes simplex virus type 1 (oHSV). We have described one particular oHSV (rQNestin34.5) that exhibits potent anti-glioma activity in animal models. Here, we hypothesized that alterations in STAT3 signaling in glioma cells may affect the replicative ability of rQNestin34.5. In fact, human U251 glioma cells engineered to either over-express STAT3 or with genetic down-regulation of STAT3 supported oHSV replication to a significantly higher or lesser degree, respectively, when compared to controls. Administration of pharmacologic agents that increase STAT3 phosphorylation/activation (Valproic Acid) or increase STAT3 levels (Interleukin 6) also significantly enhanced oHSV replication. Instead, administration of inhibitors of STAT3 phosphorylation/activation (LLL12) significantly reduced oHSV replication. STAT3 led to a reduction in interferon signaling in oHSV infected cells and inhibition of interferon signaling abolished the effect of STAT3 on oHSV replication. These data thus indicate that STAT3 signaling in malignant gliomas enhances oHSV replication, likely by inhibiting the interferon response in infected glioma cells, thus suggesting avenues for possible potentiation of oncolytic virotherapy.
doi:10.1371/journal.pone.0071932
PMCID: PMC3732216  PMID: 23936533
15.  A proprotein convertase/MMP-14 proteolytic cascade releases a novel 40 kDa vasculostatin from tumor suppressor BAI1 
Oncogene  2012;31(50):5144-5152.
Brain angiogenesis inhibitor 1 (BAI1), an orphan GPCR-type seven transmembrane receptor, was recently found mutated or silenced in multiple human cancers and can interfere with tumor growth when overexpressed. Yet, little is known about the molecular mechanisms through which this novel tumor suppressor exerts its anti-cancer effects. Here, we demonstrate that the N-terminus of BAI1 is cleaved extracellularly to generate a truncated receptor and a 40 kDa fragment that inhibits angiogenesis. We demonstrate that this novel proteolytic processing event depends on a two-step cascade of protease of activation: proprotein convertases, primarily furin, activate latent matrix metalloproteinase 14, which then directly cleaves BAI1 to release the bioactive fragment. These findings significantly augment our knowledge of BAI1 by showing a novel posttranslational mechanism regulating BAI1 activity through cancer-associated proteases, have important implications for BAI1 function and regulation, and present novel opportunities for therapy of cancer and other vascular diseases.
doi:10.1038/onc.2012.1
PMCID: PMC3355202  PMID: 22330140
16.  Gene expression profiling of the anti-glioma effect of Cilengitide 
SpringerPlus  2013;2:160.
Cilengitide (EMD121974), an inhibitor of the adhesive function of integrins, demonstrated preclinical efficacy against malignant glioma. It is speculated that cilengitide can inhibit tumor growth, invasion, and angiogenesis. However, the effects of cilengitide on these processes have not been sufficiently examined. In this study, we investigated the anti-glioma effect of cilengitide using DNA microarray analysis. U87ΔEGFR cells (human malignant glioma cell line) were used for this experiment. The cells were harvested after 16 h of cilengitide treatment, and mRNA was extracted. Gene expression and pathway analyses were performed using a DNA microarray (CodeLink™Human Whole Genome Bioarray). The expression of 265 genes was changed with cilengitide treatment. The expression of 214 genes was up-regulated by more than 4-fold and the expression of 51 genes was down-regulated by more than 4-fold compared to the controls. In pathway analysis, “apoptotic cleavage of cellular proteins” and “TNF receptor signaling pathway” were over-represented. Apoptotic-associated genes such as caspase 8 were up-regulated. Gene expression profiling revealed more detailed mechanism of the anti-glioma effect of cilengitide. Genes associated with apoptosis were over-represented following cilengitide treatment.
doi:10.1186/2193-1801-2-160
PMCID: PMC3647089  PMID: 23667810
Glioma; Integrin; Cilengitide; Gene expression profiling; Apoptosis
17.  Extracellular matrix protein CCN1 limits oncolytic efficacy in glioma 
Cancer Research  2012;72(6):1353-1362.
Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. CCN1 is an extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. In this study, we examined an hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Strikingly, we found that exogenous CCN1 in glioma ECM orchestrated a cellular antiviral response that reduced viral replication and limited cytolytic efficacy. Gene expression profiling and real time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1 exposure. This induction was accompanied by activation of the Jak/Stat signaling pathway, consistent with induction of an innate antiviral cellular response. Both effects were mediated by the binding of CCN1 to the cell surface integrin α6β1, activating its signaling and leading to rapid secretion of interferon-α, which was essential for the innate antiviral effect. Together, our findings reveal how an integrin signaling pathway mediates activation of a type-I antiviral interferon response that can limit the efficacy of oncolytic viral therapy. Further, they suggest therapeutic interventions to inhibit CCN1-integrin α6 interactions to sensitize gliomas to viral oncolysis.
doi:10.1158/0008-5472.CAN-11-2526
PMCID: PMC3366191  PMID: 22282654
CCN1; Cyr61; Glioma; Oncolytic Virus
18.  The Histone Deacetylase Inhibitor Valproic Acid Lessens NK Cell Action against Oncolytic Virus-Infected Glioblastoma Cells by Inhibition of STAT5/T-BET Signaling and Generation of Gamma Interferon 
Journal of Virology  2012;86(8):4566-4577.
Tumor virotherapy has been and continues to be used in clinical trials. One barrier to effective viral oncolysis, consisting of the interferon (IFN) response induced by viral infection, is inhibited by valproic acid (VPA) and other histone deacetylase inhibitors (HDACi). Innate immune cell recruitment and activation have been shown to be deleterious to the efficacy of oncolytic herpes simplex virus (oHSV) infection, and in this report we demonstrate that VPA limits this deleterious response. VPA, administered prior to oHSV inoculation in an orthotopic glioblastoma mouse model, resulted in a decline in NK and macrophage recruitment into tumor-bearing brains at 6 and 24 h post-oHSV infection. Interestingly, there was a robust rebound of recruitment of these cells at 72 h post-oHSV infection. The observed initial decline in immune cell recruitment was accompanied by a reduction in their activation status. VPA was also found to have a profound immunosuppressive effect on human NK cells in vitro. NK cytotoxicity was abrogated following exposure to VPA, consistent with downmodulation of cytotoxic gene expression of granzyme B and perforin at the mRNA and protein levels. In addition, suppression of gamma IFN (IFN-γ) production by VPA was associated with decreased STAT5 phosphorylation and dampened T-BET expression. Despite VPA-mediated immune suppression, mice were not at significantly increased risk for HSV encephalitis. These findings indicate that one of the avenues by which VPA enhances oHSV efficacy is through initial suppression of immune cell recruitment and inhibition of inflammatory cell pathways within NK cells.
doi:10.1128/JVI.05545-11
PMCID: PMC3318659  PMID: 22318143
19.  Phase IB Study of Gene-Mediated Cytotoxic Immunotherapy Adjuvant to Up-Front Surgery and Intensive Timing Radiation for Malignant Glioma 
Journal of Clinical Oncology  2011;29(27):3611-3619.
Purpose
Despite aggressive therapies, median survival for malignant gliomas is less than 15 months. Patients with unmethylated O6-methylguanine–DNA methyltransferase (MGMT) fare worse, presumably because of temozolomide resistance. AdV-tk, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, plus prodrug synergizes with surgery and chemoradiotherapy, kills tumor cells, has not shown MGMT dependency, and elicits an antitumor vaccine effect.
Patients and Methods
Patients with newly diagnosed malignant glioma received AdV-tk at 3 × 1010, 1 × 1011, or 3 × 1011 vector particles (vp) via tumor bed injection at time of surgery followed by 14 days of valacyclovir. Radiation was initiated within 9 days after AdV-tk injection to overlap with AdV-tk activity. Temozolomide was administered after completing valacyclovir treatment.
Results
Accrual began December 2005 and was completed in 13 months. Thirteen patients were enrolled and 12 completed therapy, three at dose levels 1 and 2 and six at dose level 3. There were no dose-limiting or significant added toxicities. One patient withdrew before completing prodrug because of an unrelated surgical complication. Survival at 2 years was 33% and at 3 years was 25%. Patient-reported quality of life assessed with the Functional Assessment of Cancer Therapy-Brain (FACT-Br) was stable or improved after treatment. A significant CD3+ T-cell infiltrate was found in four of four tumors analyzed after treatment. Three patients with MGMT unmethylated glioblastoma multiforme survived 6.5, 8.7, and 46.4 months.
Conclusion
AdV-tk plus valacyclovir can be safely delivered with surgery and accelerated radiation in newly diagnosed malignant gliomas. Temozolomide did not prevent immune responses. Although not powered for efficacy, the survival and MGMT independence trends are encouraging. A phase II trial is ongoing.
doi:10.1200/JCO.2011.35.5222
PMCID: PMC3179270  PMID: 21844505
20.  Extracellular Vesicles and Their Convergence with Viral Pathways 
Advances in Virology  2012;2012:767694.
Extracellular vesicles (microvesicles), such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. Microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. Here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. Herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. In addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. We highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. Finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. More research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies.
doi:10.1155/2012/767694
PMCID: PMC3410301  PMID: 22888349
21.  Choindroitinase ABC I-mediated enhancement of oncolytic virus spread and anti tumor efficacy 
Purpose
The inhibitory role of secreted Chondroitin-sulfate-proteoglycans (CSPGs) on Oncolytic viral (OV) therapy was examined. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove chondroitin sulfate glycoso-amino glycans from proteoglycans without any deleterious effects in vivo. We examined the effect of Chase-ABC on OV spread and efficacy.
Experimental Design
Three dimensional glioma spheroids placed on cultured brain slices were utilized to evaluate OV spread. Replication-conditional OV expressing Chase-ABC (OV-Chase) was engineered using HSQuik technology, and tested for spread and efficacy in glioma spheroids. Subcutaneous and intracranial glioma xenograft, were utilized to compare anti-tumor efficacy of OV-Chase, rHsvQ (control) and PBS. Titration of viral particles was performed from OV treated subcutaneous tumors. Glioma invasion was assessed in collagen embedded glioma spheroids in vitro, and in intracranial tumors. All statistical tests were two sided.
Results
Treatment by Chase-ABC in cultured glioma cells significantly enhanced OV spread in glioma spheroids grown on brain slices (P< 0.0001). Inoculation of subcutaneous glioma xenografts with Chase-expressing OV significantly increased viral titer (> 10 times, P=0.0008), inhibited tumor growth and significantly increased overall animal survival (P<0.006) compared to treatment with parental rHsvQ virus. Single OV-Chase administration in intracranial xenografts also resulted in longer median survival of animals compared to rHsvQ (32 versus 21 days, P<0.018). Glioma cell migration and invasion were not increased by OV-Chase treatment.
Conclusions
We conclude that degradation of glioma ECM by OV expressing bacterial Chase-ABC enhanced OV spread and anti-tumor efficacy.
doi:10.1158/1078-0432.CCR-10-2213
PMCID: PMC3140790  PMID: 21177410
22.  Deciphering the Multifaceted Relationship between Oncolytic Viruses and Natural Killer Cells 
Advances in Virology  2011;2012:702839.
Despite active research in virotherapy, this apparently safe modality has not achieved widespread success. The immune response to viral infection appears to be an essential factor that determines the efficacy of oncolytic viral therapy. The challenge is determining whether the viral-elicited immune response is a hindrance or a tool for viral treatment. NK cells are a key component of innate immunity that mediates antiviral immunity while also coordinating tumor clearance. Various reports have suggested that the NK response to oncolytic viral therapy is a critical factor in premature viral clearance while also mediating downstream antitumor immunity. As a result, particular attention should be given to the NK cell response to various oncolytic viral vectors and how their antiviral properties can be suppressed while maintaining tumor clearance. In this review we discuss the current literature on the NK response to oncolytic viral infection and how future studies clarify this intricate response.
doi:10.1155/2012/702839
PMCID: PMC3263705  PMID: 22312364
23.  Directing systemic oncolytic viral delivery to tumors via carrier cells 
Cytokine & growth factor reviews  2010;21(2-3):119-126.
doi:10.1016/j.cytogfr.2010.02.004
PMCID: PMC3109423  PMID: 20226717
24.  Impact of tumor microenvironment on oncolytic viral therapy 
Cytokine & growth factor reviews  2010;21(2-3):127-134.
Interactions between tumor cells and their microenvironment have been shown to play a very significant role in the initiation, progression, and invasiveness of cancer. These tumor-stromal interactions are capable of altering the delivery and effectiveness of therapeutics into the tumor and are also known to influence future resistance and re-growth after treatment. Here we review recent advances in the understanding of the tumor microenvironment and its response to oncolytic viral therapy. The multifaceted environmental response to viral therapy can influence viral infection, replication, and propagation within the tumor. Recent studies have unveiled the complicated temporal changes in the tumor vasculature post OV treatment, and their impact on tumor biology. Similarly, the secreted extracellular matrix in solid tumors can affect both infection and spread of the therapeutic virus. Together, these complex changes in the tumor microenvironment also modulate the activation of the innate antiviral host immune response, leading to quick and efficient viral clearance. In order to combat these detrimental responses, viruses have been combined with pharmacological adjuvants and “armed” with therapeutic genes in order to suppress the pernicious environmental conditions following therapy. In this review we will discuss the impact of the tumor environment on viral therapy and examine some of the recent literature investigating methods of modulating this environment to enhance oncolysis.
doi:10.1016/j.cytogfr.2010.02.014
PMCID: PMC2881175  PMID: 20399700
25.  Enhanced anti tumor efficacy of Vasculostatin (Vstat120) expressing oncolytic HSV-1 
Oncolytic viral therapy is a promising therapeutic modality for brain tumors. Vasculostatin (Vstat120), is the cleaved and secreted extracellular fragment of BAI1, a brain specific receptor. However, the therapeutic efficacy of Vstat120 delivery into established tumors has not been investigated. Here we tested therapeutic efficacy of combining Vstat120 gene delivery in conjunction with oncolytic viral therapy. We constructed a novel oncolytic virus RAMBO: Rapid Anti-angiogensis Mediated By Oncolytic virus, which expresses Vsta120 under the control of an immediate early viral promotor. Secreted Vstat120 was detectable in infected cells as soon as four hours post infection. Vstat120 produced by RAMBO efficiently inhibited endothelial cell migration and tube formation in vitro (P=0.0005 and P=0.0184 respectively) and inhibited angiogeneisis (P=0.007) in vivo. There was a significant suppression of tumors growth in mice bearing intracranial and subcutaneous gliomas treated with RAMBO compared to the control HSVQ treated mice (P=0.0021, and P<0.05 respectively). Statistically significant reduction in tumor vascular volume fraction and microvessel density was observed in tumors treated with RAMBO. This study is the first report of antitumor effects of Vstat120 in established tumors and supports the further development of RAMBO as a possible treatment for cancer.
doi:10.1038/mt.2009.232
PMCID: PMC2818668  PMID: 19844198
Glioma; Vasculostatin; Oncolytic virus; HSV-1; angiogenesis

Results 1-25 (34)