Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus
2010; Springer Nature; Volume: 2; Issue: 7 Linguagem: Inglês
10.1002/emmm.201000081
ISSN1757-4684
AutoresFrederik C. Roos, Andrew M. Roberts, Irene Hwang, Eduardo H. Moriyama, Andrew Evans, Stephanie S. Sybingco, Ian R. Watson, Letícia A. M. Carneiro, Craig Gedye, Stephen E. Girardin, Laurie Ailles, Michael A.S. Jewett, Michael Milosevic, Brian C. Wilson, John C. Bell, Sandy D. Der, Michael Ohh,
Tópico(s)RNA Interference and Gene Delivery
ResumoResearch Article15 July 2010Open Access Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus Frederik C. Roos Frederik C. Roos Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Department of Urology, Johannes Gutenberg University, Mainz, Germany Equal contribution Search for more papers by this author Andrew M. Roberts Andrew M. Roberts Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Equal contribution Search for more papers by this author Irene I. L. Hwang Irene I. L. Hwang Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Equal contribution Search for more papers by this author Eduardo H. Moriyama Eduardo H. Moriyama Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Andrew J. Evans Andrew J. Evans Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Stephanie Sybingco Stephanie Sybingco Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Ian R. Watson Ian R. Watson Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Cancer Research Program and Division of Haematology-Oncology, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Leticia A. M. Carneiro Leticia A. M. Carneiro Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Craig Gedye Craig Gedye Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada Search for more papers by this author Stephen E. Girardin Stephen E. Girardin Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Laurie E. Ailles Laurie E. Ailles Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada Search for more papers by this author Michael A. S. Jewett Michael A. S. Jewett Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Michael Milosevic Michael Milosevic Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Brian C. Wilson Brian C. Wilson Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author John C. Bell John C. Bell Center for Cancer Therapeutics, Ottawa Health Research Institute, Ottawa, Ontario, Canada Search for more papers by this author Sandy D. Der Sandy D. Der Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Michael Ohh Corresponding Author Michael Ohh [email protected] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Frederik C. Roos Frederik C. Roos Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Department of Urology, Johannes Gutenberg University, Mainz, Germany Equal contribution Search for more papers by this author Andrew M. Roberts Andrew M. Roberts Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Equal contribution Search for more papers by this author Irene I. L. Hwang Irene I. L. Hwang Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Equal contribution Search for more papers by this author Eduardo H. Moriyama Eduardo H. Moriyama Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Andrew J. Evans Andrew J. Evans Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Stephanie Sybingco Stephanie Sybingco Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Ian R. Watson Ian R. Watson Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Cancer Research Program and Division of Haematology-Oncology, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Leticia A. M. Carneiro Leticia A. M. Carneiro Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Craig Gedye Craig Gedye Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada Search for more papers by this author Stephen E. Girardin Stephen E. Girardin Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Laurie E. Ailles Laurie E. Ailles Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada Search for more papers by this author Michael A. S. Jewett Michael A. S. Jewett Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Michael Milosevic Michael Milosevic Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author Brian C. Wilson Brian C. Wilson Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada Search for more papers by this author John C. Bell John C. Bell Center for Cancer Therapeutics, Ottawa Health Research Institute, Ottawa, Ontario, Canada Search for more papers by this author Sandy D. Der Sandy D. Der Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Michael Ohh Corresponding Author Michael Ohh [email protected] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Frederik C. Roos1,2, Andrew M. Roberts1, Irene I. L. Hwang1, Eduardo H. Moriyama3, Andrew J. Evans1,4, Stephanie Sybingco1, Ian R. Watson1,5, Leticia A. M. Carneiro1, Craig Gedye6, Stephen E. Girardin1, Laurie E. Ailles6, Michael A. S. Jewett4, Michael Milosevic3, Brian C. Wilson3, John C. Bell7, Sandy D. Der1 and Michael Ohh *,1 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada 2Department of Urology, Johannes Gutenberg University, Mainz, Germany 3Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada 4Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, Ontario, Canada 5Cancer Research Program and Division of Haematology-Oncology, Hospital for Sick Children, Toronto, Ontario, Canada 6Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada 7Center for Cancer Therapeutics, Ottawa Health Research Institute, Ottawa, Ontario, Canada *Tel: +1-416-946-7922; Fax: +1-416-978-5959 EMBO Mol Med (2010)2:275-288https://doi.org/10.1002/emmm.201000081 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF-κB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre-emptively limiting viral replication. Recent evidence has shown that hypoxia-inducible factor (HIF) increases NF-κB-mediated anti-apoptotic response in clear-cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel–Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF-κB-dependent gene expression profile concomitant with a lack of interferon-mediated anti-viral response in VHL-null CCRCC, and that multiple established CCRCC cell lines, as well as early-passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF-κB dramatically reverses CCRCC cellular susceptibility to EMCV-induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre-clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF-κB-survival signature. The paper explained PROBLEM: Clear-cell renal cell carcinoma (CCRCC), the most common form of kidney cancer, is among the most resistant of cancers to radiation and chemotherapy. The inactivation of the VHL tumour suppressor protein, the principal negative regulator of the transcription factor known as HIF, causes the vast majority of CCRCC. There is no effective treatment for advanced CCRCC. RESULTS: Emerging evidence has shown that HIF whose overexpression is intimately linked to poor prognosis potentiates the activity of a critical cellular survival pathway mediated by NF-κB. Taking advantage of this property, this report describes the selective killing of CCRCC cells by EMCV in a manner that is dependent upon activation of HIF and the NF-κB-mediated survival pathway. EMCV acts as an oncolytic virus and suppresses human CCRCC tumour growth in a mouse dorsal skin window-chamber model system. IMPACT: This pre-clinical study proposes the use of EMCV as an oncolytic virus aimed at eradicating kidney tumours and potentially other tumours with elevated HIF and NF-κB pathways. Cancers that are particularly resistant to conventional cancer therapies may be paradoxically more susceptible to EMCV-induced killing since this virus exploits the very nature of cancer cells to evade cell death to exacerbate its own virulence. INTRODUCTION Apoptosis is an effective host defence mechanism against invading microbes (Teodoro & Branton, 1997). While many viruses have evolved to trigger apoptosis via several mechanisms, including viral disruption of cellular metabolism and cell cycle and recognition by cytotoxic T cells (Shen & Shenk, 1995), some viruses have instead evolved to evade host programmed cell death to promote viral replication and spread. For example, viruses encode proteins such as adenovirus E1B, baculovirus p35, cowpox virus CrmA and viral FLICE-inhibitory proteins (v-FLIPs) that directly inhibit caspases (Bertin et al, 1997; Boulakia et al, 1996; Bump et al, 1995; Ray et al, 1992; Thome et al, 1997). Mutant viruses lacking these anti-apoptotic genes trigger premature apoptotic death of host cells and consequently produce lower yields of progeny virus (Brooks et al, 1995; Hershberger et al, 1992; Pilder et al, 1984). These lines of evidence support a causal relationship between programmed cell death and viral virulence. NF-κB is a transcription factor that regulates host immune and inflammatory responses, and influences viral replication (Baeuerle & Henkel, 1994; Wang et al, 1996). Viral infection or proinflammatory cytokines such as TNF-α promote rapid E3 ubiquitin ligase-mediated degradation of inhibitory IκBα allowing the release of NF-κB to the nucleus. NF-κB transactivates numerous genes including those encoding interleukins, cytokines, acute phase proteins, and anti-apoptotic proteins (Hayden & Ghosh, 2004). In addition to the activation of the NF-κB-mediated survival response, TNF-α also triggers caspase-mediated apoptotic response whereby the eventual fate of a cell depends on the overall balance between these opposing pathways (Qi & Ohh, 2003). Cells deficient in NF-κB p65 subunit or expressing constitutively stable IκBα are therefore unable to mount NF-κB-dependent survival response and hence are highly susceptible to TNF-α-induced caspase-dependent apoptosis (Beg & Baltimore, 1996; Van Antwerp et al, 1996). These observations support the general notion that NF-κB activation is a protective response by the host to an invading pathogen. Paradoxically, however, some pathogens have strategies to potentiate NF-κB to exploit its anti-apoptotic functions to enhance their own replication and spread. For example, NF-κB p50 subunit knockout mice exhibit accelerated apoptosis of encephalomyocarditis virus (EMCV)-infected cells, which dramatically limits viral replication and consequently survive EMCV infection that otherwise readily kills wild-type (WT) mice (Schwarz et al, 1998; Sha et al, 1995). These results suggest that NF-κB-mediated anti-apoptotic response is required for unrestricted EMCV replication, spread and virulence. Evasion of apoptosis is also a cardinal feature of cancer. For example, clear-cell renal cell carcinoma (CCRCC) is not only the most common form of kidney cancer, but also one of the most resistant tumours to conventional cancer treatments that exhibits increased NF-κB activity and expression of anti-apoptotic NF-κB-target proteins such as survivin, c-IAP1/2 and c-FLIP (Qi & Ohh, 2003; Yang et al, 2007). CCRCC cells overexpress hypoxia-inducible factor (HIF)-α subunit due to the functional inactivation of von Hippel–Lindau (VHL) tumour suppressor protein, which serves as a substrate-specifying component of an E3 ubiquitin ligase ECV (Elongins BC/Cullin 2/VHL) that catalyses oxygen-dependent polyubiquitin-mediated destruction of prolyl-hydroxylated HIF-α. Under hypoxia, the unmodified HIF-α escapes destructive recognition by ECV and associates with its constitutively stable partner HIF-β to form an active heterodimeric HIF transcription factor. HIF binds to hypoxia-responsive elements (HREs) located in the promoter/enhancer regions of numerous hypoxia-inducible genes to initiate various adaptive responses to hypoxia, such as anaerobic metabolism, erythropoiesis and angiogenesis (Kaelin, 2002; Roberts & Ohh, 2008). VHL has been implicated in facilitating TNF-α-induced apoptosis by suppressing the expression of NF-κB-dependent anti-apoptotic genes (Qi & Ohh, 2003). Therefore, functional loss of VHL observed in the vast majority of CCRCC increases anti-apoptotic protein expression, rendering these tumour cells more resistant to normal cell death processes. Recently, An and Rettig demonstrated that VHL-mediated suppression of NF-κB activity is dependent on HIF (An & Rettig, 2005), while Yang et al showed that VHL serves as an adaptor molecule that binds and promotes the inhibitory phosphorylation of the NF-κB agonist Card9 by casein kinase 2. Downregulation of Card9 in VHL-null CCRCC normalized NF-κB activity and sensitivity to cytokine-induced cell death (Yang et al, 2007). These reports establish an association between HIF and VHL to the NF-κB survival pathway. EMCV is a single positive-stranded RNA picornavirus with a large host range, infecting numerous mammals and birds. However, only a small number of animal species appear to be adversely affected, including swine, non-human primates and mice. Importantly, a causal relationship between EMCV infection of humans and illness has never been established (Brewer et al, 2001; Moran et al, 2005). Here, we asked whether the hallmark of cancer cells to evade apoptosis could be exploited for tumour-specific oncolytic killing via EMCV that requires elevated anti-apoptotic properties of host cells for viral replication, spread and virulence. Using multiple genetically engineered established CCRCC cell lines, early-passage primary CCRCC cultured cells and CCRCC xenografts in a murine model, we provide pre-clinical evidence that NF-κB-dependent replication and virulence of EMCV targets tumour cells that exhibit elevated HIF status for selective and effective killing. RESULTS Suppression of NF-κB activity in MEFs and CCRCC cells impairs EMCV virulence Virulence of EMCV is positively associated with the NF-κB-mediated survival pathway. In particular EMCV is highly pathogenic to mice, causing myocarditis and dilated cardiomyopathy, invariably killing normal healthy mice whereas NF-κB p50−/− mice survive EMCV infections (Sha et al, 1995). Schwarz et al subsequently showed that mouse embryonic fibroblasts (MEFs) derived from p50−/− or p65−/− mice are resistant to EMCV-induced cytotoxicity, and revealed that NF-κB-mediated upregulation of anti-apoptotic signalling is responsible for increased EMCV viral replication and the resulting lytic death of WT MEFs. In the absence of NF-κB activity, p50−/− and p65−/− cells were able to undergo rapid apoptosis upon EMCV infection prior to viral replication, thus pre-emptively preventing progeny viral overload and spread (Schwarz et al, 1998). Together, these studies indicate that the inactivation of NF-κB transcription factor via loss of components, p50 or p65, results in severely attenuated EMCV virulence. Indirect activation of NF-κB pathway due to aberrant oncogenic signalling is a common phenomenon in many types of cancer, which increases the capacity for tumour cells to evade apoptosis and gain a survival advantage over normal, untransformed cells. We asked whether indirect perturbation of NF-κB is sufficient to influence the susceptibility of cells to EMCV-induced cytotoxicity. NEMO (also known as IKK-γ) is a critical component of a tripartite IκBα-Kinase (IKK) complex required for phosphorylation and subsequent degradation of IκBα, thereby activating NF-κB. Therefore, loss of NEMO results in decreased NF-κB-mediated signalling due to the constitutive stabilization of IκBα (Kim et al, 2003). NEMO−/− MEFs showed markedly reduced levels of cytotoxicity than WT MEFs upon EMCV infection as measured by propidium iodide staining at regular intervals (Fig 1A), which, consistent with the observation in p50−/− and p65−/− MEFs, suggests that NF-κB inactivation ultimately protects cells from EMCV-induced death. Similar observations were noted in parallel experiments using Annexin V and Trypan Blue exclusion cell viability assays (data not shown). These results support the notion that the functionality of NF-κB can profoundly affect cellular susceptibility to EMCV-induced killing, which may therefore provide therapeutic rationale for an oncolytic EMCV-based approach in the treatment of cancers with elevated NF-κB signalling. Figure 1. NF-κB pathway influences cellular susceptibility to EMCV-induced cytotoxicity. A.. NEMO promotes susceptibility of MEFs to EMCV-induced death. Equal amounts of total cell lysates from WT and NEMO−/− MEFs were immunoblotted with anti-NEMO and anti-Vinculin antibodies (upper panel). WT and NEMO−/− MEFs were infected with EMCV (MOI = 0.1) and viable cells were counted at 2 h intervals post-infection by Annexin V-FITC/propidium iodide staining. Experiments were conducted in triplicate and error bars represent standard errors (lower graph). Two-way ANOVA was applied for statistical analysis between treatments and time points. * and *** denote p < 0.05 and p < 0.001, respectively. B.. NEMO promotes susceptibility of 786-O CCRCC cells to EMCV-induced death. 786-O cells transfected with NEMO-specific siRNA (siNEMO) or scrambled non-targeting siRNA (siCON) (left panel) were infected with or without EMCV (MOI = 0.1), and viable cells counted 18 h post-infection by Trypan Blue exclusion assay (right graph). C.. Activated NF-κB pathway promotes susceptibility of 786-O CCRCC cells to EMCV-induced death. 786-O cells were treated with or without JSH-23 (10 µM) in the presence or absence of LPS (10 µg/ml) and the level of nuclear NF-κB visualized by immunoblotting (left panel). 786-O cells treated with or without JSH-23 were challenged with EMCV (MOI = 0.1) and viable cells were counted 18 h post-infection by Trypan Blue exclusion assay (right graph). C1 + C2 denote two splice isoforms of nuclear restricted pre-mRNA binding protein hnRNP. Experiments were performed in triplicate and error bars represent standard errors, independent Student's t-test was used to analyse difference between groups. Download figure Download PowerPoint We next asked whether NF-κB influenced EMCV virulence in VHL-null 786-O CCRCC cells, which are known to exhibit elevated NF-κB activity (An & Rettig, 2005), via molecular manipulation of NEMO. 786-O cells with transient siRNA-mediated knockdown of endogenous NEMO (786-O + siNEMO) were resistant to EMCV virulence relative to 786-O cells transfected with scrambled siRNA (786-O + siCON) (Fig 1B). In parallel, treatment of 786-O cells with a small molecule JSH-23, an aromatic diamine that inhibits nuclear translocation of NF-κB (Shin et al, 2004) significantly reduced both NF-κB nuclear localization upon LPS treatment and susceptibility to EMCV virulence (Fig 1C). These results suggest that the susceptibility of CCRCC cells to EMCV virulence is significantly influenced by NF-κB activity. Notably, comparable levels of endogenous NEMO expression were observed between patient-derived CCRCC tumour and matched normal kidney tissue samples, as well as between 786-O (VHL−/−; HIF1α−/−) CCRCC cells or isogenically matched HA-tagged WT VHL-reconstituted cells (786-VHL) and normal renal proximal tubule epithelial cells (RPTEC) (Supporting Information Fig 1). Loss of VHL enhances NF-κB-mediated survival signalling upon EMCV challenge We asked whether the loss of VHL influences susceptibility of CCRCC cells, which frequently harbour loss-of-function mutation or loss of VHL, to EMCV. 786-O or 786-VHL cells were challenged with EMCV and analysed for NF-κB-dependent gene expression profile by Affymetrix array and real-time semi-quantitative RT-PCR analyses. EMCV challenge induced NF-κB-regulated genes such as IL-8, IL-6 and TNF-α to a significantly greater extent in 786-O cells as compared to 786-VHL cells (Table 1 and Fig 2). The markedly enhanced NF-κB-mediated gene expression observed upon EMCV challenge in the absence of VHL is likely due to the pre-existing activation, and therefore readiness, of NF-κB-mediated signalling pathway resulting from VHL loss (An & Rettig, 2005; Qi & Ohh, 2003). Table 1. NF-κB-dependent genes are preferentially induced by EMCV in 786-O cells compared to 786-VHL cells Affymetrix probe Gene name Gene title 7B6-O VSV 786-VHL VSV 786-0 EMCV 786-VHL EMCV 211506_s_at IL8 Interleukin-8 1.23 0.54 17.15 6.50 205207_at IL6 Interleukin-6 (interferon, beta 2) 3.48 1.87 6.50 2.64 231779_at IRAK2 Interleukin-1 receptor-associated kinase 2 1.87 1.07 4.92 1.41 201502_s_at NFKBIA Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 2.30 2.14 2.83 1.41 227345_at TNFRSF10D Tumor necrosis factor receptor superfamily, member 10d 1.07 0.76 2.83 1.52 209294_x_at TNFRSF10B Tumor necrosis factor receptor superfamily, member 10b 1.32 1.15 2.30 1.74 203927_at NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon 1.41 1.00 2.14 1.00 231775_at TNFRSF10A Tumor necrosis factor receptor superfamily, member 10a 1.07 0.93 2.00 1.23 Affymetrix microarray expression analysis of NF-κB-regulated genes induced upon EMCV or VSV challenge in 786-O cells and 786-VHL cells. The complete gene expression data have been deposited on the ArrayExpress database maintained by the European Bioinformatics Institute (accession number: E-MTAB-294). Figure 2. Loss of VHL in CCRCC cells accentuates NF-κB-mediated survival signalling upon EMCV challenge. A.. Loss of VHL in 786-O cells accentuates NF-κB-mediated transcription upon EMCV challenge. Steady-state 1L8, 1L6 and TNF-α mRNA levels were measured by real-time PCR in 786-O (open bars) and 786-VIIL (solid bars) cells infected with EMCV (MOI = 0.1) for 8 h. Experiments were performed in triplicate and error bars represent standard errors. B.. Heatmap representation NF-κB-dependent and IFN/IFN-stimulated gene expression in 786-O and 786-VHL cells challenged with EMCV or VSV. Relative gene expression levels of NF-κB-dependent (left panel) and IFN/IFN-stimulated (right panel) genes in 786-O and 786-VHL cells challenged with EMCV or VSV. Actual expression levels as determined by Affymetrix microarray expression analysis arc shown in Tables 1 and 2. Download figure Download PowerPoint Previously, we demonstrated that 786-O cells mounted a stronger anti-viral interferon-mediated response than 786-VHL cells upon vesicular stomatitus virus (VSV) challenge, and thereby protecting 786-O cells against VSV-induced virulence (Hwang et al, 2006). In contrast, EMCV infection induced a modest to negligible interferon response (Table 2). EASE analysis of transcripts preferentially induced by VSV in 786-O cells compared to 786-VHL cells yielded 'interferon induction' as it is most enriched SwissProt category (EASE score = 1.10E−19; Fisher's Exact Test score = 1.06E−21). However, EMCV-infected 786-O cells showed no significant enrichment of 'interferon induction' category (EASE score = 1). In addition, while VSV infection of 786-O cells for 8 h induced a 74-fold increase in IFN-β transcript levels, EMCV infection had a negligible influence on IFN-β expression (0.93-fold change) (Table 2). Moreover, unlike EMCV, VSV did not generate further NF-κB signalling response irrespective of VHL status (Table 1). These results indicate that EMCV and VSV cause strikingly different VHL-dependent responses in CCRCC cells. The lack of interferon-mediated anti-viral response in combination with elevated NF-κB-mediated anti-apoptotic response in 786-O cells upon EMCV challenge suggest that the loss of VHL will likely boost EMCV replication and render CCRCC cells susceptible to EMCV-induced cytotoxicity. Table 2. EMCV induces markedly weaker IFN/IFN-stimulated gene expression than VSV Affymetrix gene Gene title 786-O VSV 786-VHL VSV 786-O EMCV 786-VHL EMCV Interferon induction (SwissProt) 204533_A⌉CXCL10 Chemokine (C-X-C motif) ligand 10 17.15 2.64 0.50 0.27 205552_S_OAS1 2′,5′-Oligoadenylate synthetase 1, 40/46 kDa 17.15 6.96 1.23 1.15 204747_A⌉IFIT4 Interferon-induced protein with tetratricopeptide repeats 4 14.93 5.66 1.32 0.71 231577_S_GBP1 Guanylate binding protein 1, interferon-inducible, 67 kDa 12.13 2.30 1.32 1.00 205660_A⌉OASL 2′-5′-Oligoadenylate synthetase-like 11.31 4.59 1.15 0.76 204994_A⌉MX2 Myxovirus (influenza virus) resistance 2 (mouse) 9.85 2.14 1.00 0.71 214022_S_IFITM1 Interferon induced transmembrane protein 1 (9–27) 6.96 2.30 0.76 0.66 204972_A⌉0AS2 2′-5′-Oligoadenylate synthetase 2, 69/71 kDa 6.50 2.14 0.71 1.00 202531_A⌉IRF1 Interferon regulatory factor 1 5.28 2.46 2.14 1.15 208966_X_IFI16 Interferon, gamma-inducible protein 16 4.92 2.00 1.00 1.23 234987_A⌉SAMHD1 SAM domain and HD domain 1 4.29 2.00 0.93 0.93 203275_A⌉IRF2 Interferon regulatory factor 2 3.25 1.15 1.41 1.00 202748_A⌉GBP2 Guanylate binding protein 2, interferon-inducible 2.64 0.87 1.23 1.07 Affymetrix microarray expression analysis of IFN and genes identified as being involved in 'Interferon Induction' by EASE induced upon EMCV or VSV challenge in 786-O and 786-VHL cells. The complete gene expression data have been deposited on the ArrayExpress database maintained by the European Bioinformatics Institute (accession number: E-MTAB-294). Loss of VHL enhances EMCV replication and increases CCRCC cell cytotoxicity We next asked whether VHL influences EMCV viral replication. Lysates from EMCV-challenged 786-O and 786-VHL cells were added to L929 mouse fibroblast monolayers, which were then monitored for cytotoxicity in which an increase in L929 cytotoxicity would be indicative of an increase in EMCV titre. As predicted, 786-O cells with strong hypoxic signature, as indicated by elevated expression of hypoxia-inducible GLUT-1 (Fig 3A, left panel), and correspondingly enhanced capacity for NF-κB activation produced orders of magnitude higher EMCV titres than in 786-VHL cells with low NF-κB activity, demonstrating enhanced EMCV replication in CCRCC cells devoid of VHL (Fig 3A, right panel). EMCV challenge produced similar orders of magnitude higher EMCV titres in another distinct VHL−/− CCRCC cell line ectopically expressing empty plasmid (RCC4-MOCK) than in WT VHL reconstituted isogenic counterpart (RCC4-VHL) (Fig 3B). Figure 3. Loss of VHL in CCRCC cells enhances EMCV replication. A.. Loss of VHL in 786-O cells enhances EMCV replication. 786-O and 786-VHL cells were lysed and immunoblotted with anti-HA, anti-GLUT-1 and anti-α-Tubulin antibodies (left panel). Cells were challenged with EMCV (MOI = 0.01) and cumulative virus titre was evaluated at 3 h intervals post-infection (right graph). Two-way ANOVA was applied for statistical analysis between treatments and time points. * and *** denote p < 0.05 and p < 0.001, respectively. B.. Loss of VHL in RCC4 cells enhances EMCV replication. RCC4-MOCK and RCC4-VHL cells were lysed and immunoblotted with
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