Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. Cysteine rich 61 (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 a 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. Furthermore, they suggest therapeutic interventions to inhibit CCN1–integrin α6 interactions to sensitize gliomas to viral oncolysis. Cancer Res; 72(6); 1353–62. ©2012 AACR.
Glioblastoma multiforme (GBM) is the most common primary brain tumor, and despite aggressive therapy involving tumor resection, chemotherapy, and radiation treatment, median survival of patients remains less than 15 months from diagnosis (1). Oncolytic viruses (OV) are biological therapeutics that selectively replicate in and kill tumor cells. These viruses have shown promising results in preclinical models (2), and their safety and efficacy is currently being investigated in clinical trials. Despite these advances, the impact of changes in the tumor microenvironment on OV therapeutic efficacy has not been very well studied.
We have previously described a dose dependent and rapid induction of the secreted angiogenic inducer cysteine rich 61 (CCN1) in the tumor microenvironment following OV therapy (3). CCN1 is a member of the growth factor inducible immediate early family CCN, named as such for its first 3 members cysteine rich 61, connective tissue growth factor, and nephroblastoma overexpressed (4). It is a secreted protein which typically localizes in the extracellular matrix (ECM) and on the cell surface (5), in which it binds integrin receptors to modulate a variety of cellular functions including adhesion, migration, and proliferation (6). In brain tumors, CCN1 is overexpressed in 68% (27 of 40) of GBM specimens and in cell lines derived from high-grade gliomas (7). Its increased expression in the mucosa of patients with colorectal cancer has also implicated it in “priming for carcinogenesis” (8), and its oncogenic potential is largely accredited to its activation of integrin-linked kinase-mediated β-catenin–TCF/LEF and AKT (9).
Apart from its induction in glioma cells infected with herpes simplex virus 1 (HSV-1)–derived OVs, CCN1 has also been found to be dysregulated in cells after infection with coxsackievirus B3 and adenovirus type 12, suggesting that it may play a role in viral infection of mammalian cells (10, 11). Here, we evaluated the impact of CCN1 expression on OV efficacy. Our findings indicate that CCN1 limits OV replication and cytotoxicity due to its significant activation and enhancement of the innate antiviral type-I IFN response in cells. Furthermore, our studies reveal that this IFN response is activated by CCN1 binding to integrin α6β1 on glioma cells, which results in the rapid and early secretion of IFNα and activation of the Jak/Stat signaling pathway. The results from this study show a novel role for CCN1 and integrin α6β1 in regulating cellular innate defense responses against viral infection and indicate a need for patient selection based on gene expression profiling for therapeutic interventions.
Materials and Methods
Cells and viruses
Human LN229, U343, Gli36ΔEGFR-H2B-RFP, U251T2, and U251T3 glioma cell lines are maintained as described (3). EGFR-transduced baby hamster kidney JiEGFR cells are maintained as described (12). Tet-regulated CCN1-expressing clones Cy-1 and Cy-2 were established as described (13). For radiation studies, cells were irradiated at 10 gy with RS-2000 Biological Irradiator. HSV-1–derived OVs, rHSVQ1, rHsvQ1-IE4/5-Luc, and 34.5ENVE, have been described previously (14–16).
All animal experiments were conducted in accordance with the Subcommittee on Research Animal Care of The Ohio State University guidelines. Six- to 8-week-old female athymic nude mice were used for all studies.
For CCN1 effects on viral progeny, mice were implanted s.c. with 4 × 106 Cy-1 or LN229 cells into the rear flank and monitored for tumor growth. When tumors reached 100 mm3, mice were randomized and fed sucrose ± doxycycline (1 mg/mL) in drinking water. Two days post-doxycycline treatment initiation, mice were administered rHSVQ1 (1 × 106 pfu) by direct intratumoral injection and sacrificed 48 hours postinfection; tumors were harvested for the number of infectious virus particles and analyzed by a standard plaque assay.
For effects of viral progeny on tumor cell growth, mice were implanted s.c. with 1.5 × 107 U251T3 cells into the rear flank. When tumors reached an average of 250 mm3, mice were administered 34.5ENVE virus by direct intratumoral injection with the indicated dose. Tumor volume was calculated by the following formula: volume = 0.5LW2 as described (17).
Antibodies and reagents
Reagents used in this study were obtained from the following sources: cilengitide (Merck), valproic acid (VPA) and laminin (Sigma), fibronectin (Calbiochem), vitronectin (Promega), CCN1 protein (Cell Sciences). Antibodies were obtained from the following sources: CCN1 (Novus Biologicals); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and ITGA6 (Abcam); STAT1 and PSTAT1 (Cell Signaling); STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2, and IFNαR2 (Millipore); sheep anti-mouse HRP (GE Healthcare); goat anti-rabbit HRP; immunoglobulin G negative control (DAKO). IFNα levels were measured from cell supernatants with PBL Interferon Verikine Human IFNα ELISA Kit.
Real time PCR
RNA was isolated with RNeasy Mini Kit (QIAGEN). For quantitative real-time PCR, cDNA was made with Superscript First-Strand Synthesis System (Invitrogen). Real-time continuous detection of PCR product was achieved using SYBR Green (Applied Biosystems). GAPDH was used as an internal control. Primers were designed with the Primer Express Program (Applied Biosystems; Supplementary Table S1).
Total RNA from Cy-1 cells incubated ± doxycycline for 24 hours was isolated with RNeasy Mini Kit (QIAGEN). Samples were then submitted to The Ohio State University Microarray Shared Resource Center for microarray analysis using the Affymetrix GeneChip Analysis. The microarray data from this publication have been submitted to the Gene Expression Omnibus database (accession number GSE29384).
Results are presented as mean ± SEM. Statistical analysis was carried out by unpaired Student t test using GraphPad Prism 5.01 software. P < 0.05 was considered statistically significant. Affymetrix GeneChip was used for gene expression study. Signal intensities were quantified by Affymetrix software.
CCN1 gene expression is upregulated by virus but not by chemotherapy or radiation treatment
Apart from increased CCN1 gene expression in glioma cells post-OV infection, its induction has also been described in H19-7 cells after treatment with etoposide, in UV-irradiated human skin fibroblasts and in HeLa cells infected with Coxsackievirus B3 virus (10, 18, 19). Here, we tested whether induction of CCN1 in glioma cells infected with oncolytic HSV-1 represents a general response to glioma cell killing. Figure 1A and B shows that while LN229 glioma cells infected with rHSVQ1 led to a significant increase of CCN1 mRNA, its expression was not increased after radiation or temozolomide treatment. To determine whether this response could be generalized to other viruses, we examined changes in its expression in LN229 cells infected with 3 different viruses in addition to wild-type HSV-1: vesicular stomatitis virus (VSV), adenovirus (Ad), and Newcastle Disease virus (NDV). Figure 1C shows a significant induction of CCN1 in glioma cells after infection with all the viruses tested indicating that its induction may represent a general response of glioma cells to viral infection.
Extracellular CCN1 expression inhibits viral transgene expression, replication, and oncolysis
To investigate the impact of induction of CCN1 expression on viral therapy, we analyzed its effect on OV transgene expression in glioma cells transiently expressing CCN1 (Gli36ΔEGFR-H2B-RFP and U251T2 cells) and in tet-inducible glioma cells (Cy-1 and Cy-2). Figure 2A and B and Supplementary Fig. S1A show a significant reduction in viral transgene expression upon both transient and tet-inducible induction of CCN1 expression (Supplementary Fig. S1B and S1C and Supplementary Table S2) and this reduction is dose dependent (Supplementary Fig. S2A). No change was observed in parental LN229 glioma cells treated with doxycycline (Supplementary Fig. S2B).
To evaluate whether the reduction in OV infection/replication was a result of secreted CCN1 in the ECM, we seeded U251T2 and LN229 glioma cells on CCN1/bovine serum albumin (BSA)–coated plates before infection with rHsvQ1-IE4/5-Luc virus. Confocal fluorescent microscopy revealed reduced GFP-positive cells when seeded on purified CCN1 compared with BSA (Fig. 2C and D). Quantification of OV-expressed luciferase indicated a significant reduction of viral transgene expression in cells seeded on CCN1 matrix compared with control (Supplementary Fig. S3A and S3B). To examine the role of endogenous CCN1 on OV replication, we infected glioma cells in the presence or absence of CCN1-neutralizing antibody. Figure 2E shows that inhibition of physiologic levels of CCN1 enhances viral transgene expression in 3 different glioma cell lines. Furthermore, CCN1-mediated reduction in viral transgene expression in doxycycline-induced Cy-1 cells was rescued in the presence of CCN1-neutralizing antibody, indicating that CCN1 acting on the cell surface of glioma cells mediates the OV inhibition (Fig. 2F).
We next evaluated the impact of CCN1 expression on viral replication by measuring the total amount of infectious viral particles released by Cy-1 glioma cells in vitro. Figure 3A shows a significant reduction in viral titers in cells upon CCN1 induction. Consistent with reduced virus replication, we also found a reduction in the ability of OV to kill glioma cells expressing CCN1 (Fig. 3B). To test the in vivo relevance of these findings, we examined the impact of CCN1 induction on virus replication in subcutaneous tumors. Mice-bearing Cy-1 tumors were fed sucrose water ± doxycycline to induce CCN1 expression, 2 days before infection with rHSVQ1. Two days post-OV infection, viral progeny was isolated and quantified. We found tumoral expression of CCN1 led to a significant reduction of viral progeny by 5.6-fold (Fig. 3C); a difference which reduces viral antitumor efficacy in vivo (Supplementary Fig. S4). Collectively, these results show reduced virus replication and reduced killing of glioma cells with increased levels of CCN1 both in vitro and in vivo.
Transcript profiling uncovered CCN1-mediated induction of type-I IFN response
The ECM has been shown to influence cellular gene expression through its interaction with cell surface receptors (20). Transcript profiling of Cy-1 glioma cells induced to express CCN1 revealed a significant induction of the antiviral type-I IFN pathway (Fig. 4A). To identify functional networks and gene ontologies, we analyzed the upregulated gene expression data using Ingenuity Pathway Analysis software. Investigating key biological functions linked to CCN1 gene expression, we found the main functions of genes upregulated with CCN1 were as follows: IFN signaling, activation of IFN regulatory factor (IRF) by cytosolic pattern recognition receptors, and recognition of bacteria and viruses by pattern recognition receptors (Fig. 4B). Ingenuity's Top Network Analysis revealed a highly significant relationship between the genes differentially expressed by CCN1 induction and regulation of the antimicrobial response, inflammatory response, and infection mechanism in glioma cells (Fig. 4C). Interestingly both Ingenuity Pathway Analysis (IPA) and a detailed PubMed analysis did not reveal a published link between type-I IFN activation and CCN1 expression in ECM.
Real-time quantitative PCR analysis was conducted to verify induction of a subset of the type-I IFN responsive genes involved in the antiviral defense response in these cells (Table 1). Statistically significant induction of IFNs α and β along with downstream regulatory genes such as signal transducers and activators of transcription 1 and 2 (Stat1 and Stat2), double-stranded RNA-dependent protein kinase (PKR), IRF1, 3, and 7, and 2′,5′-oligoadenylate synthetase 2 (OAS2) was observed. These genes were further upregulated in Cy-1 cells–expressing CCN1 following infection with rHSVQ1 suggesting an enhanced activation of the type-I IFN response by CCN1 (Table 1). Consistent with this, Western blot analysis of Cy-1 cell lysates revealed increased phosphorylation of both Stat1 and Stat2 in cells induced to express CCN1 in the presence and absence of OV infection, suggesting CCN1 both activates and exacerbates the innate cellular antiviral response. No difference was found in phosphorylation status of Stat1 or Stat2 in control LN229 cells treated with doxycycline (Fig. 5A and B).
|.||Without OV .||.||.||With OV .||.||.|
|Gene name .||No Dox .||Dox .||Fold induced .||P .||No Dox .||Dox .||Fold induced .||P .|
|.||Without OV .||.||.||With OV .||.||.|
|Gene name .||No Dox .||Dox .||Fold induced .||P .||No Dox .||Dox .||Fold induced .||P .|
Abbreviations: STAT, signal transducer and activator; Dox, doxycycline.
To test whether the observed CCN1-mediated antiviral effects were dependent on activation of the type-I IFN pathway, we compared viral transgene expression in cells expressing CCN1 in the presence of VPA, a histone deacetylase inhibitor known to interfere with the transcriptional activation of type-I IFN responsive genes (21, 22). Figure 5C shows that VPA treatment rescued CCN1-mediated inhibition of viral transgene expression.
CCN1-mediated OV inhibition is dependent on α6β1 integrin receptor–mediated IFNα secretion
CCN1 is a multifunctional, secreted ECM protein that has been shown to bind to multiple cell surface receptors including integrins αvβ3, αvβ5, and α6β1. To determine the cell surface receptor through which CCN1 is mediating its antiviral effects, we investigated the potential contribution of these receptors. We first evaluated the ability of cRGD (Cilengitide; αvβ3 antagonist) and LM609 (a function blocking monoclonal antibody against αvβ3) to rescue virus inhibition in doxycycline-induced Cy-1 cells. Figure 6A and B show that neither agent could rescue CCN1-mediated OV repression. Consistent with this result, LN229 glioma cells plated on fibronectin-coated plates (a known αvβ3-activating ligand) also had no effect on OV transgene expression (Fig. 6C).
We next assessed the potential role of integrin αvβ5 in CCN1-mediated OV inhibition. Figure 6D shows that treatment of glioma cells with P1F6 (an αvβ5 function blocking antibody) did not rescue CCN1-mediated reduction of OV. Moreover, activation of cell surface αvβ5 by vitronectin (a known αvβ5-activating ligand), also did not affect OV transgene expression (Fig. 6E).
CCN1 binds to and activates integrin α6β1 on fibroblasts, vascular smooth muscle cells, and vascular endothelial cells (23–25). More recently, glioblastoma stem cells (GSC) were also found to express the integrin α6 chain of this heterodimeric receptor (26). To investigate whether CCN1-mediated OV inhibition was due to the activation of integrin α6β1 on glioma cells, we measured the impact of function blocking monoclonal antibodies against α6 and β1 on viral infection. Figure 6F shows that the inhibition in OV transgene expression observed when Cy-1 cells express CCN1 is rescued in the presence of function blocking monoclonal antibodies against either α6 or β1. Consistent with this, glioma cells plated on laminin (a known α6β1-activating ligand) leads to a significant inhibition of OV transgene expression (Fig. 6G). This ability of laminin to inhibit viral transgene expression is rescued in the presence of a function blocking antibody against integrin α6, indicating that CCN1-mediated activation of integrin α6β1 on glioma cells leads to the induction of an antiviral defense response (Fig. 6H). Supplementary Figure S5A shows presence of integrin α6 on all glioma cell lines tested.
Integrin-mediated cell–matrix interactions are known to play a role in protein secretion (27–29), and among these, integrin α6β1 has been shown to mediate insulin secretion in primary rat β cells (30, 31). To further delineate the underlying mechanism behind integrin α6β1 activation of the type-I IFNs, we carried out an ELISA looking for changes in the IFNα secretion pattern in the presence of CCN1. A time course analysis with Cy-1 cells induced to express CCN1 indicated that this protein is induced within minutes after treatment with doxycycline (Fig. 6I). Interestingly, minutes after protein induction, we observed a rapid burst in the secretion of IFNα (Fig. 6J) independent of its gene expression (Supplementary Fig. S5B) and independent of doxycycline treatment (data not shown). This suggests that CCN1 protein induction mediates a rapid type-I IFN secretion in glioma cells. In addition, CCN1-mediated OV transgene inhibition was rescued by IFNα2 receptor blocking antibody indicating that secreted IFNα was required for this antiviral effect in vitro (Fig. 6K). Consistent with this, CCN1 did not have an antiviral effect on U87ΔEGFR cells, which have a homozygous deletion of the entire IFNA/IFNW gene cluster and of the IFNB1 gene (Supplementary Fig. S5C; refs. 32–34).
To examine whether CCN1 induced by OV infection could activate this antiviral response in adjacent uninfected cells, we cultured JiEGFR cells, which are resistant to HSV infection (12; Supplementary Fig. S6), in the presence of LN229 cells infected with OV. Figure 6L and M shows increased phosphorylation of Stat1 in JiEGFR cells. More significantly, this increased phosphorylation is rescued in the presence of CCN1-neutralizing antibodies (Fig. 6N) indicating that endogenous CCN1 induced after OV infection could activate Jak/Stat signaling in adjacent uninfected cells.
Collectively, these results indicate that increased expression of CCN1 in the tumor microenvironment leads to the activation of integrin α6β1 on glioma cells, resulting in the secretion of IFNα and activation of an antiviral response in the tumor microenvironment which ultimately limits OV infection and replication (Supplementary Fig. S7).
CCN1 is a pleiotropic ECM molecule which binds several cell surface receptors and modulates cell signaling events affecting diverse cellular functions including proliferation, adhesion, and migration. In this study, we report the induction of CCN1 gene expression in glioma cells infected with several different viruses. We further show that CCN1 in the tumoral ECM binds to cell surface α6β1 integrin receptors to activate an innate antiviral defense response by the secretion of IFNα. Collectively, these results suggest that secretion of CCN1 upon infection orchestrates an “alarm signal” in the tumor microenvironment which activates an antiviral state in adjacent uninfected cells leading to increased resistance to viral infection/replication (Fig. 6L–N). To our knowledge, this is the first report linking integrin binding and activation by extracellular CCN1 to secretion of IFNα and activation of the antiviral type-I IFN response. Although CCN1's role as a proinflammatory molecule is beginning to be realized (35), its effect on the type-I IFN response is quite novel, and this is the first study linking an inhibitory role of CCN1 to OV therapy. This study has several implications for biological therapies and viral infections.
CCN1's role in tumor biology has been extensively studied, and depending on the tissue type has been found both pro- and antitumorigenic (36). Apart from negatively modulating the cellular response to OV therapy, the expression of CCN1 protein in breast, prostate, and ovarian cancer correlates with a poor prognosis (37–39). Conversely, its expression in lung, endometrial, and gastric cancer has been associated with a better prognosis and outcome (40–42). Though the reason underpinning CCN1's opposing effect in different tissue has not been elucidated, it may depend, in part, on the context in which CCN1 is expressed differing by the presence of coactivators and repressors and the receptor expression profiles present in different tissues.
Here, we show that CCN1 activates a type-I IFN proinflammatory cascade in glioma cells by binding to and activating the α6β1 integrin receptor and inducing secretion of IFNα. We show that CCN1 expression not only upregulates the type-I IFNs α and β, but also several downstream mediators of the type-I IFN response known to play key roles in the cellular antiviral defense response such as PKR and OAS (43, 44). These results suggest that while expression of CCN1 leads to increased angiogenesis and invasion in the tumors, it also interferes with oncolytic viral therapy, and inhibition of this pathway may provide opportunities to enhance OV antitumor efficacy.
Recently, integrin α6 has been recognized as an enrichment marker for GSCs (26) and was found to be coexpressed with CD133 (a widely accepted glioma stem cell marker) in GBM biopsies. Apart from increased tumorigenicity, glioma stem cells have been shown resistant to both radiation and chemotherapy (45). In U87 glioma cells, it has been shown that stable cell surface expression of integrin α6β1 leads to both enhanced proliferation and decreased apoptosis in vitro and in vivo (46). The results from our study indicate that CCN1-mediated activation of integrin α6 contributes to the reduced efficacy of viral oncolytic therapy, and it will be interesting to understand how the CCN1–integrin α6 interaction plays a role in glioma therapeutic resistance.
In conclusion, this is the first study to reveal the effect of a secreted matricellular integrin binding protein on the initiation of an innate type-I IFN cellular defense response to virus infection. This study suggests that therapeutic interventions which inhibit the CCN1–integrin α6 interaction may sensitize glioma to chemo and radiation therapies and viral oncolysis. Future studies will evaluate the extent to which expression of CCN1 and/or integrin α6 receptor on tumors can serve as a predictor of patient response to oncolytic viral therapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work was supported by RO1NS064607-01, RO1CA150153-01, and P30NS045758.
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