Human cytomegalovirus US28 found in glioblastoma promotes an invasive and angiogenic phenotype

Abstract

Human cytomegalovirus (HCMV) infections are seen often in glioblastoma multiforme (GBM) tumors, but whether the virus contributes to GBM pathogenesis is unclear. In this study, we explored an oncogenic role for the G-protein-coupled receptor-like protein US28 encoded by HCMV that we found to be expressed widely in human GBMs. Immunohistochemical and reverse transcriptase PCR approaches established that US28 was expressed in approximately 60% of human GBM tissues and primary cultures examined. In either uninfected GBM cells or neural progenitor cells, thought to be the GBM precursor cells, HCMV infection or US28 overexpression was sufficient to promote secretion of biologically active VEGF and to activate multiple cellular kinases that promote glioma growth and invasion, including phosphorylated STAT3 (p-STAT3) and endothelial nitric oxide synthase (e-NOS). Consistent with these findings, US28 overexpression increased primary GBM cell invasion in Matrigel. Notably, this invasive phenotype was further enhanced by exposure to CCL5/RANTES, a US28 ligand, associated with poor patient outcome in GBM. Conversely, RNA interference-mediated knockdown of US28 in human glioma cells persistently infected with HCMV led to an inhibition in VEGF expression and glioma cell invasion in response to CCL5 stimulation. Analysis of clinical GBM specimens further revealed that US28 colocalized in situ with several markers of angiogenesis and inflammation, including VEGF, p-STAT3, COX2, and e-NOS. Taken together, our results indicate that US28 expression from HCMV contributes to GBM pathogenesis by inducing an invasive, angiogenic phenotype. In addition, these findings argue that US28-CCL5 paracrine signaling may contribute to glioma progression and suggest that targeting US28 may provide therapeutic benefits in GBM treatment.

Figure 1. HCMV US28 transcript and protein are expressed in human GBMs

A–B. Primary GBM-derived cultures were processed for US28 immunofluorescence in the absence (A) or presence (B) of a blocking peptide. Nuclei are counterstained with propidium iodide. Bar= 100μm. C–F. Consecutive (5μm) paraffin sections obtained from a different GBM patient sample were processed for US28 (C, D), VEGF (E), and COX-2 (F) immunohistochemistry. Counterstaining, hematoxylin. Bar= 100μm. G. RT-PCR for US28 was performed using cDNA from several GBM cases. HCMV infected neural precursor cells (NPC+CMV) served as positive control. Several cases show a US28 band of the correct size. HCMV UL56 detection is also shown. Rab 14 was used to verify equal loading. NC= Negative control.

Figure 2. US28-CCL5 signaling promotes glioblastoma invasiveness

A. NPCs infected with HCMV Towne and TR (1MOI, 72 h) were profiled using a HCMV DNA microarray containing all predicted open reading frames for Ad169/Toledo strains. Expression levels of HCMV transcripts are displayed as fold increase over uninfected control. B. RNA from HCMV-treated and control NPCs were profiled using Affymetrix Gene 1.0 ST DNA arrays. The heatmap shows the 30 most upregulated and 30 most downregulated human transcripts in HCMV-infected NPCs vs mock. CCL5 was induced over 40 fold by HCMV treatment (arrow). C. Kaplan-Meyer curves showing the relationship between levels of CCL5 transcript and survival probability in patients with glioblastoma (log-rank p-value upregulated vs all other samples, p=0.001523, REMBRANDT data base, NCI). D. Human glioma cells (U251 and U87) and two primary glioblastoma-derived cultures (designated GBM#1 and GBM #2) transfected with US28 or control vector were subjected to Matrigel invasion assays in the absence or presence of CCL5 (50ng/ml). ** p< 0.005, ANOVA. E. CCL5 levels measured by ELISA in mock-treated U87 cells, or HCMV infected +/− CCL5 neutralizing antibody. **, p< 0.005 ANOVA. F. Mock and HCMV-infected U87 cells were subjected to Matrigel invasion assays. Mean number of cells/filter are shown for each condition. ** p<0.005 ANOVA. US28 KD was achieved using two siRNA duplexes in combination (siRNA1+2). Data from one representative experiment are shown. Each condition was performed in triplicate and experiments were repeated three times.

Figure 3. US28 induces activation of cellular kinases involved in glioma pathogenesis

AB. HCMV (Towne, 1MOI) and Mock- treated NPCs (A) and glioma cells (B) were profiled using a human phosphor-kinase antibody array. C. Densitometry measurements were performed per manufacturer’s instructions. Percentage change in phosphorylation levels between HCMV/US28 treated and control cells are shown. One (out of two) representative experiment is shown. D. Double immunofluorescence for US28 and the indicated proteins in NPCs transduced with LXSN-US28 for 48h. Right panels represent IgG staining controls. Nuclei were counter-stained with propidium iodide. Bar = 50μm.

Figure 4. US28 promotes glioma angiogenesis

A. NPC transduced with either LXSN-HA-US28 or Ad-US28 and control LXSN/Mock treated cells were processed for immunofluorescence. Right panels- NPCs that express US28 (green) secrete VEGF (blue), as demonstrated by co-localization of the two markers. Nuclei are stained with propidium iodide. Bar= 100μm. B. NPC, U251, U87, and a primary GBM line ( 4121) were treated with HCMV (Towne, TR, 1 MOI), transduced with Ad-US28, or treated with EGF (50ng/ml) in serum free media. Supernatants were used in an ELISA for VEGF. Samples were assayed in quadruplicate and the experiment was repeated twice. Comparisons between treated and mock within the same cell line, were analyzed using ANOVA *, p=0.02, **, p<0.002. C. NPC-derived supernatants were tested in HUVEC tube formation assays. Complete endothelial cell growth media was used as a positive control. Representative photomicrographs are shown. Each condition was assayed in six wells of a 24 well plate and the experiment was repeated twice. Bar= 100μm. D. Average numbers of branch points and endothelial cell lumens are shown from one representative experiment. Comparisons were analyzed using ANOVA. *, p<0.02 in all cases.

Figure 5. US28 Knockdown in HCMV infected glioma cells inhibits VEGF secretion and subsequent angiogenesis

A. Immunofluorescence was used for detection of US28 (green) and VEGF (red) in U87 cells persistently infected with HCMV treated either with control siRNA (upper panels) or siRNAs1+2 targeting US28. Nuclei were counterstained with DAPI. Bar = 50 μm. B. Cumulative distribution of mean pixel intensity per cell obtained from immunofluorescent detection of US28 and VEGF in U87 cells treated with either targeting (1+2) or control siRNAs. Kolmogorov-Smirnov test was used to determine significance of differences in the fluorescence intensity measured in > 100 cells/condition, p=0.0001. C. VEGF levels were measured using ELISA in U87 glioma cells and primary 4121 GBM cells uninfected or HCMV –infected in the presence of either control or US28 targeting siRNA1, siRNA2, or siRNA 1+2. Differences were significant, * p=0.05; ** p=0.002 student T-test. D. Quantification of HUVEC branches and lumens formed in each of the indicated conditions. **, p<0.02 ANOVA. E. Representative photomicrographs of HUVEC tube formation assay in the presence of various types of conditioned media, as indicated. Bar= 100μm. HUVEC tube formation assays were repeated three times, each condition was run in quadruplicate.

Figure 6. HCMV US28 co-localizes with markers of invasiveness and angiogenesis in situ

A–F. Primary glioblastoma derived cells were processed for immunofluorescence using antibodies against US28 (A and D), VEGF (B) and e-NOS (E). Co-localization of US28 and the two markers of angiogenesis is shown by the merged photo-micrographs in panels C and F. Nuclei are counterstained using propidium iodide. Bar, 100μm. G–L. Consecutive paraffin sections (5μm apart) from a glioblastoma specimen were stained for US28, VEGF, e-NOS, and COX2 and developed using HRP-DAB. Arrows indicate cells positive for several markers in the same area. Counterstaining, hematoxylin. Bar= 50μm. M. The diagram summarizes the autocrine and paracrine signaling pathways through which US28 promotes GBM growth, invasion, and angiogenesis.