Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor

<div><p>Glioblastoma multiforme (GBM) is a highly malignant primary central nervous system neoplasm characterized by tumor cell invasion, robust angiogenesis, and a mean survival of 15 months. Human cytomegalovirus (HCMV) infection is present in >90% of GBMs, although the role the virus plays in GBM pathogenesis is unclear. We report here that HCMV pp71, a viral protein previously shown to promote cell cycle progression, is present in a majority of human GBMs and is preferentially expressed in the CD133+, cancer stem-like cell population. Overexpression of pp71 in adult neural precursor cells resulted in potent induction of stem cell factor (SCF), an important pro-angiogenic factor in GBM. Using double immunofluorescence, we demonstrate in situ co-localization of pp71 and SCF in clinical GBM specimens. pp71 overexpression in both normal and transformed glial cells increased SCF secretion and this effect was specific, since siRNA mediated knockdown of pp71 or treatment with the antiviral drug cidofovir resulted in decreased expression and secretion of SCF by HCMV-infected cells. pp71- induced upregulation of SCF resulted in downstream activation of its putative endothelial cell receptor, c-kit, and angiogenesis as measured by increased capillary tube formation <i>in vitro</i>. We demonstrate that pp71 induces a pro-inflammatory response via activation of NFΚB signaling which drives SCF expression. Furthermore, we show that pp71 levels and NFKB activation are selectively augmented in the mesenchymal subtype of human GBMs, characterized by worst patient outcome, suggesting that HCMV pp71-induced paracrine signaling may contribute to the aggressive phenotype of this human malignancy.</p></div

pp71 stimulation of SCF secretion requires activation of the NFKB signaling pathway.

<p><b>A</b>: U87 cells stably transduced with a retrovirus encoding pp71 (pLXSN-pp71) or control (pLXSN) were used for transcriptome profiling using an Affymetrix Gene 1.0 ST array. Ingenuity pathway analysis (IPA) for differentially expressed genes identified several targets of the NFKB signaling pathway as being upregulated (fold-increase in expression is displayed next to the molecule icon). <b>B</b>: NPCs transduced with rAD-pp71, vector control, or HCMV- infected were co-immunostained for pp71 and markers of NFKB activation (p100/p52, Cox2, and RelB). Cells were counterstained with DAPI. <b>C</b>: Primary GSC neurospheres were treated as indicated and total RNA was analyzed by TaqMan for SCF, CXCL12, IL8, and Myb expression levels. Rab14 levels were used for normalization. The percent change in expression relative to untreated control is displayed. The experiment was repeated three times with similar results. <b>D</b>: U87 cells were transfected with an SCF-promoter driven luciferase construct and then treated as indicated. Luminescence readouts from a representative experiment are shown. Each condition was run in triplicate and the experiment was repeated three times. Positive (RPL13) and negative (scrambled) control promoter driven luciferase constructs were used. Differences were not statistically significant by student t-test.</p

pp71 preferentially activates non-canonical NFKB signaling in endogenously infected glioblastoma tissues.

<p><b>A</b>: Primary GBM tissue was processed using a subcellular protein fractionation kit. An equivalent amount of protein from each fraction was analyzed by western blot for pp71, p65/RelA, p100/p52, Sox2 and histone. <b>B</b>: Primary GBMs classified as proneural or mesenchymal using TaqMan analysis of differentially expressed transcripts (described in C) were analyzed by western blot for pp71, p100/p52, NIK, Actin, Olig2, and CD44. <b>C</b>: TaqMan was performed on GBM cDNA using probes to known mesenchymal (CEBP, CHI3L1, TWIST) and proneural (OLIG2, PDGFRα, Sox11) genes. The results show ΔΔCt for each marker relative to Rab14 in the tissues analyzed.</p

SCF induced by pp71 in NPCs and glioma cells is biologically active.

<p><b>A</b>: HUVECs stimulated with either recombinant human SCF or conditioned medium from NPCs transduced with rAD-GFP, rAD-pp71, or rAD-pp71+ SCF neutralizing antibody were analyzed by western blot for phosphor-c-Kit, total c-kit, phosphor-p38, total p38, and actin. <b>B</b>: HUVECs were grown overnight in gel matrix supplemented as indicated. Capillary tubes that formed in each condition were visualized by microscopy (top panel), counted and plotted (bottom panel). *p = <0.05 as determined by student t-test when rAD-pp71 was compared to rAD-GFP and when rAD-pp71+Nab was compared to rAD-pp71. Each condition was run in duplicate and the experiment was repeated three times. <b>C</b>: HUVEC cells were subjected to a 4 h transwell migration assay using either 20,000 U87 cells treated as indicated (dark grey bars) or supernatant obtained from the same cells (light grey bars) on the bottom wells. For negative and positive control we used serum-free medium or rhSCF (1 ug/mL) respectively. HUVECs that migrated to the bottom of the well were stained and counted. *p = 0.015 for cells and 0.032 for supernatant when compared to untreated control, or 0.058 and 0.075 when compared to rAD-GFP, respectively by student t-test. Each condition was run in triplicate and the experiment was repeated twice. <b>D</b>: Frozen GBM tissue sections were immunostained for pp71 alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068176#pone-0068176-g002" target="_blank">figure 2F</a>) or co-immunostained for pp71 (green) and the endothelial cell marker CD31 (red). Tissues were counterstained with DAPI. Two regions from a representative example from a GBM sample are shown. Magnification of the region of interest is shown in the last panel. Bar = 200µm. Negative controls can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068176#pone-0068176-g002" target="_blank">figure 2F</a>.</p

U87-pp71 IPA pathway analysis summary.

<p>Chart summarizing fold changes for the top 20 significantly altered genes in pp71 expressing versus control U87 cells (left panel). The top biological function alterations as determined by IPA software when compared to the Ingenuity knowledge base with associated p-values are listed (right panel).</p

Detection of pp71 RNA and protein in primary glioma specimens.

<p><b>A:</b> RNA was extracted from 20 different primary brain tissues, synthesized into cDNA, and amplified using pp71 and Rab14-specific PCR primers. RNA from U251 glioma cells mock infected or infected with HCMV Towne strain and commercially available RNA samples from normal fetal and adult human brain were used as controls. The N.C. negative control PCR contained water in place of cDNA. <b>B</b>: Several cDNA samples described in A were analyzed by TaqMan using primers and probes specific for the pp71 gene and normalized to Rab14. Copy number was determined using Ad169 viral DNA standard curve. <b>C</b>: Western blot analysis for pp71 from 10 different primary brain tissues. Lysates from normal human astrocytes and mock-infected or HCMV-infected U87 glioma cells were used as controls. <b>D</b>: Cells from 3 freshly resected GBM were sorted using CD133 labeled antibody and analyzed by RT-PCR for pp71 and Rab14. Negative control (NC) is PCR performed with water instead of cDNA. <b>E</b>: HCMV-infected primary GBM cell line (3832) was sorted using CD133 labeled antibody at 72 hours post-infection and analyzed by TaqMan for pp71 compared to a mock-treated control. The relative expression of pp71 normalized to Rab14 is displayed for 3 independent experiments, *p<0.05 by student t-test.</p

pp71 stimulates expression and secretion of the pro-angiogenic cytokine SCF.

<p><b>A</b>: NPC cultures were transduced with adenoviruses expressing either pp71 or GFP as a negative control and total RNA was harvested, reverse transcribed into cDNA, and analyzed using a quantitative PCR array. Fold-change in expression of 24 out of 87 cellular genes in pp71-transduced cells is displayed <b>B</b>: Conditioned medium from serum-starved NPCs, U87 cells, or normal human astrocytes treated as indicated were analyzed by ELISA for human SCF. *p<0.05 by student t-test when compared to untreated control; when compared to rAD-GFP control p = 0.037 for NHAs, 0.09 for NPCs, and 0.094 for U87s. <b>C</b>: Top panel: NPCs mock-infected or infected with HCMV TR strain for 5 days were immunostained for pp71 (red), SCF (green) and counterstained with DAPI (blue). Bottom panel: NPCs mock-infected or transduced with rAD-GFP or rAD-pp71 were immunostained for the pp71 associated HA tag (green – mock and rAD-pp71 only), human SCF (blue) and counterstained propidium iodide (red). <b>D</b>: Several cDNA samples as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068176#pone-0068176-g001" target="_blank">figure 1A</a> were analyzed by TaqMan for SCF expression. Copy number was determined using a human brain cDNA standard curve and is plotted relative to the presence of pp71 transcripts in the same specimens. <b>E</b>: Primary GBM cells were immunostained for pp71 (green) and SCF (blue). Secondary antibody only was a negative control (IgG-green) and counterstain was performed with propidium iodide (red). <b>F</b>: Frozen tissue sections from primary GBMs were processed by immunostaining for pp71 only (green) or pp71 and SCF (red, red arrow). Areas of co-localization in the merged image are yellow (yellow arrow). Tissues were counterstained with DAPI. Controls for immunofluorescent staining of frozen tissue sections were performed with secondary antibody only (anti-mouse IgG 488 and anti-rabbit IgG 594). <b>G</b>: The Pearson coefficient for green (pp71) and red (SCF) pixels were calculated in both co-stained and single-stained samples from 7 cells and plotted (quantification was performed as described in reference ²¹). pp71 and SCF staining was highly correlated (r = 0.69) and significantly different from single stained controls (*, p = 0.001 for pp71 and 0.002 for SCF).</p

pp71 status, HCMV serology and IF analysis of primary GBM samples.

<p>Serology for IgG and IgM was performed and charted relative to pp71 expression status as determined by RT-PCR, TaqMan and western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068176#pone-0068176-g001" target="_blank">figure 1</a>. Flash frozen tissue sections were analyzed for the presence of pp71, SCF and CD31 by immunofluorescence (IF) in selected tumor cases, as indicated. GBM patient samples without available plasma were labeled N/A. Tumors were classified as low grade astrocytoma (LGA) or GBM by pathology, and the GBM samples were further subclassified as proneural (PN), mesenchymal (MES), or classical (CLAS) based on their gene expression signatures (data not shown and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068176#pone-0068176-g006" target="_blank">figure 6C</a>). BC refers to a non-tumor surgical specimen from a blood clot. ND signifies not determined.</p