A Cell-Surface Membrane Protein Signature for Glioblastoma.

We present a systems strategy that facilitated the development of a molecular signature for glioblastoma (GBM), composed of 33 cell-surface transmembrane proteins. This molecular signature, GBMSig, was developed through the integration of cell-surface proteomics and transcriptomics from patient tumors in the REMBRANDT (n = 228) and TCGA datasets (n = 547) and can separate GBM patients from control individuals with a Matthew\u27s correlation coefficient value of 0.87 in a lock-down test. Functionally, 17/33 GBMSig proteins are associated with transforming growth factor β signaling pathways, including CD47, SLC16A1, HMOX1, and MRC2. Knockdown of these genes impaired GBM invasion, reflecting their role in disease-perturbed changes in GBM. ELISA assays for a subset of GBMSig (CD44, VCAM1, HMOX1, and BIGH3) on 84 plasma specimens from multiple clinical sites revealed a high degree of separation of GBM patients from healthy control individuals (area under the curve is 0.98 in receiver operating characteristic). In addition, a classifier based on these four proteins differentiated the blood of pre- and post-tumor resections, demonstrating potential clinical value as biomarkers

Publisher Correction: Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

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 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

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