Detection of human cytomegalovirus in normal and neoplastic breast epithelium

Abstract Introduction Human cytomegalovirus (HCMV) establishes a persistent life-long infection, and can cause severe pathology in the fetus and the immunocompromised host[1]. Breast milk is the primary route of transmission in humans worldwide, and breast epithelium is thus a likely site of persistent infection and/or reactivation, though this phenomenon has not previously been demonstrated. Increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. We hypothesized that persistent HCMV infection occurs in normal adult breast epithelium and that persistent viral expression might be associated with normal and neoplastic ductal epithelium. Methods Surgical biopsy specimens of normal breast (n = 38) breast carcinoma (n = 39) and paired normal breast from breast cancer patients (n = 21) were obtained. Specimens were evaluated by immunohistochemistry, in situ hybridization, PCR and DNA sequencing for evidence of HCMV antigens and nucleic acids. Results We detected HCMV expression specifically in glandular epithelium in 17/27 (63%) of normal adult breast cases evaluated. In contrast, HCMV expression was evident in the neoplastic epithelium of 31/32 (97%) patients with ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) cases evaluated (p = 0.0009). Conclusions These findings are the first to demonstrate that persistent HCMV infection occurs in breast epithelium in a significant percentage of normal adult females. HCMV expression was also evident in neoplastic breast epithelium in a high percentage of normal and neoplastic breast tissues obtained from breast cancer patients, raising the possibility that viral infection may be involved in the neoplastic process. </jats:sec

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

SCF induction by HCMV can be blocked by pp71 knockdown and Cidofovir.

<p><b>A</b>: NPCs infected with HCMV (TR) were transfected with either negative control siRNA or a pp71-specific siRNA (designated A and B), pp71 protein levels were measured 72 hours later using western blot analysis (left panel). IE1 expression was determined to ensure specificity of the knockdown. <b>B</b>: Conditioned medium samples from the cells described in A were analyzed by ELISA for SCF. The percent decrease in SCF is displayed. (* p = 0.07 for pp71 B siRNA compared to negative control siRNA). <b>C</b>: HCMV positive, primary GBM cells transfected with control non-targeting siRNA or a siRNA specific to pp71 were analyzed by western blot for pp71 and actin 72h later. Percentage of pp71 knockdown (12.5%) from the western blot in C was calculated by normalization to actin (top panel). Conditioned medium from the same samples was analyzed by ELISA for secreted human SCF (bottom panel). *p = 0.003 by student t-test when pp71 siRNA was compared to negative control siRNA treatment. <b>D</b>: U87 cells mock or HCMV-infected were treated with 10uM cidofovir (CDV) or vehicle for 72 hours. RNA was collected and analyzed by TaqMan for pp71 and SCF and expression was normalized to Rab14 (top panel). Conditioned medium from the cells in E were analyzed by ELISA in triplicate. The percent decrease in SCF after CDV treatment compared to vehicle control is displayed (bottom panel). *p<0.05 as determined by a student t-test. <b>E</b>: Endogenously infected primary GBM cells (CPMC-145) were sorted for CD133 and the positive fraction was sub-cultured and treated with vehicle only or 10 uM CDV for 72 hours. RNA was collected and analyzed by TaqMan for pp71, US28 (another viral gene), SCF and Rab14.</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

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

Diagram represents proposed signaling pathways between HCMV and the IL6-BMXp-STAT3 axis in long-term HCMV infected human glioma stem cells.

<p>Diagram represents proposed signaling pathways between HCMV and the IL6-BMXp-STAT3 axis in long-term HCMV infected human glioma stem cells.</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