Human cytomegalovirus infection is correlated with enhanced cyclooxygenase-2 and 5-lipoxygenase protein expression in breast cancer.

PURPOSE: While enhanced expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) and their derived metabolites is associated with breast cancer (BC) risk, the precise link between BC carcinogenesis and enhanced inflammatory activity remains to be clarified. Human Cytomegalovirus (HCMV) may induce expression of COX-2 and 5-LO and is frequently found in breast cancer biopsies. Thus, we investigated whether there is an association between HCMV proteins and expression of COX-2 and 5-LO in human BC tissue and BC cell lines. MATERIALS AND METHODS: Paraffin embedded biopsies obtained from 49 patients with breast cancer and 26 tissue samples from adjacent, benign breast tissues were retrospectively examined for HCMV-immediate early (IE), HCMV-Late (LA), COX-2, and 5-LO proteins by immunohistochemistry. In vitro, uninfected and HCMV-infected BC cell lines were examined for COX-2 and 5-LO transcripts and proteins by PCR and flow cytometry. RESULTS: Extensive expression of COX-2, 5-LO and HCMV-IE proteins were preferentially detected in BC samples. We found a statistically significant concordant correlation between extensive HCMV-IE and COX-2 (P < 0.0001) as well as with HCMV-IE and 5-LO (P = 0.0003) in infiltrating BC. In vitro, HCMV infection induced COX-2 and 5-LO transcripts and COX-2 proteins in MCF-7 cells (P =0.008, P =0.018, respectively). In MDA-MB-231 cells that already had high base line levels of COX-2 expression, HCMV induced both COX-2 and 5-LO proteins but not transcripts. CONCLUSION: Our findings demonstrate a significant correlation between extensive HCMV-IE protein expression and overexpression of COX-2 and 5-LO in human breast cancer.

5­Azacytidine treatment results in nuclear exclusion of DNA methyltransferase­1, as well as reduced proliferation and invasion in human cytomegalovirus­infected glioblastoma cells.

Glioblastoma (GBM) is the most aggressive form of brain tumor in adults, with a devastating outcome. Emerging evidence shows that human cytomegalovirus (HCMV) proteins and nucleic acids are present in GBM tissues. DNA methylation is important for the initiation and progression of cancer and is an established host response against invading nucleic acids. The expression and localization of DNA methyltransferase 1 (DNMT­1) was assessed, and the effects of DNA methylation inhibitor 5­azacytidine (5AZA) were analyzed in the context of the viral replication, proliferation and invasion capacities of HCMV­infected GBM U343MG cells. In addition, the expression of various HCMV proteins and DNMT­1 was examined in GBM tissue specimens obtained from five patients. DNMT­1 was localized in the nucleus of cells expressing HCMV­immediate early, whereas in cells expressing HCMV­glycoprotein gB (gB), extranuclear/cytoplasmic localization was observed. This was also observed in vitro in U343MG cells. In addition, DNMT­1 was localized to the extranuclear/cytoplasmic space of cells lining blood vessel walls within the GBM tumors. Treatment of infected U343MG cells with 5AZA did not affect viral replication, but reduced cell invasion and proliferation (P=0.05 and P<0.0001, respectively). However, 5AZA treatment of uninfected cells did not affect cell invasion (P=0.09), but proliferation was significantly reduced (P<0.0001). These findings may be of importance in further investigations aimed at using DNA methylation and viral inhibitors in GBM therapy.

Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer.

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P = .002, P < .0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P = .04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5 months, P = .03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P = .03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.

Overexpression of endothelin B receptor in glioblastoma: a prognostic marker and therapeutic target?

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with median survival of 12-15 months. Owing to uncertainty in clinical outcome, additional prognostic marker(s) apart from existing markers are needed. Since overexpression of endothelin B receptor (ETBR) has been demonstrated in gliomas, we aimed to test whether ETBR is a useful prognostic marker in GBM and examine if the clinically available endothelin receptor antagonists (ERA) could be useful in the disease treatment. METHODS: Data from The Cancer Genome Atlas and the Gene Expression Omnibus database were analyzed to assess ETBR expression. For survival analysis, glioblastoma samples from 25 Swedish patients were immunostained for ETBR, and the findings were correlated with clinical history. The druggability of ETBR was assessed by protein-protein interaction network analysis. ERAs were analyzed for toxicity in in vitro assays with GBM and breast cancer cells. RESULTS: By bioinformatics analysis, ETBR was found to be upregulated in glioblastoma patients, and its expression levels were correlated with reduced survival. ETBR interacts with key proteins involved in cancer pathogenesis, suggesting it as a druggable target. In vitro viability assays showed that ERAs may hold promise to treat glioblastoma and breast cancer. CONCLUSIONS: ETBR is overexpressed in glioblastoma and other cancers and may be a prognostic marker in glioblastoma. ERAs may be useful for treating cancer patients.

Increased cytomegalovirus replication by 5-Azacytidine and viral-induced cytoplasmic expression of DNMT­1 in medulloblastoma and endothelial cells.

Among all brain tumors diagnosed in children, medulloblastomas (MBs) are associated with a poor prognosis. The etiology of MB is not fully understood, yet the impact of epigenetic alterations of oncogenes has previously been established. During the past decade, the human cytomegalovirus (HCMV) has been detected in several types of cancer, including MB. Since DNA methylation occurs in the cell nucleus and this is considered a host defence response, we studied the impact of HCMV infection on DNA methyltransferase (DNMT­1) in MB (D324) cells, human umbilical vein endothelial cells (HUVECs) as well as in MB tissue sections. We hypothesized that infection and DNMT­1 intracellular localization are linked. Uninfected and HCMV­infected D324 cells and HUVECs were analyzed for HCMV immediate early (HCMV­IE) protein, HCMV­glycoprotein B (HCMV­gB) and DNMT­1 using immunofluorescence staining and quantitative ELISA. DNMT­1 localized to the nucleus of uninfected and HCMV­IE- expressing D324 cells and HUVECs, but accumulated in the extra nuclear space in all HCMV­gB-positive cells. Inhibition of HCMV late protein expression by Cymevene® (ganciclovir) prevented the cytoplasmic localization of DNMT­1. Treatment of HCMV­ infected D324 cells and HUVECs with the methylation inhibitor 5-Azacytidine (5AZA), significantly increased HCMV­IE and HCMV­gB gene transcription and protein expression. Immunohistochemical staining of DNMT­1 and HCMV proteins in MB cancer tissue sections revealed both nuclear and cytoplasmic DNMT­1 localization. In conclusion, DNMT­1 resides in the cytoplasm of HCMV­gB-expressing HUVECs and D324 cells. Increased viral protein synthesis in 5AZA-treated cells suggests that HCMV replication may benefit from a DNA methyltransferase-free cellular environment. Our findings emphasize the importance of assessing potential viral activation in the treatment of MB patients with epigenetic drugs.

Low Expression of Estrogen Receptor-α and Progesterone Receptor in Human Breast Cancer Tissues Is Associated With High-Grade Human Cytomegalovirus Protein Expression.

BACKGROUND: The underlying mechanisms for breast cancer (BC) are largely unknown. We investigated possible correlations between the expression levels of human cytomegalovirus (HCMV) proteins and established histopathological markers of BC, including expression of estrogen receptor (ER)-α, the progesterone receptor (PgR), and HER2. MATERIALS AND METHODS: We retrospectively examined paraffin-embedded biopsy specimens of BC (n = 62), ductal carcinoma in situ (n = 19), and adjacent normal breast tissue (n = 42) for HCMV immediate-early protein (IE), HCMV late antigen, HCMV DNA and RNA, and investigated possible correlations between them and expression of ER-α, PgR, and HER2. RESULTS: HCMV DNA and RNA were detected in all examined infiltrating BCs. High-grade positivity for HCMV-IE was detected in 77% of infiltrating BCs, 39% of ductal carcinomas in situ, and 7% of tumor-free breast tissue samples. HCMV expression correlated inversely with ER-α (P = .02) and PgR (P = .003) expression. HER2 expression was also reduced in HCMV-positive samples without reaching a level of statistical significance (P = .09). CONCLUSION: The negative correlation between high-grade expression HCMV-IE and hormone receptor expression suggests a role for HCMV in hormone receptor-negative BC tumors, possibly by forcing BC cells into a more aggressive phenotype.

Human Cytomegalovirus-Encoded Receptor US28 Is Expressed in Renal Allografts and Facilitates Viral Spreading In Vitro.

BACKGROUND: Renal transplantation is the preferred treatment for patients with end-stage renal disease. Human cytomegalovirus (HCMV) activation is associated with decreased renal graft function and survival. Human cytomegalovirus encodes several immune modulatory proteins, including the G protein-coupled receptor US28, which scavenges human chemokines and modulates intracellular signaling. METHODS: Our aim was to identify the expression and localization of US28 in renal allograft biopsies by immunohistochemistry and determine its role in viral spreading in vitro. RESULTS: Immunohistochemistry revealed US28 in 31 of 34 renal transplant biopsies from HCMV-seropositive donors. Expression was independent of HCMV viremia or IgG serostatus. US28 was predominantly expressed in the cytoplasm of vascular smooth muscle cells (VSMCs) and tubular epithelial cells, with a median positivity of 20% and 40%, respectively. Also, US28-positive cells were present within arterial neointima. In contrast to US28, HCMV-encoded immediate early antigen was detected in less than 5% of VSMCs, tubular epithelial cells, interstitial endothelium, interstitial inflammatory infiltrates, and glomerular cells.Primary VSMCs were infected with green fluorescent protein-tagged wild type or US28-deficient HCMV. The viral spreading of US28-deficient HCMV, via culture medium or cell-to-cell transmission, was significantly impeded as shown by green fluorescent protein (ie, infected) cell quantification and quantitative real-time polymerase chain reaction. Additionally, the number and size of foci was smaller. CONCLUSIONS: In summary, HCMV-encoded US28 was detected in renal allografts from HCMV-positive donors independent of viremia and serostatus. Also, US28 facilitates HCMV spreading in VSMCs in vitro. Because the vasculature is affected in chronic renal transplant dysfunction, US28 may provide a potential target for therapeutic intervention.

Human cytomegalovirus may promote tumour progression by upregulating arginase-2.

BACKGROUND: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.

Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

Background. Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation. Methods. Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays. Results. Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs. Conclusions. Human CMV may contribute to CVD via ETBR induction.

Detection of circulating hcmv-miR-UL112-3p in patients with glioblastoma, rheumatoid arthritis, diabetes mellitus and healthy controls.

BACKGROUND: microRNAs (miRNA) are 18-22 nucleotides long non-coding RNAs that regulate gene expression at a post-transcriptional level. Human cytomegalovirus (HCMV) encodes at least 26 known mature miRNAs. hcmv-miR-UL112-3p (miR-UL112-3p) is the most well characterized HCMV miRNA, which is suggested to play role in establishment and maintenance of viral latency. Elevated miR-UL112-3p levels have been reported to be present in plasma of patients with hypertension. OBJECTIVES: In this study, we aimed to quantify miR-UL112-3p levels in the plasma/serum of patients with Diabetes Mellitus (DM; from the DIGAMI-2 cohort), Glioblastoma multiforme (GBM), Rheumatoid Arthritis (RA) and Healthy Controls (HC). STUDY DESIGN: Total RNA was isolated from plasma/serum samples of 87 patients and controls, a TaqMan miRNA assay was performed to detect miR-UL112-3p and the copy numbers were normalized to 10 ng of total RNA. HCMV IgG and IgM were analysed using ELISA. RESULTS: HCMV miR-UL112-3p was detected in 14/27 (52%) of DM, 5/20 (25%) of GBM, 1/20 (5%) of RA patients and in 2/20 (10%) of HC, respectively. Anti-HCMV IgG was detected in 85%, 65%, 75% of patients and 70% of HC, respectively. Anti-HCMV IgM was found only in one GBM patient of 87 examined patients and controls. CONCLUSIONS: A higher prevalence of miR-UL112-3p was detected in DM and GBM patients than in RA patients and HC. Elevated levels of miR-UL112-3p and higher prevalence of HCMV IgG were observed in DM patients. Whether the presence of circulating miR-UL112-3p denotes a biomarker of HCMV latency or active replication in patients warrants further investigation.