Focal-to-bilateral tonic-clonic seizures and High-grade CMV-infection are poor survival predictors in Tumor-related Epilepsy Adult-type diffuse gliomas-A single-center study and literature review.

Introduction: Previous studies have reported a correlation between a high-grade CMV-infection and an unfavorable prognosis in glioblastoma (GB). Coversely, epilepsy has been associated with a more favorable outcome in GB patients. Despites epilepsy and CMV share similar molecular mechanisms in GB tumoral microenvironment, the correlation between Tumor-Related-Epilepsy (TRE) and CMVinfection remains unexplored. The aim of our study is to examine the correlation between the dregree of CMV infection and seizure types on the survival of TRE Adult-type-diffuse-glioma. To achieve this objective, we conducted a comprehensive literature review to assess our results regarding previous publications. Methods: We conducted a retrospective-observational study on TRE Adult-type-diffuse-gliomas treated at a single center in Mexico from 2010 to 2018. Tumor tissue and cDNA were analyzed by immunochemistry (IHC) for CMV (IE and LA antigens) at the Karolinska Institute in Sweden, and RT-PCR for CMV-gB in Torreon Mexico, respectively. Bivariate analysis (X2-test) was performed to evaluate the association between subtypes of Adult-type-diffuse-glioma (IDH-mut grade 4 astrocytoma vs. IDH-wt glioblastoma) and the following variables: type of hemispheric involvement (mesial vs. neocortical involvement), degree of CMV infection (<25%vs. >25% infected-tumoral cells) and seizure types [Focal awareness, focal impaired awareness, and FBTCS]. Kaplan Meier and Cox analyses were performed to determine the risk, p < 0.05 was considered statistically significant. Results: Sixty patients with TRE Adult type diffuse gliomas were included (80% IDH-wt glioblastoma and 20% IDH-mut grade 4astrocytomas). The mean age was 61.5 SD ± 18.4, and 57% were male. Fifty percent of the patients presented with mesial involvement of the hemysphere. Seizure types included focal awareness (15%), focal impaired awareness (43.3%), and FBTCS (41.7%). Ninety percent of cases were treated with Levetiracetam and 33.3% presented Engel-IA postoperative seizure control. More than 90% of samples were positive for CMV-immunohistochemistry (IHC). However, all cDNA analyzed by RT-PCR return negative results. The median of overall survival (OS) was 15 months. High-grade CMV-IE infection (14 vs. 25 months, p<0.001), mesial involvement (12 vs. 18 months, p<0.001), and FBTCS were associated with worse OS (9 vs.18 months for non-FBTCS). Multivariate analysis demonstrated that high-grade CMV infection (HR = 3.689, p=0.002) and FBTCS (HR=7.007, p<0.001) were independent unfavorable survival factors. Conclusions: CMV induces a proinflammatory tumoral microenvironment that contributes to the developmet of epilepsy. Tumor progression could be associated not only with a higher degree of CMV infection but also to epileptogenesis, resulting in a seizure phenotype chracterized by FBTCS and poor survival outcomes. This study represents the first survival analysis in Latin America to include a representative sample of TRE Adult-type diffuse gliomas considering CMV-infection-degree and distinguishing features (such as FBTCS) that might have potential clinical relevance in this group of patients. Further prospective studies are required to validate these results.

Non-classical HLA-E restricted CMV 15-mer peptides are recognized by adaptive NK cells and induce memory responses.

Introduction: Human cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive. Methods: We employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses. Results: Here, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs. Discussion: These findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.

Human cytomegalovirus infection enhances 5­lipoxygenase and cycloxygenase­2 expression in colorectal cancer.

Colorectal cancer (CRC) is one of the most common and fatal types of cancer. Inflammation promotes CRC development, however, the underlying etiological factors are unknown. Human cytomegalovirus (HCMV), a virus that induces inflammation and other cancer hallmarks, has been detected in several types of malignancy, including CRC. The present study investigated whether HCMV infection was associated with expression of the pro­inflammatory enzymes 5­lipoxygenase (5­LO) and cyclooxygenase­2 (COX­2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146 individual paraffin­embedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki­67 index and EGFR by immunohistochemistry (IHC). The cores were further analyzed by IHC for the expression of two HCMV proteins (Immediate Early, IE and pp65) and the inflammatory markers 5­LO and COX­2. The CRC cell lines Caco­2 and LS­174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or anti­inflammatory drug celecoxib (CCX) and analyzed by reverse transcription­quantitative PCR and immunofluorescence for 5­LO, COX­2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in ~90% of the CRC cases tested; this was correlated with COX­2, 5­LO and KI­67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. In vitro, HCMV infection upregulated 5­LO and COX­2 transcript and proteins in both Caco­2 and LS­174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX­2, 5­LO, HCMV IE and pp65 in infected cells. HCMV was widely expressed in CRC and may promote inflammation and serve as a potential new target for CRC therapy.

Higher Human Cytomegalovirus (HCMV) Specific IgG Antibody Levels in Plasma Samples from Patients with Metastatic Brain Tumors Are Associated with Longer Survival.

Background: Human cytomegalovirus (HCMV) has been detected in tissue samples from patients with glioblastoma but little is known about the systemic immunological response to HCMV in these patients. Objectives: To investigate the presence and clinical significance of HCMV antibodies levels in plasma samples obtained from patients with brain tumors. Materials and Methods: HCMV-specific IgG and IgM antibody levels were determined in 59 plasma samples collected from brain tumor patients included in a prospective study and in 114 healthy individuals. We examined if the levels of HCMV specific antibodies varied in patients with different brain tumor diagnoses compared to healthy individuals, and if antibody levels were predictive for survival time. Results: HCMV specific IgG antibodies were detected by ELISA in 80% and 89% of patients with GBM and astrocytoma grades II-III, respectively, in all samples (100%) from patients with secondary GBM and brain metastases, as well as in 80% of healthy donors (n = 114). All plasma samples were negative for HCMV-IgM. Patients with brain metastases who had higher plasma HCMV-IgG titers had longer survival times (p = 0.03). Conclusions: HCMV specific IgG titers were higher among all brain tumor patient groups compared with healthy donors, except for patients with secondary GBM. Higher HCMV specific IgG levels in patients with brain metastases but not in patients with primary brain tumors were associated with prolonged survival time.

Human Cytomegalovirus Protein Expression Is Correlated with Shorter Overall Survival in Breast Cancer Patients: A Cohort Study.

Background: Human cytomegalovirus (HCMV) is increasingly suggested to be involved in human carcinogenesis and onco-modulation due to its ability to contribute to all hallmarks of cancer. Growing evidence demonstrates a link between HCMV infection and various malignancies, including breast cancer, which incidence and mortality are still on the rise. The etiology of breast cancer remains mostly unclear, leaving 80% of breast cancer cases considered to be sporadic. Identifying novel risk- and prognostic factors for improved breast cancer treatment and increased survival rates, were the objectives of this study. Methods: Automated immunohistochemical staining results for HCMV proteins in 109 breast tumors and lymph node metastasis were correlated with clinical follow-up data (>10 years). Statistical analyses for median Overall Survival (OS) were performed. Results: Survival analyses revealed shorter median OS for patients with HCMV-IE positive tumors of 118.4 months compared to 202.4 months for HCMV-IE negative tumors. A higher number of HCMV-LA positive cells in the tumors was also associated with a shorter OS in patients (146.2 months vs. 151.5 months). Conclusions: Our findings suggest a link between HCMV-infections and breast cancer prognosis, which paves the way for potential novel clinical intervention and targeted therapy that may prolong the overall survival of selected patients with breast cancer.

Platelet p110ß mediates platelet-leukocyte interaction and curtails bacterial dissemination in pneumococcal pneumonia.

Phosphatidylinositol 3-kinase catalytic subunit p110ß is involved in tumorigenesis and hemostasis. However, it remains unclear if p110ß also regulates platelet-mediated immune responses, which could have important consequences for immune modulation during anti-cancer treatment with p110ß inhibitors. Thus, we investigate how platelet p110ß affects inflammation and infection. Using a mouse model of Streptococcus pneumoniae-induced pneumonia, we find that both platelet-specific p110ß deficiency and pharmacologic inhibition of p110ß with TGX-221 exacerbate disease pathogenesis by preventing platelet-monocyte and neutrophil interactions, diminishing their infiltration and enhancing bacterial dissemination. Platelet p110ß mediates neutrophil phagocytosis of S. pneumoniae in vitro and curtails bacteremia in vivo. Genetic deficiency or inhibition of platelet p110ß also impairs macrophage recruitment in an independent model of sterile peritonitis. Our results demonstrate that platelet p110ß dysfunction exacerbates pulmonary infection by impeding leukocyte functions. Thereby, our findings provide important insights into the immunomodulatory potential of PI3K inhibitors in bacterial infection.

Valganciclovir as Add-on to Second-Line Therapy in Patients with Recurrent Glioblastoma.

Glioblastoma invariably recurs despite aggressive and multimodal first-line treatment and no standardized second-line therapy exists. We previously reported that treatment with the antiviral drug valganciclovir as an add-on to standard therapy significantly prolonged overall survival in 102 patients with newly diagnosed glioblastoma compared to contemporary controls. Here we present the results of retrospective survival analyses including patients with glioblastoma that initiated valganciclovir therapy after recurrence. Twenty-nine patients with recurrent glioblastoma received valganciclovir as an add-on to second-line therapy at Karolinska University Hospital. Contemporary controls were 109 patients with glioblastoma who received similar second-line therapy at our institution. We retrospectively analyzed survival data of these patients. Patients with recurrent glioblastoma who received valganciclovir had longer median overall survival after recurrence than controls (12.1 vs. 7.4 months, respectively, p = 0.0028). The drug was well tolerated. Both patients who underwent re-operation and patients that were not re-operated after recurrence benefitted significantly from valganciclovir therapy. Valganciclovir prolonged survival after recurrence both in patients with an unmethylated and methylated MGMT promoter gene. Valganciclovir was safe to use and prolonged median survival after recurrence for patients with recurrent glioblastoma, re-operated or not after recurrence, and with methylated or unmethylated MGMT promoter gene.

Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.

Viral infections enhance cancer risk and threaten host genome integrity. Although human cytomegalovirus (HCMV) proteins have been detected in a wide spectrum of human malignancies and HCMV infections have been implicated in tumorigenesis, the underlying mechanisms remain poorly understood. Here, we employed a range of experimental approaches, including single-molecule DNA fiber analysis, and showed that infection by any of the four commonly used HCMV strains: AD169, Towne, TB40E or VR1814 induced replication stress (RS), as documented by host-cell replication fork asymmetry and formation of 53BP1 foci. The HCMV-evoked RS triggered an ensuing host DNA damage response (DDR) and chromosomal instability in both permissive and non-permissive human cells, the latter being particularly relevant in the context of tumorigenesis, as such cells can survive and proliferate after HCMV infection. The viral major immediate early enhancer and promoter (MIEP) that controls expression of the viral genes IE72 (IE-1) and IE86 (IE-2), contains transcription-factor binding sites shared by promoters of cellular stress-response genes. We found that DNA damaging insults, including those relevant for cancer therapy, enhanced IE72/86 expression. Thus, MIEP has been evolutionary shaped to exploit host DDR. Ectopically expressed IE72 and IE86 also induced RS and increased genomic instability. Of clinical relevance, we show that undergoing standard-of-care genotoxic radio-chemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV protein markers after tumor recurrence. Collectively, these results are consistent with our proposed concept of HCMV hijacking transcription-factor binding sites shared with host stress-response genes. We present a model to explain the potential oncomodulatory effects of HCMV infections through enhanced replication stress, subverted DNA damage response and induced genomic instability.

Presence of the Human Cytomegalovirus in Glioblastomas-A Systematic Review.

Glioblastoma is a malignant brain tumor with a dismal prognosis. The standard treatment has not changed in the past 15 years as clinical trials of new treatment protocols have failed. A high prevalence of the human cytomegalovirus (HCMV) in glioblastomas was first reported in 2002. The virus was found only in the tumor and not in the surrounding healthy brain tissue. Many groups have confirmed the presence of the HCMV in glioblastomas, but others could not. To resolve this discrepancy, we systematically reviewed 645 articles identified in different databases. Of these, 81 studies included results from 247 analyses of 9444 clinical samples (7024 tumor samples and 2420 blood samples) by different techniques, and 81 articles included 191 studies that identified the HCMV in 2529 tumor samples (36% of all tumor samples). HCMV proteins were often detected, whereas HCMV nucleic acids were not reliably detected by PCR methods. Optimized immunohistochemical techniques identified the virus in 1391 (84,2%) of 1653 samples. These data suggest that the HCMV is highly prevalent in glioblastomas and that optimized immunohistochemistry techniques are required to detect it.

Detection of Human Cytomegalovirus Proteins in Paraffin-Embedded Breast Cancer Tissue Specimens-A Novel, Automated Immunohistochemical Staining Protocol.

Emerging evidence supports a significant association between human cytomegalovirus (HCMV) and human malignancies, suggesting HCMV as a human oncomodulatory virus. HCMV gene products are found in >90% of breast cancer tumors and seem to be correlated with more aggressive disease. The definitive diagnosis of HCMV relies on identification of virus inclusions and/or viral proteins by different techniques including immunohistochemical staining. In order to reduce biases and improve clinical value of HCMV diagnostics in oncological pathology, automation of the procedure is needed and this was the purpose of this study. Tumor specimens from 115 patients treated for primary breast cancer at Akershus University Hospital in Norway were available for the validation of the staining method in this retrospective study. We demonstrate that our method is highly sensitive and delivers excellent reproducibility for staining of HCMV late antigen (LA), which makes this method useful for future routine diagnostics and scientific applications.

NUDT15-mediated hydrolysis limits the efficacy of anti-HCMV drug ganciclovir.

Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV.

Evidence of human cytomegalovirus infection and expression of 5-lipoxygenase in borderline ovarian tumors.

Borderline ovarian tumors (BOTs) belong to a group of tumors that are distinctly different from ovarian carcinomas. There is an increased risk of BOTs in patients with pelvic inflammatory disease. Human cytomegalovirus (HCMV) has been detected in ovarian cancer tissue specimens. This virus favors the inflammatory milieu by inducing expression of the potent inflammatory factor 5-lipoxygenase (5LO), which stimulates cellular viability, cellular proliferation and activates antiapoptotic signaling pathways. Here, we aimed to examine presence of HCMV and 5LO in BOTs. Expression levels of HCMV proteins (IE and pp65) and 5LO were examined in paraffin embedded BOT tissue sections by immunohistochemistry staining and HCMV immunoglobulin M and immunoglobulin G (IgG) levels were determined by serology in blood samples obtained from 15 patients with BOTs identified in a prospective study at Karolinska University Hospital. Extensive expression of HCMV-IE, pp65, and 5LO were detected in 87%, 40%, and 90% of examined BOT tissue sections, respectively. HCMV-IgG prevalence and antibody levels were significantly higher in patients with BOT compared to age matched healthy women (83.3% vs. 65,6%, respectively, p = .01). Whether HCMV can induce inflammation and affect the pathogenesis of BOTs should therefore be further investigated.

Valganciclovir as Add-On to Standard Therapy in Secondary Glioblastoma.

Patients with glioblastoma have a very poor prognosis despite aggressive therapeutic strategies. Cytomegalovirus has been detected in >90% of glioblastoma tumors. This virus can affect tumor progression and may represent a novel glioblastoma therapy target. We report, here, a retrospective survival analysis of patients with secondary glioblastoma who were treated with the anti-viral drug valganciclovir at Karolinska University Hospital in Stockholm. We performed survival analyses of eight patients with secondary glioblastoma who were treated with a standard dose of valganciclovir as an add-on to second-line therapy after their disease progression to glioblastoma. Thirty-six patients with secondary glioblastoma admitted during the same time period who received similar treatment and care served as contemporary controls. The patients treated with valganciclovir showed an increased median overall survival after progression to glioblastoma compared with controls (19.1 versus 12.7 months, p = 0.0072). This result indicates a potential positive effect of valganciclovir in secondary glioblastoma, which is in agreement with our previous observation that valganciclovir treatment improves the outcomes of patients with newly diagnosed glioblastoma. Larger randomized studies are warranted to prove this hypothesis.

Valganciclovir as Add-on to Standard Therapy in Glioblastoma Patients.

PURPOSE: Several groups have reported a prevalence of human cytomegalovirus (CMV) in glioblastoma close to 100%. Previously, we reported that treatment with the antiviral drug valganciclovir as an add-on to standard therapy significantly prolonged survival in 50 patients with glioblastoma. Here, we present an updated retrospective analysis that includes an additional 52 patients. EXPERIMENTAL DESIGN: From December 2006 to November 2019, 102 patients with newly diagnosed glioblastoma received valganciclovir as an add-on to standard therapy. No additional toxicity was observed. Contemporary controls were 231 patients with glioblastoma who received similar baseline therapy. RESULTS: Patients with newly diagnosed glioblastoma receiving valganciclovir had longer median overall survival (OS 24.1 vs. 13.3 months, P < 0.0001) and a 2-year survival rate (49.8% vs. 17.3%) than controls. Median time-to-tumor progression was also longer than in controls; 9.9 (0.7-67.5 months) versus 7.3 (1.2-49 months), P = 0.0003. Valganciclovir improved survival in patients with radical or partial resection and an unmethylated or methylated MGMT promoter gene. CONCLUSIONS: Valganciclovir prolonged median OS of patients with newly diagnosed glioblastoma (with methylated or unmethylated MGMT promoter gene) and was safe to use.

Human Cytomegalovirus Infection Induces High Expression of Prolactin and Prolactin Receptors in Ovarian Cancer.

One of the potential biomarkers for ovarian cancer patients is high serum level of prolactin (PRL), which is a growth factor that may promote tumor cell growth. The prolactin receptor (PRLR) and human cytomegalovirus (HCMV) proteins are frequently detected in ovarian tumor tissue specimens, but the potential impact of HCMV infection on the PRL system have so far not been investigated. In this study, HCMV's effects on PRL and PRLR expression were assessed in infected ovarian cancer cells (SKOV3) by PCR and Western blot techniques. The levels of both PRL and PRLR transcripts as well as the corresponding proteins were highly increased in HCMV-infected SKOV3 cells. Tissue specimens obtained from 10 patients with ovarian cancer demonstrated high expression of PRLR, HCMV-IE, and pp65 proteins. Extensive expression of PRLR was detected in all examined ovarian tumor tissue specimens except for one from a patient who had focal expression of PRLR and this patient was HCMV-negative in her tumor. In conclusion, PRL and PRLR were induced to high levels in HCMV-infected ovarian cancer cells and PRLR expression was extensively detected in HCMV-infected ovarian tissue specimens. Highly induced PRL and PRLR by HCMV infection may be of relevance for the oncomodulatory role of this virus in ovarian cancer.

Cancer cell stemness, responses to experimental genotoxic treatments, cytomegalovirus protein expression and DNA replication stress in pediatric medulloblastomas.

Despite recent progress in research on brain tumors, including identification of cancer stem-like cells (CSCs), little is known about the interplay of stemness with the commonly observed infection by the human cytomegalovirus (HCMV) and the widespread features of replication stress in these malignancies. To shed more light on these outstanding issues, here we combine immunohistochemical analysis of archival clinical specimens from a cohort of 25 human pediatric medulloblastomas, complemented by functional experiments and analytical approaches to examine three medulloblastoma cell lines. In the clinical samples, we find consistent, yet individually variable subsets of CSCs expressing the stem-cell markers CD133 and CD15, and a candidate marker VEGFR2, across the spectrum of endogenous DNA damage (γH2AX), expression of HCMV immediate early and late proteins, proliferation rate (Ki67) or molecular class of MB. Contrary to MB cell lines DAOY and D324, the D283 cells showed pronounced phenotypic features of stemness, associated with enhanced endogenous DNA damage, exceptionally high susceptibility to infection with HCMV, unorthodox signaling pathway response to ionizing radiation and hyperactive response to hydroxyurea-induced replication stress. Notably, single-molecule DNA fiber analysis revealed aberrantly slow replication fork progression, pronounced fork asymmetry and inability to timely recover from drug-induced fork stalling in stem-like D283 cells, all hallmarks of pronounced chronic replication stress and propensity to genomic instability. These findings provide insights into human medulloblastoma stemness phenotypes, with various susceptibilities to infection by HCMV and impact on replication fork (mal)function, with implications for better understanding pathogenesis and responses to treatment in pediatric brain malignancies.Abbreviations: CSC: cancer stem-like cells; FBS: fetal bovine serum; HCMV: human cytomegalovirus; MB: medulloblastoma; MBSC: medulloblastoma stem cells; MOI: multiplicity of infection; PBS: phosphate-buffered saline; RPA: replication protein A; RS: replication stress; SHH: sonic hedgehog; VEGFR2: vascular endothelia growth factor receptor 2.

A Review of the Potential Role of Human Cytomegalovirus (HCMV) Infections in Breast Cancer Carcinogenesis and Abnormal Immunity.

Previously recognized classical human onco-viruses can regulate complex neoplastic events, and are estimated to play a role during carcinogenesis in 15-20% of cancer cases. Although the DNA and gene products of several viruses have been found in breast tumors, none of the classical onco-viruses have definitely been linked to the initiation of breast cancer. However, recent evidence shows that human cytomegalovirus (HCMV) gene products are found in >90% of tumors and metastases of breast cancers, and their increased expression can be correlated to a more aggressive breast cancer phenotype. Supporting the active role of HCMV in breast cancer, a specific HCMV strain, HCMV-DB, was recently shown to exert oncogenic transformational activity in breast epithelial cells in vitro, and to give rise to fast-growing, triple-negative breast tumors when injected into immune deficient mice. The same observation holds true for clinical studies implying increased HCMV protein expression in triple negative breast cancer biopsies. In addition to functionally being able to hijack tumor-promoting cellular events, HCMV is known to exhibit a wide range of immunosuppressive effects, which can have radical impact on the tumor microenvironment. HCMV infected cells can avoid recognition and elimination by the immune system by orchestrating polarization of immunosuppressive type II macrophages, preventing antigen presentation, by expressing T cell inhibitory molecules, and possibly, by the induction of regulatory T (Treg) cell responses. These actions would be especially deleterious for the antigenic activation and proliferation of tumor specific CD8+ cytotoxic T lymphocytes (CTLs), whose effector functions have recently been targeted by successful, experimental immunotherapy protocols. The recognition of alternative causes and drivers of breast cancer is a pivotal research topic for the development of diagnostics and novel, effective preventive and therapeutic strategies targeting both tumor cells and their microenvironments.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

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.