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.

The diagnostic value of insulin-like growth factor 1 (IGF1) for GH deficiency (GHD) in adults is not optimal. Molecular profiling could be used for biomarker discovery. The aim of this pilot study was to compare the serum metabolome between GHD patients and healthy controls, and identification of potential markers for diagnosis and/or for individual GH dosing. A total of ten patients with GHD, median age of 55 years and BMI of 27 kg/m(2), were compared with ten healthy age- and gender-matched controls. The serum metabolic profiles were generated using gas chromatography-coupled mass spectroscopy on fasting samples taken in the morning from the controls and at baseline and during 6 months of GH replacement in the patients with GHD. The difference in low-molecular weight compounds (LMC) distinguished the healthy controls from GHD patients. Among 285 measured metabolites, 13 were identified as being most important in differentiating GHD patients from controls. Of these, 11 could not be structurally annotated but many were classified as lipids. The difference in the LMC pattern persisted despite normalisation of IGF1 following GH replacement. GH replacement increased the levels of specific fatty acid compounds and decreased the levels of certain amino acids. No metabolite changed in response to GH treatment, to the same extent as IGF1. The measurement of 285 metabolites resulted in a unique pattern in GHD, but changes in the metabolite patterns during GH treatment were limited. The utility of metabolomics to find new markers in GHD and GH replacement remains to be further elucidated.

Recent studies suggest that SOCS2 is involved in the regulation of TLR signaling. In this study, we found that the expression of SOCS2 is regulated in human monocyte-derived DC by ligands stimulating TLR2, 3, 4, 5, 8 and 9 signaling. SOCS2 induction by LPS was dependent on the type I IFN regulated transcription factors IRF1 and IRF3 as shown by using silencing RNAs for IRFs. Blocking endogenous type I IFN signaling, by neutralizing antibodies to the receptor IFNAR2, abolished SOCS2 mRNA expression after TLR4 stimulation. Transcription factors STAT3, 5 and 6 displayed putative binding sites in the promoter regions of the human SOCS2 gene. Subsequent silencing experiments further supported that STAT3 and STAT5 are involved in LPS induced SOCS2 regulation. In mice we show that SOCS2 mRNA induction is 45% lower in bone marrow derived macrophages derived from MyD88(-/-) mice, and do not increase in BMMs from IRF3(-/-) mice after BCG infection. In conclusion, our results suggest that TLR4 signaling indirectly increases SOCS2 in late phase mainly via the production of endogenous type I IFN, and that subsequent IFN receptor signaling activates SOCS2 via STAT3 and STAT5.

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that approximately 35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.

Patients diagnosed with high grade serous ovarian adenocarcinoma have a poor prognosis. Recently human cytomegalovirus (HCMV) has been detected in several tumors. Here, we evaluated HCMV in ovarian cancer tissue specimens obtained at pre- and postchemotherapy tumor resection.Available paraffin embedded ovarian cancer tissues from matched pre- and postchemotherapy tumor resection specimens (i.e., diagnostic excisional biopsy prechemotherapy; DEBPC) and neoadjuvant chemotherapy followed by interval debulking surgery (NACT + IDS) from 10 patients with stage IIIC-IV high grade serous ovarian carcinoma (HGS) diagnosed between years 2007 and 2008 at Karolinska University Hospital were examined for HCMV immediate-early protein (HCMV-IE), tegument protein pp65, and nucleic acid (β2.7) by immunohistochemistry and in situ hybridization.HCMV-IE and pp65 were detected in 8/10 (80%), 4/9 (44%) and in 4/10 (40%), 5/8 in ovarian cancer tissue specimens from DEBPC and NACT + IDS, respectively. HCMV-β2.7 was detected in all available tissue sections obtained from DEBPC and NACT + IDS. Patients with HCMV-IE or pp65 positive cells in their ovarian tumors at IDS after NACT had a median overall survival of 23.4 and 18.2 months, respectively, compared to 29.6 and 54 months, respectively, in those who did not express HCMV proteins in their tumors.In conclusion, HCMV proteins and nucleic acids are frequently detected at different levels in HGS ovarian carcinoma. Despite the limitation of our study, shorter median overall survival of patients with HCMV-IE and pp65 in their tumor highlights the need to further investigate the role of HCMV in ovarian cancer patients.

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.

Ovarian cancer (OC) is characterized by a high morbidity and mortality, highlighting a great need for a better understanding of biological mechanisms that affect OC progression and improving its early detection methods. This study investigates effects of prolactin (PRL) on ovarian cancer cells, analyzes PRL receptors (PRLR) in tissue micro arrays and relates PRLR expression to survival of ovarian cancer. A database, composed of transcript profiles from OC, was searched for PRLR expression and results were put in relation to survival. Expression of PRLR in OC tissue sections and OC cell lines SKOV3, OV2008 and OVSAHO was assessed using immunohistochemistry, western blots and quantitative real-time PCR. The biological function of PRLR was evaluated by proliferation, colony formation and wound healing assays. Levels of PRLR mRNA are related to survival; in epithelial OC a high PRLR mRNA expression is related to a shorter survival. Analysis of a tissue micro array consisting of 84 OC showed that 72% were positive for PRLR immuno-staining. PRLR staining tended to be higher in OC of high grade tumors compared to lower grades. PRLR mRNA and protein can further be detected in OC cell lines. Moreover, in vitro treatment with PRL significantly activated the JAK/STAT pathway. PRLR expression is associated with OC survivals. PRL and its receptor may play an onco-modulatory role and promote tumor aggressiveness in OC. Alternatively, increased PRLR levels may form a base for the development of PRLR antagonist or PRLR antagonist-drug conjugate to increase selective uptake of anti-cancer drugs.

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 pro‑inflammatory enzymes 5‑lipoxygenase (5‑LO) and cyclooxygenase‑2 (COX‑2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146 individual paraffin‑embedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki‑67 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 5‑LO and COX‑2. The CRC cell lines Caco‑2 and LS‑174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or anti‑inflammatory drug celecoxib (CCX) and analyzed by reverse transcription‑quantitative PCR and immunofluorescence for 5‑LO, COX‑2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in ~90% of the CRC cases tested; this was correlated with COX‑2, 5‑LO and KI‑67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. In vitro, HCMV infection upregulated 5‑LO and COX‑2 transcript and proteins in both Caco‑2 and LS‑174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX‑2, 5‑LO, 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.

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.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is highly variable and could be mediated by a cross-protective pre-immunity. We identified 14 cross-reactive peptides between SARS-CoV-2 and influenza A H1N1, H3N2, and human herpesvirus (HHV)-6A/B with potential relevance. The H1N1 peptide NGVEGF was identical to a peptide in the most critical receptor binding motif in SARS-CoV-2 spike protein that interacts with the angiotensin converting enzyme 2 receptor. About 62%-73% of COVID-19-negative blood donors in Stockholm had antibodies to this peptide in the early pre-vaccination phase of the pandemic. Seasonal flu vaccination enhanced neutralizing capacity to SARS-CoV-2 and T cell immunity to this peptide. Mathematical modeling taking the estimated pre-immunity levels to flu into account could fully predict pre-Omicron SARS-CoV-2 outbreaks in Stockholm and India. This cross-immunity provides mechanistic explanations to the epidemiological observation that influenza vaccination protected people against early SARS-CoV-2 infections and implies that flu-mediated cross-protective immunity significantly dampened the first SARS-CoV-2 outbreaks.

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.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (KD = 2.3±0.2 nM) and mouse serum albumin (KD = 0.38±0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21±3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

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.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2-/- mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway.

The active form of vitamin D(3), 1α,25-dihydroxyvitamin D(3)(1α,25(OH)(2)D(3)), has anti-proliferative and anti-invasive activities in prostate cancer cells. Because of 1α,25(OH)(2)D(3) therapeutic potential in treating cancers, numerous analogues have been synthesized with an attempt to increase anti-proliferative and/or decrease calcemic properties. Among these analogues, 19-nor-1α,25(OH)(2)D(2) while being less calcemic has equivalent potency as 1α,25(OH)(2)D(3) in several in vitro and in vivo systems. We recently showed that 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (MART-10) was at least 500-fold and 10-fold more active than 1α,25(OH)(2)D(3) in inhibiting the proliferation of an immortalized normal prostate PZ-HPV-7 cells and the invasion of androgen insensitive PC-3 prostate cancer cells, respectively. In this study, we further investigated the effects of MART-10 and 1α,25(OH)(2)D(3) on the dose- and time-dependent induction of CYP24A1 gene expression in PC-3 prostate cancer cells. We found that MART-10 induced CYP24A1 gene expression at a lower concentration with a longer duration compared to 1α,25(OH)(2)D(3), suggesting that MART-10 is less susceptible to CYP24A1 degradation. Molecular docking model of human CYP24A1 and MART-10 indicates that its side chain is far away from the heme ion and is less likely to be hydroxylated by the enzyme. Furthermore, MART-10 was a more potent inhibitor of PC-3 cell proliferation and invasion compared to 1α,25(OH)(2)D(3). In addition, MART-10 down-regulated matrix metalloproteinase-9 (MMP-9) expression which could be one mechanism whereby MART-10 influences cancer cell invasion. Finally, we observed that subcutaneous administration of MART-10 up-regulated the CYP24A1 mRNA expression in rat kidneys without affecting their plasma calcium levels. Thus, our findings demonstrate that MART-10 is biologically active in vivo and may be an effective vitamin D analogue for clinical trials to treat prostate cancer.