Virus transcript levels and cell growth rates after naturally occurring HPV16 integration events in basal cervical keratinocytes.

Cervical carcinogenesis is characterized by a clonal selection process in which the high-risk human papillomavirus (HRHPV) genome usually changes from the extra-chromosomal (episomal) state seen in productive infections to DNA that is integrated into host chromosomes. However, it is not clear whether all HRHPV integration events provide cells with a selective growth advantage compared with the episome-containing cells from which they originate. It is also unclear whether selection of cells containing a particular integrant from a mixed population simply reflects the highest levels of virus oncogene expression or has additional determinants. These early events in cervical carcinogenesis cannot readily be addressed by cross-sectional studies of clinical samples. We used the W12 model system to generate a panel of cervical squamous cell clones that were derived from an identical background under non-competitive conditions and differed only by the genomic site of HPV16 integration. Compared with the 'baseline' episome-containing cells from which they were isolated, only 9/17 clones (53%) showed significantly greater growth rates and only 7/17 (41%) showed significantly greater expression of the major virus oncogenes E7/E6. There were significant variations in levels of HPV16 transcription per DNA template, changes that were associated with histone modifications in the integrated virus chromatin. Cell growth rates showed only weak and non-significant associations with protein and mRNA levels for E7, E6, and the mean E7/E6 values. We conclude that HPV16 integration in basal cervical cells does not necessarily lead to increased levels of virus oncogenes, or to a competitive growth advantage, when compared with the initiating episome-containing cells

Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells.

Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of 'master regulators' for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins.Cancer Research UK (Programme Grant A13080)This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep2983

Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma.

Oncostatin M receptor (OSMR) is commonly over-expressed in advanced cervical squamous cell carcinoma (SCC), producing a significantly worse clinical outcome. Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness. Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of cervical and oral SCC. TGM2 depletion in cervical SCC cells abrogated OSM-induced migration on fibronectin-coated surfaces and invasiveness through extracellular matrix, while ectopic expression of TGM2 increased cell motility and invasiveness. Confocal microscopy and co-immunoprecipitation assays showed that TGM2 interacted with integrin-α5β1 in the presence of fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin-α5β1 and fibronectin were also over-expressed in cervical and oral SCC, where levels correlated with those of OSMR and TGM2. This combined tissue and in vitro study demonstrates for the first time that stimulation of over-expressed OSMR in cervical SCC cells activates TGM2/integrin-α5β1 interactions and induces pro-malignant changes. We conclude that an OSMR/TGM2/integrin-α5β1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting

A high-throughput computational framework for identifying significant copy number aberrations from array comparative genomic hybridisation data.

Reliable identification of copy number aberrations (CNA) from comparative genomic hybridization data would be improved by the availability of a generalised method for processing large datasets. To this end, we developed swatCGH, a data analysis framework and region detection heuristic for computational grids. swatCGH analyses sequentially displaced (sliding) windows of neighbouring probes and applies adaptive thresholds of varying stringency to identify the 10% of each chromosome that contains the most frequently occurring CNAs. We used the method to analyse a published dataset, comparing data preprocessed using four different DNA segmentation algorithms, and two methods for prioritising the detected CNAs. The consolidated list of the most commonly detected aberrations confirmed the value of swatCGH as a simplified high-throughput method for identifying biologically significant CNA regions of interest

Circulating microRNAs as biomarkers to assist the management of the malignant germ-cell-tumour subtype choriocarcinoma.

Germ-cell-tumours (GCTs) are heterogeneous and management is complex. The current conventional biomarkers, alpha-fetoprotein and human-chorionic-gonadotropin (HCG), have limited utility for diagnosis/follow-up as secretion is restricted to specific malignant-GCT subtypes and long half-life can make interpretation and clinical decision-making challenging. We sought to identify circulating microRNAs that reflected choriocarcinoma disease activity more accurately than HCG in a metastatic primary mediastinal nonseminomatous-GCT (PMNSGCT) case with elevated diagnostic serum HCG (>250,000U/L), consistent with pure choriocarcinoma. We undertook comprehensive microRNA profiling (n=754 microRNAs) using two 384-well TaqMan Low-Density-Array cards in 16 serum samples; 10 from PMNSGCT diagnosis/follow-up and six controls. Key findings underwent confirmatory qRT-PCR. We identified a serum panel of choriocarcinoma-specific ‘chromosome-19-microRNA-cluster’ (C19MC) microRNAs that were highly elevated at diagnosis but fell rapidly on treatment and normalised before the second full chemotherapy course. We also re-confirmed serum elevation of the previously identified malignant-GCT marker miR-371a-3p at diagnosis. These circulating microRNA markers reflected choriocarcinoma disease activity more accurately than serum HCG and real-time knowledge would have assisted clinical decision-making. With further study, these microRNA markers will facilitate future management of such patients and are likely to result in improved outcomes.Acknowledgment of research support: The authors acknowledge grant funding from the St. Baldrick’s Foundation [reference 358099], the Isaac Newton Trust [reference 15.40f], the Medical Research Council [reference MR/R001146/1] and Addenbrooke’s Charitable Trust [reference 23/17 B (iv)]. We are grateful for support from the Max Williamson Fund and from Christiane and Alan Hodson, in memory of their daughter Olivia. The funders were not involved in study design, data collection or interpretation, or decision to submit for publication

A high-throughput computational framework for identifying significant copy number aberrations from array comparative genomic hybridisation data.

Reliable identification of copy number aberrations (CNA) from comparative genomic hybridization data would be improved by the availability of a generalised method for processing large datasets. To this end, we developed swatCGH, a data analysis framework and region detection heuristic for computational grids. swatCGH analyses sequentially displaced (sliding) windows of neighbouring probes and applies adaptive thresholds of varying stringency to identify the 10% of each chromosome that contains the most frequently occurring CNAs. We used the method to analyse a published dataset, comparing data preprocessed using four different DNA segmentation algorithms, and two methods for prioritising the detected CNAs. The consolidated list of the most commonly detected aberrations confirmed the value of swatCGH as a simplified high-throughput method for identifying biologically significant CNA regions of interest.Peer Reviewe

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified. Furthermore, using our 'HPV16-specific Region Capture Hi-C' technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a 'cancer-causing gene' is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.This work was supported by Cancer Research UK (www.cancerresearchuk.org) Programme Award (A13080) to NC. ELAD was supported by a PhD studentship from The Pathological Society of GB & NI (www.pathsoc. org) awarded to IJG and NC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The still under-investigated role of cognitive deficits in PML diagnosis

Background: Despite cognitive deficits frequently represent the first clinical manifestations of Progressive Multifocal Leukoencephalopathy (PML) in Natalizumab-treated MS patients, the importance of cognitive deficits in PML diagnosis is still under-investigated. The aim of the current study is to investigate the cognitive deficits at PML diagnosis in a group of Italian patients with PML. Methods: Thirty-four PML patients were included in the study. The demographic and clinical data, the lesion load and localization, and the longitudinal clinical course was compared between patients with (n = 13) and without (n = 15) cognitive deficit upon PML suspicion (the remaining six patients were asymptomatic). Clinical presentation of cognitive symptoms was described in detail. Result: After symptoms detection, the time to diagnosis resulted to be shorter for patients presenting with cognitive than for patients with non cognitive onset (p = 0.03). Within patients with cognitive onset, six patients were presenting with language and/or reading difficulties (46.15%); five patients with memory difficulties (38.4%); three patients with apraxia (23.1%); two patients with disorientation (15.3%); two patients with neglect (15.3%); one patients with object agnosia (7.7%), one patient with perseveration (7.7%) and one patient with dementia (7.7%). Frontal lesions were less frequent (p = 0.03), whereas temporal lesions were slightly more frequent (p = 0.06) in patients with cognitive deficits. The longitudinal PML course seemed to be more severe in cognitive than in non cognitive patients (F = 2.73, p = 0.03), but differences disappeared (F = 1.24, p = 0.29) when balancing for the incidence of immune reconstitution syndrome and for other treatments for PML (steroids, plasma exchange (PLEX) and other therapies (Mefloquine, Mirtazapine, Maraviroc). Conclusion: Cognitive deficits at PML onset manifest with symptoms which are absolutely rare in MS. Their appearance in MS patients should strongly suggest PML. Clinicians should be sensitive to the importance of formal neuropsychological evaluation, with particular focus on executive function, which are not easily detected without a formal assessment

Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis.

BACKGROUND: Chronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase. METHODS: Liver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro. RESULTS: 13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to in vitro induction of cellular senescence, which had no effect. CONCLUSION: Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence