Natural Killer Cell Dysfunction in Premenopausal BRCA1 Mutation Carriers: A Potential Mechanism for Ovarian Carcinogenesis.

BACKGROUND: Tissue-specificity for fimbrial fallopian tube ovarian carcinogenesis remains largely unknown in BRCA1 mutation carriers. We aimed to assess the cell autonomous and cell-nonautonomous implications of a germline BRCA1 mutation in the context of cancer immunosurveillance of CD3- CD56+ natural killer (NK) cells. METHODS: Premenopausal BRCA1 mutation carriers versus age-matched non-carriers were compared. Daily urinary 5ß-pregnanediol levels were used to determine progesterone metabolomics across an ovarian cycle. Using peripherally acquired NK cells the cell-mediated cytotoxicity of tumor targets (OVCAR-3, K-562) was determined using live cellular impedance (xCELLigence®) and multicolor flow cytometry. Hypoxia-inducible factor 1-alpha (HIF-1α) immunohistochemistry of cancer-free fallopian tube specimens allowed a comparison of proximal versus distal portions. Utilizing these findings the role of environmental factors relevant to the fimbrial fallopian tube (progesterone, hypoxia) on NK cell functional activity were studied in an ovarian phase-specific manner. RESULTS: BRCA1 mutation carriers demonstrate a differential progesterone metabolome with a phase-specific reduction of peripheral NK cell functional activity. Progesterone exposure further impairs NK cell-mediated cytotoxicity in a dose-dependent manner, which is reversed with the addition of mifepristone (1.25 µM). The fimbrial fallopian tube demonstrated significantly higher HIF-1α staining, particularly in BRCA1 mutation carriers, reflecting a site-specific 'hypoxic niche'. Exposure to hypoxic conditions (1% O2) can further impair tumor cytotoxicity in high-risk carriers. CONCLUSIONS: Phase-specific differential NK cell activity in BRCA1 mutation carriers, either systemically or locally, may favor site-specific pre-invasive carcinogenesis. These cumulative effects across a reproductive lifecycle in high-risk carriers can have a detrimental effect further supporting epidemiological evidence for ovulation inhibition.

MICA: a multi-omics method to predict gene regulatory networks in early human embryos.

Recent advances in single-cell omics have transformed characterisation of cell types in challenging-to-study biological contexts. In contexts with limited single-cell samples, such as the early human embryo inference of transcription factor-gene regulatory network (GRN) interactions is especially difficult. Here, we assessed application of different linear or non-linear GRN predictions to single-cell simulated and human embryo transcriptome datasets. We also compared how expression normalisation impacts on GRN predictions, finding that transcripts per million reads outperformed alternative methods. GRN inferences were more reproducible using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared with alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2γ in early human embryos. Overall, our comparative analysis of GRN prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation.

Antiprogestins reduce epigenetic field cancerization in breast tissue of young healthy women.

BACKGROUND: Breast cancer is a leading cause of death in premenopausal women. Progesterone drives expansion of luminal progenitor cells, leading to the development of poor-prognostic breast cancers. However, it is not known if antagonising progesterone can prevent breast cancers in humans. We suggest that targeting progesterone signalling could be a means of reducing features which are known to promote breast cancer formation. METHODS: In healthy premenopausal women with and without a BRCA mutation we studied (i) estrogen and progesterone levels in saliva over an entire menstrual cycle (n = 20); (ii) cancer-free normal breast-tissue from a control population who had no family or personal history of breast cancer and equivalently from BRCA1/2 mutation carriers (n = 28); triple negative breast cancer (TNBC) biopsies and healthy breast tissue taken from sites surrounding the TNBC in the same individuals (n = 14); and biopsies of ER+ve/PR+ve stage T1-T2 cancers and healthy breast tissue taken from sites surrounding the cancer in the same individuals (n = 31); and (iii) DNA methylation and DNA mutations in normal breast tissue (before and after treatment) from clinical trials that assessed the potential preventative effects of vitamins and antiprogestins (mifepristone and ulipristal acetate; n = 44). RESULTS: Daily levels of progesterone were higher throughout the menstrual cycle of BRCA1/2 mutation carriers, raising the prospect of targeting progesterone signalling as a means of cancer risk reduction in this population. Furthermore, breast field cancerization DNA methylation signatures reflective of (i) the mitotic age of normal breast epithelium and (ii) the proportion of luminal progenitor cells were increased in breast cancers, indicating that luminal progenitor cells with elevated replicative age are more prone to malignant transformation. The progesterone receptor antagonist mifepristone reduced both the mitotic age and the proportion of luminal progenitor cells in normal breast tissue of all control women and in 64% of BRCA1/2 mutation carriers. These findings were validated by an alternate progesterone receptor antagonist, ulipristal acetate, which yielded similar results. Importantly, mifepristone reduced both the TP53 mutation frequency as well as the number of TP53 mutations in mitotic-age-responders. CONCLUSIONS: These data support the potential usage of antiprogestins for primary prevention of poor-prognostic breast cancers. TRIAL REGISTRATION: Clinical trial 1 Mifepristone treatment prior to insertion of a levonorgestrel releasing intrauterine system for improved bleeding control - a randomized controlled trial, clinicaltrialsregister.eu, 2009-009014-40 ; registered on 20 July 2009. Clinical trial 2 The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations, clinicaltrials.gov, NCT01898312 ; registered on 07 May 2013. Clinical trial 3 A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk, clinicaltrialsregister.eu, 2015-001587-19 ; registered on 15 July 2015.

Susceptibility to hormone-mediated cancer is reflected by different tick rates of the epithelial and general epigenetic clock.

BACKGROUND: A variety of epigenetic clocks utilizing DNA methylation changes have been developed; these clocks are either tissue-independent or designed to predict chronological age based on blood or saliva samples. Whether discordant tick rates between tissue-specific and general epigenetic clocks play a role in health and disease has not yet been explored. RESULTS: Here we analyze 1941 cervical cytology samples, which contain a mixture of hormone-sensitive cervical epithelial cells and immune cells, and develop the WID general clock (Women's IDentification of risk), an epigenetic clock that is shared by epithelial and immune cells and optimized for cervical samples. We then develop the WID epithelial clock and WID immune clock, which define epithelial- and immune-specific clocks, respectively. We find that the WID-relative-epithelial-age (WID-REA), defined as the difference between the epithelial and general clocks, is significantly reduced in cervical samples from pre-menopausal women with breast cancer (OR 2.7, 95% CI 1.28-5.72). We find the same effect in normal breast tissue samples from pre-menopausal women at high risk of breast cancer and show that potential risk reducing anti-progesterone drugs can reverse this. In post-menopausal women, this directionality is reversed. Hormone replacement therapy consistently leads to a significantly lower WID-REA in cancer-free women, but not in post-menopausal women with breast or ovarian cancer. CONCLUSIONS: Our findings imply that there are multiple epigenetic clocks, many of which are tissue-specific, and that the differential tick rate between these clocks may be an informative surrogate measure of disease risk.

Inference of tissue relative proportions of the breast epithelial cell types luminal progenitor, basal, and luminal mature.

Single-cell analysis has revolutionised genomic science in recent years. However, due to cost and other practical considerations, single-cell analyses are impossible for studies based on medium or large patient cohorts. For example, a single-cell analysis usually costs thousands of euros for one tissue sample from one volunteer, meaning that typical studies using single-cell analyses are based on very few individuals. While single-cell genomic data can be used to examine the phenotype of individual cells, cell-type deconvolution methods are required to track the quantities of these cells in bulk-tissue genomic data. Hormone receptor negative breast cancers are highly aggressive, and are thought to originate from a subtype of epithelial cells called the luminal progenitor. In this paper, we show how to quantify the number of luminal progenitor cells as well as other epithelial subtypes in breast tissue samples using DNA and RNA based measurements. We find elevated levels of cells which resemble these hormone receptor negative luminal progenitor cells in breast tumour biopsies of hormone receptor negative cancers, as well as in healthy breast tissue samples from BRCA1 (FANCS) mutation carriers. We also find that breast tumours from carriers of heterozygous mutations in non-BRCA Fanconi Anaemia pathway genes are much more likely to be hormone receptor negative. These findings have implications for understanding hormone receptor negative breast cancers, and for breast cancer screening in carriers of heterozygous mutations of Fanconi Anaemia pathway genes.

Comodularity and detection of co-communities.

This paper introduces the notion of comodularity, to cocluster observations of bipartite networks into co-communities. The task of coclustering is to group together nodes of one type with nodes of another type, according to the interactions that are the most similar. The measure of comodularity is introduced to assess the strength of co-communities, as well as to arrange the representation of nodes and clusters for visualization, and to define an objective function for optimization. We demonstrate the usefulness of our proposed methodology on simulated data, and with examples from genomics and consumer-product reviews.

Single-cell Co-expression Subnetwork Analysis.

Single-cell transcriptomic data have rapidly become very popular in genomic science. Genomic science also has a long history of using network models to understand the way in which genes work together to carry out specific biological functions. However, working with single-cell data presents major challenges, such as zero inflation and technical noise. These challenges require methods to be specifically adapted to the context of single-cell data. Recently, much effort has been made to develop the theory behind statistical network models. This has lead to many new models being proposed, and has provided a thorough understanding of the properties of existing models. However, a large amount of this work assumes binary-valued relationships between network nodes, whereas genomic network analysis is traditionally based on continuous-valued correlations between genes. In this paper, we assess several established methods for genomic network analysis, we compare ways that these methods can be adapted to the single-cell context, and we use mixture-models to infer binary-valued relationships based on gene-gene correlations. Based on these binary relationships, we find that excellent results can be achieved by using subnetwork analysis methodology popular amongst network statisticians. This methodology thereby allows detection of functional subnetwork modules within these single-cell genomic networks.

Epigenetic reprogramming of fallopian tube fimbriae in BRCA mutation carriers defines early ovarian cancer evolution.

The exact timing and contribution of epigenetic reprogramming to carcinogenesis are unclear. Women harbouring BRCA1/2 mutations demonstrate a 30-40-fold increased risk of high-grade serous extra-uterine Müllerian cancers (HGSEMC), otherwise referred to as 'ovarian carcinomas', which frequently develop from fimbrial cells but not from the proximal portion of the fallopian tube. Here we compare the DNA methylome of the fimbrial and proximal ends of the fallopian tube in BRCA1/2 mutation carriers and non-carriers. We show that the number of CpGs displaying significant differences in methylation levels between fimbrial and proximal fallopian tube segments are threefold higher in BRCA mutation carriers than in controls, correlating with overexpression of activation-induced deaminase in their fimbrial epithelium. The differentially methylated CpGs accurately discriminate HGSEMCs from non-serous subtypes. Epigenetic reprogramming is an early pre-malignant event integral to BRCA1/2 mutation-driven carcinogenesis. Our findings may provide a basis for cancer-preventative strategies.

Intra-Gene DNA Methylation Variability Is a Clinically Independent Prognostic Marker in Women's Cancers.

We introduce a novel per-gene measure of intra-gene DNA methylation variability (IGV) based on the Illumina Infinium HumanMethylation450 platform, which is prognostic independently of well-known predictors of clinical outcome. Using IGV, we derive a robust gene-panel prognostic signature for ovarian cancer (OC, n = 221), which validates in two independent data sets from Mayo Clinic (n = 198) and TCGA (n = 358), with significance of p = 0.004 in both sets. The OC prognostic signature gene-panel is comprised of four gene groups, which represent distinct biological processes. We show the IGV measurements of these gene groups are most likely a reflection of a mixture of intra-tumour heterogeneity and transcription factor (TF) binding/activity. IGV can be used to predict clinical outcome in patients individually, providing a surrogate read-out of hard-to-measure disease processes.

Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest.

Glioblastoma (GBM) is an aggressive brain tumor whose growth is driven by stemcell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stemcells (GSCs) and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Herewe find only a subset ofGSCcultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy forGBM.

Cognitive flexibility and long-term depression (LTD) are impaired following ß-catenin stabilization in vivo.

The cadherin/ß-catenin adhesion complex is a key mediator of the bidirectional changes in synapse strength which are believed to underlie complex learning and memory. In the present study, we demonstrate that stabilization of ß-catenin in the hippocampus of adult mice results in significant impairments in cognitive flexibility and spatial reversal learning, including impaired extinction during the reversal phase of the Morris water maze and deficits in a delayed nonmatch to place T-maze task. In accordance with these deficits, ß-catenin stabilization was found to abolish long-term depression by stabilizing cadherin at the synaptic membrane and impairing AMPA receptor endocytosis, while leaving basal synaptic transmission and long-term potentiation unaffected. These results demonstrate that the ß-catenin/cadherin adhesion complex plays an important role in learning and memory and that aberrant increases in synaptic adhesion can have deleterious effects on cognitive function.

A DNA methylation network interaction measure, and detection of network oncomarkers.

Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. DNA methylation patterns offer a route to develop prognostic measures based directly on DNA measurements, rather than less-stable RNA measurements. A novel DNA methylation-based measure of the co-ordinated interactive behaviour of genes is developed, in a network context. It is shown that this measure reflects well the co-regulatory behaviour linked to gene expression (at the mRNA level) over the same network interactions. This measure, defined for pairs of genes in a single patient/sample, associates with overall survival outcome independent of known prognostic clinical features, in several independent data sets relating to different cancer types. In total, more than half a billion CpGs in over 1600 samples, taken from nine different cancer entities, are analysed. It is found that groups of gene-pair interactions which associate significantly with survival identify statistically significant subnetwork modules. Many of these subnetwork modules are shown to be biologically relevant by strong correlation with pre-defined gene sets, such as immune function, wound healing, mitochondrial function and MAP-kinase signalling. In particular, the wound healing module corresponds to an increase in co-ordinated interactive behaviour between genes for worse prognosis, and the immune module corresponds to a decrease in co-ordinated interactive behaviour between genes for worse prognosis. This measure has great potential for defining DNA-based cancer biomarkers. Such biomarkers could naturally be developed further, by drawing on the rapidly expanding knowledge base of network science.

Corruption of the intra-gene DNA methylation architecture is a hallmark of cancer.

Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test [Formula: see text] in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers.

The intersections of NMDAR-dependent synaptic plasticity and cell survival.

The discovery of a requirement for N-methyl d-aspartate receptor (NMDAR) activation in long-term potentiation (LTP) set off an explosion of interest in the mechanisms of NMDAR-dependent synaptic plasticity. Meanwhile other research has advanced our understanding of how NMDAR activation regulates neuronal death and survival. Surprisingly, there have been few attempts to correlate these important areas of research. Here we review current knowledge of the various mechanisms of NMDAR-dependent synaptic plasticity that are shared with neuronal survival and death, while drawing comparisons with the proneurotrophin/neurotrophin receptor and intracellular signaling systems. Our conclusion is that NMDAR-dependent LTP and long-term depression (LTD) share many common mechanisms with cell survival and cell death, respectively. The intersections of plasticity and cell survival may represent novel avenues for neuroprotection. This article is part of the Special Issue entitled 'Glutamate Receptor-Dependent Synaptic Plasticity'.

Illuminating synapse-specific homeostatic plasticity.

Homeostatic plasticity can globally scale the strength of all synapses on a neuron, but whether a similar bidirectional homeostatic scaling can also operate independently at individual synapses was unknown until now. Here, Man and colleagues demonstrate that single synapses show an input-specific homeostatic downregulation of synaptic efficacy in response to increased activity.

Slice orientation and muscarinic acetylcholine receptor activation determine the involvement of N-methyl D-aspartate receptor subunit GluN2B in hippocampal area CA1 long-term depression.

BACKGROUND: The contribution of different GluN2 subunits of the N-methyl D-aspartate (NMDA) receptor to the induction of bidirectional hippocampal synaptic plasticity is a controversial topic. As both supporting and refuting evidence for the hypothesis of subunit specialization in opposing directions of plasticity has accumulated since it was first proposed a few years ago, we hypothesize that differences in experimental conditions may have in part contributed to some of the inconsistent results from these studies. Here we investigate the controversial hypothesis that long-term depression (LTD) is preferentially induced by GluN2B-containing NMDA receptors in area CA1 of hippocampal slices. RESULTS: We find that brain slices from 2-3 week old rats prepared in the sagittal orientation have GluN2B-independent LTD whereas slices prepared in the coronal orientation have GluN2B-dependent LTD. There was no difference between the orientations in the fraction of the NMDAR EPSC sensitive to a GluN2B-selective antagonist, leading us to believe that the intracellular signaling properties of the NMDARs were different in the two preparations. Coronal slices had greater association of LTD-related intracellular signaling protein RasGRF1 with GluN2B relative to sagittal slices. Antagonism of muscarinic acetylcholine receptors (mAChRs) in the sagittal slices returned LTD to a GluN2B-dependent form and increased the association of GluN2B with RasGRF1. CONCLUSIONS: These results suggest a novel form of NMDAR modulation by mAChRs and clarify some disagreement in the literature.

Postsynaptic TrkC and presynaptic PTPσ function as a bidirectional excitatory synaptic organizing complex.

Neurotrophin receptor tyrosine kinases (Trks) have well-defined trophic roles in nervous system development through kinase activation by neurotrophins. Yet Trks have typical cell-adhesion domains and express noncatalytic isoforms, suggesting additional functions. Here we discovered noncatalytic TrkC in an unbiased hippocampal neuron-fibroblast coculture screen for proteins that trigger differentiation of neurotransmitter release sites in axons. All TrkC isoforms, but not TrkA or TrkB, function directly in excitatory glutamatergic synaptic adhesion by neurotrophin-independent high-affinity trans binding to axonal protein tyrosine phosphatase receptor PTPσ. PTPσ triggers and TrkC mediates clustering of postsynaptic molecules in dendrites, indicating bidirectional synaptic organizing functions. Effects of a TrkC-neutralizing antibody that blocks TrkC-PTPσ interaction and TrkC knockdown in culture and in vivo reveal essential roles of TrkC-PTPσ in glutamatergic synapse formation. Thus, postsynaptic TrkC trans interaction with presynaptic PTPσ generates bidirectional adhesion and recruitment essential for excitatory synapse development and positions these signaling molecules at the center of synaptic pathways.

Differential roles of NR2A and NR2B-containing NMDA receptors in LTP and LTD in the CA1 region of two-week old rat hippocampus.

The role of NMDA receptors in the induction of long-term potentiation (LTP) and long-term depression (LTD) is well established but which particular NR2 subunits are involved in these plasticity processes is still a matter of controversy. We have studied the effects of subtype selective NMDA receptor antagonists on LTP induced by high frequency stimulation (100 Hz for 1s) and LTD induced by low frequency stimulation (1 Hz for 15 min) in the CA1 region of hippocampal slices from 14 day old Wistar rats. Against recombinant receptors in HEK293 cells NVP-AAM077 (NVP) was approximately 14-fold selective for NR2A vs NR2B receptors, whilst Ro 25-6981 (Ro) was highly selective for NR2B receptors. On NMDA receptor-mediated EPSCs from Schaffer collaterals in CA1 neurones, NVP and Ro both reduced the amplitude but differentially affected the time constant of decay. The data are compatible with the selective effect of NVP (0.1 microM) and Ro (4 microM) on native NR2A and NBR2B receptors, respectively. NVP reduced both LTP and LTD whereas Ro reduced only LTP. Thus, LTP was reduced by 63% at 0.1 microM NVP and almost completely at 0.4 microM whereas 5 microM Ro reduced LTP by 45%. These data are consistent with a role for both NR2A and NR2B in the induction of LTP, under our experimental conditions. In comparison, LTD was unaffected by Ro (5 microM) even in the presence of a glutamate uptake inhibitor threo-beta-benzylaspartic acid (TBOA) to increase the concentration of glutamate at NR2B containing receptors. NVP (0.2-0.4 microM), however, produced a concentration dependent inhibition of LTD which was complete at 0.4 microM. The lack of effect of 0.1 microM NVP on LTD contrasts with its marked effect on LTP and raises the possibility that different NVP-sensitive NR2 subunit-containing NMDA receptors are required for LTP and LTD in this preparation.