A novel Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis.

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knockdown of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases ATP demand and glycolysis in breast cancer cells, likely by upregulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and the movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

Is it possible to measure good science?

Metrics play a vital part in the valuation and funding of research for scientists worldwide. We review the challenges that metrics pose in providing a fair and equitable system for research funding. We highlight the attempts with declarations, including the San Francisco Declaration on Research Assessment (SF-DORA), to improve the research environment and specific impacts that metric choice can have on the evaluation and progression of Early Career Lecturers (ECLs). While there is much evidence that metrics will never be entirely satisfactory, we conclude there are opportunities that would benefit ECLs and reason for optimism for researchers.

Detection of subtype-specific breast cancer surface protein biomarkers via a novel transcriptomics approach.

BACKGROUND: Cell-surface proteins have been widely used as diagnostic and prognostic markers in cancer research and as targets for the development of anticancer agents. So far, very few attempts have been made to characterize the surfaceome of patients with breast cancer, particularly in relation with the current molecular breast cancer (BRCA) classification. In this view, we developed a new computational method to infer cell-surface protein activities from transcriptomics data, termed 'SURFACER'. METHODS: Gene expression data from GTEx were used to build a normal breast network model as input to infer differential cell-surface proteins activity in BRCA tissue samples retrieved from TCGA versus normal samples. Data were stratified according to the PAM50 transcriptional subtypes (Luminal A, Luminal B, HER2 and Basal), while unsupervised clustering techniques were applied to define BRCA subtypes according to cell-surface proteins activity. RESULTS: Our approach led to the identification of 213 PAM50 subtypes-specific deregulated surface genes and the definition of five BRCA subtypes, whose prognostic value was assessed by survival analysis, identifying a cell-surface activity configuration at increased risk. The value of the SURFACER method in BRCA genotyping was tested by evaluating the performance of 11 different machine learning classification algorithms. CONCLUSIONS: BRCA patients can be stratified into five surface activity-specific groups having the potential to identify subtype-specific actionable targets to design tailored targeted therapies or for diagnostic purposes. SURFACER-defined subtypes show also a prognostic value, identifying surface-activity profiles at higher risk.

How Individuals Who Self-Harm Manage Their Own Risk-'I Cope Because I Self-Harm, and I Can Cope with my Self-Harm'.

Self-harm is a complex and idiosyncratic behaviour. This article focuses on how those who self-harm manage their own risk. Utilising opportunity sampling, ten members of a self-harm support group were interviewed about how they risk manage their self-harm and the data analysed using interpretative phenomenological analysis. The analysis showed that all participants were actively involved in risk management of their self-harm. Through a process of managing consequences, exercising control in the process, and an awareness of the social context. It is posited that people who self-harm should be viewed as actively engaging with the risks of self-harm whilst it is a coping mechanism, as opposed to passive or ignoring. This understanding can be integrated into current risk management plans within services and invites a more dynamic conversation of self-harm between services users and services. Effective risk management involves good relationships between individuals who self-harm and clinicians, services which promote positive risk taking as opposed to defensive practice, and true collaboration between services and service users.

Web tools to fight pandemics: the COVID-19 experience.

The current outbreak of COVID-19 has generated an unprecedented scientific response worldwide, with the generation of vast amounts of publicly available epidemiological, biological and clinical data. Bioinformatics scientists have quickly produced online methods to provide non-computational users with the opportunity of analyzing such data. In this review, we report the results of this effort, by cataloguing the currently most popular web tools for COVID-19 research and analysis. Our focus was driven on tools drawing data from the fields of epidemiology, genomics, interactomics and pharmacology, in order to provide a meaningful depiction of the current state of the art of COVID-19 online resources.

Intestinal region-specific Wnt signalling profiles reveal interrelation between cell identity and oncogenic pathway activity in cancer development.

BACKGROUND: Cancer results from the accumulation of mutations leading to the acquisition of cancer promoting characteristics such as increased proliferation and resistance to cell death. In colorectal cancer, an early mutation leading to such features usually occurs in the APC or CTNNB1 genes, thereby activating Wnt signalling. However, substantial phenotypic differences between cancers originating within the same organ, such as molecular subtypes, are not fully reflected by differences in mutations. Indeed, the phenotype seems to result from a complex interplay between the cell-intrinsic features and the acquired mutations, which is difficult to disentangle when established tumours are studied. METHODS: We use a 3D in vitro organoid model to study the early phase of colorectal cancer development. From three different murine intestinal locations we grow organoids. These are transformed to resemble adenomas after Wnt activation through lentiviral transduction with a stable form of ß-Catenin. The gene expression before and after Wnt activation is compared within each intestinal origin and across the three locations using RNA sequencing. To validate and generalize our findings, we use gene expression data from patients. RESULTS: In reaction to Wnt activation we observe downregulation of location specific genes and differentiation markers. A similar effect is seen in patient data, where genes with significant differential expression between the normal left and right colon are downregulated in the cancer samples. Furthermore, the signature of Wnt target genes differs between the three intestinal locations in the organoids. The location specific Wnt signatures are dominated by genes which have been lowly expressed in the tissue of origin, and are the targets of transcription factors that are activated following enhanced Wnt signalling. CONCLUSION: We observed that the region-specific cell identity has a substantial effect on the reaction to Wnt activation in a simple intestinal adenoma model. These findings provide a way forward in resolving the distinct biology between left- and right-sided human colon cancers with potential clinical relevance.

Data generation and network reconstruction strategies for single cell transcriptomic profiles of CRISPR-mediated gene perturbations.

Recent advances in single-cell RNA-sequencing (scRNA-seq) in combination with CRISPR/Cas9 technologies have enabled the development of methods for large-scale perturbation studies with transcriptional readouts. These methods are highly scalable and have the potential to provide a wealth of information on the biological networks that underlie cellular response. Here we discuss how to overcome several key challenges to generate and analyse data for the confident reconstruction of models of the underlying cellular network. Some challenges are generic, and apply to analysing any single-cell transcriptomic data, while others are specific to combined single-cell CRISPR/Cas9 data, in particular barcode swapping, knockdown efficiency, multiplicity of infection and potential confounding factors. We also provide a curated collection of published data sets to aid the development of analysis strategies. Finally, we discuss several network reconstruction approaches, including co-expression networks and Bayesian networks, as well as their limitations, and highlight the potential of Nested Effects Models for network reconstruction from scRNA-seq data. This article is part of a Special Issue entitled: Transcriptional Profiles and Regulatory Gene Networks edited by Dr. Dr. Federico Manuel Giorgi and Dr. Shaun Mahony.

Novelty in science should not come at the cost of reproducibility.

The pressures of a scientific career can end up incentivising an all-or-nothing approach to cross the finish line first. While competition can be healthy and drives innovation, the current system fails to encourage scientists to work reproducibility. This sometimes leaves those individuals who come second to correct mistakes in published research without being rewarded. Instead, we need a culture that rewards reproducibility and holds it as important as the novelty of the result. Here, I draw on my own journey in the oestrogen receptor research field to highlight this and suggest ways for the 'first past the post' culture to be challenged.

Correction to: VULCAN integrates ChIP-seq with patient-derived co-expression networks to identify GRHL2 as a key co-regulator of ERa at enhancers in breast cancer.

Following publication of the original article [1], the authors reported that Figs. 4 and 5 had mistakenly been transposed. Please find the correct Figs. 4 and 5 below. The original article [1] has been corrected.

VULCAN integrates ChIP-seq with patient-derived co-expression networks to identify GRHL2 as a key co-regulator of ERa at enhancers in breast cancer.

BACKGROUND: VirtUaL ChIP-seq Analysis through Networks (VULCAN) infers regulatory interactions of transcription factors by overlaying networks generated from publicly available tumor expression data onto ChIP-seq data. We apply our method to dissect the regulation of estrogen receptor-alpha activation in breast cancer to identify potential co-regulators of the estrogen receptor's transcriptional response. RESULTS: VULCAN analysis of estrogen receptor activation in breast cancer highlights the key components of the estrogen receptor complex alongside a novel interaction with GRHL2. We demonstrate that GRHL2 is recruited to a subset of estrogen receptor binding sites and regulates transcriptional output, as evidenced by changes in estrogen receptor-associated eRNA expression and stronger estrogen receptor binding at active enhancers after GRHL2 knockdown. CONCLUSIONS: Our findings provide new insight into the role of GRHL2 in regulating eRNA transcription as part of estrogen receptor signaling. These results demonstrate VULCAN, available from Bioconductor, as a powerful predictive tool.

Genome-wide Estrogen Receptor-α activation is sustained, not cyclical.

Estrogen Receptor-alpha (ER) drives 75% of breast cancers. Stimulation of the ER by estra-2-diol forms a transcriptionally-active chromatin-bound complex. Previous studies reported that ER binding follows a cyclical pattern. However, most studies have been limited to individual ER target genes and without replicates. Thus, the robustness and generality of ER cycling are not well understood. We present a comprehensive genome-wide analysis of the ER after activation, based on 6 replicates at 10 time-points, using our method for precise quantification of binding, Parallel-Factor ChIP-seq. In contrast to previous studies, we identified a sustained increase in affinity, alongside a class of estra-2-diol independent binding sites. Our results are corroborated by quantitative re-analysis of multiple independent studies. Our new model reconciles the conflicting studies into the ER at the TFF1 promoter and provides a detailed understanding in the context of the ER's role as both the driver and therapeutic target of breast cancer.

KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice.

Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.

A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes.

Understanding the dynamics of endogenous protein-protein interactions in complex networks is pivotal in deciphering disease mechanisms. To enable the in-depth analysis of protein interactions in chromatin-associated protein complexes, we have previously developed a method termed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins). Here, we present a quantitative multiplexed method (qPLEX-RIME), which integrates RIME with isobaric labelling and tribrid mass spectrometry for the study of protein interactome dynamics in a quantitative fashion with increased sensitivity. Using the qPLEX-RIME method, we delineate the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydroxytamoxifen. Furthermore, we identify endogenous ERα-associated proteins in human Patient-Derived Xenograft tumours and in primary human breast cancer clinical tissue. Our results demonstrate that the combination of RIME with isobaric labelling offers a powerful tool for the in-depth and quantitative characterisation of protein interactome dynamics, which is applicable to clinical samples.

Parallel factor ChIP provides essential internal control for quantitative differential ChIP-seq.

A key challenge in quantitative ChIP combined with high-throughput sequencing (ChIP-seq) is the normalization of data in the presence of genome-wide changes in occupancy. Analysis-based normalization methods were developed for transcriptomic data and these are dependent on the underlying assumption that total transcription does not change between conditions. For genome-wide changes in transcription factor (TF) binding, these assumptions do not hold true. The challenges in normalization are confounded by experimental variability during sample preparation, processing and recovery. We present a novel normalization strategy utilizing an internal standard of unchanged peaks for reference. Our method can be readily applied to monitor genome-wide changes by ChIP-seq that are otherwise lost or misrepresented through analytical normalization. We compare our approach to normalization by total read depth and two alternative methods that utilize external experimental controls to study TF binding. We successfully resolve the key challenges in quantitative ChIP-seq analysis and demonstrate its application by monitoring the loss of Estrogen Receptor-alpha (ER) binding upon fulvestrant treatment, ER binding in response to estrodiol, ER mediated change in H4K12 acetylation and profiling ER binding in patient-derived xenographs. This is supported by an adaptable pipeline to normalize and quantify differential TF binding genome-wide and generate metrics for differential binding at individual sites.

XL-MS: Protein cross-linking coupled with mass spectrometry.

With the continuing trend to study larger and more complex systems, the application of protein cross-linking coupled with mass spectrometry (XL-MS) provides a varied toolkit perfectly suited to achieve these goals. By freezing the transient interactions through the formation of covalent bonds, XL-MS provides a vital insight into both the structure and organization of proteins in a wide variety of conditions. This review covers some of the established methods that underpin the field alongside the more recent developments that hold promise to further realize its potential in new directions.

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

Hekate: software suite for the mass spectrometric analysis and three-dimensional visualization of cross-linked protein samples.

Chemical cross-linking of proteins combined with mass spectrometry provides an attractive and novel method for the analysis of native protein structures and protein complexes. Analysis of the data however is complex. Only a small number of cross-linked peptides are produced during sample preparation and must be identified against a background of more abundant native peptides. To facilitate the search and identification of cross-linked peptides, we have developed a novel software suite, named Hekate. Hekate is a suite of tools that address the challenges involved in analyzing protein cross-linking experiments when combined with mass spectrometry. The software is an integrated pipeline for the automation of the data analysis workflow and provides a novel scoring system based on principles of linear peptide analysis. In addition, it provides a tool for the visualization of identified cross-links using three-dimensional models, which is particularly useful when combining chemical cross-linking with other structural techniques. Hekate was validated by the comparative analysis of cytochrome c (bovine heart) against previously reported data. Further validation was carried out on known structural elements of DNA polymerase III, the catalytic α-subunit of the Escherichia coli DNA replisome along with new insight into the previously uncharacterized C-terminal domain of the protein.

Bicaudal-D uses a parallel, homodimeric coiled coil with heterotypic registry to coordinate recruitment of cargos to dynein.

Cytoplasmic dynein is the major minus end-directed microtubule motor in eukaryotes. However, there is little structural insight into how different cargos are recognized and linked to the motor complex. Here we describe the 2.2 Å resolution crystal structure of a cargo-binding region of the dynein adaptor Bicaudal-D (BicD), which reveals a parallel coiled-coil homodimer. We identify a shared binding site for two cargo-associated proteins-Rab6 and the RNA-binding protein Egalitarian (Egl)-within a region of the BicD structure with classical, homotypic core packing. Structure-based mutagenesis in Drosophila provides evidence that occupancy of this site drives association of BicD with dynein, thereby coupling motor recruitment to cargo availability. The structure also contains a region in which, remarkably, the same residues in the polypeptide sequence have different heptad registry in each chain. In vitro and in vivo analysis of a classical Drosophila dominant mutation reveals that this heterotypic region regulates the recruitment of dynein to BicD. Our results support a model in which the heterotypic segment is part of a molecular switch that promotes release of BicD autoinhibition following cargo binding to the neighboring, homotypic coiled-coil region. Overall, our data reveal a pivotal role of a highly asymmetric coiled-coil domain in coordinating the assembly of cargo-motor complexes.

Architecture of the Pol III-clamp-exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair.

DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp ß and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III-clamp-exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.