On-chip Raman spectroscopy of live single cells for the staging of oesophageal adenocarcinoma progression.

The absence of early diagnosis contributes to oesophageal cancer being the sixth most common cause of global cancer-associated deaths, with a 5-year survival rate of < 20%. Barrett's oesophagus is the main pre-cancerous condition to adenocarcinoma development, characterised by the morphological transition of oesophageal squamous epithelium to metaplastic columnar epithelium. Early tracking and treatment of oesophageal adenocarcinoma could dramatically improve with diagnosis and monitoring of patients with Barrett's Oesophagus. Current diagnostic methods involve invasive techniques such as endoscopies and, with only a few identified biomarkers of disease progression, the detection of oesophageal adenocarcinoma is costly and challenging. In this work, single-cell Raman spectroscopy was combined with microfluidic techniques to characterise the development of oesophageal adenocarcinoma through the progression of healthy epithelial, Barrett's oesophagus and oesophageal adenocarcinoma cell lines. Principal component analysis and linear discriminant analysis were used to classify the different stages of cancer progression. with the ability to differentiate between healthy and cancerous cells with an accuracy of 97%. Whilst the approach could also separate the dysplastic stages from healthy or cancer with high accuracy-the intra-class separation was approximately 68%. Overall, these results highlight the potential for rapid and reliable diagnostic/prognostic screening of Barrett's Oesophagus patients.

E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer.

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ∼5% of all human cancers worldwide, including most cervical cancer cases and a growing number of ano-genital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited proliferation in vitro , whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we show that the LASP1 SH3 domain is essential for LASP1-mediated proliferation in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulated LASP1 mRNA levels by binding to its 3'UTR. Finally, we demonstrated that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival role in cervical cancer, thus identifying a potential therapeutic target in these cancers.

The emerging role of receptor tyrosine kinase phase separation in cancer.

Receptor tyrosine kinase (RTK)-mediated signal transduction is fundamental to cell function and drives important cellular outcomes which, when dysregulated, can lead to malignant tumour growth and metastasis. The initiation of signals from plasma membrane-bound RTKs is subjected to multiple regulatory mechanisms that control downstream effector protein recruitment and function. The high propensity of RTKs to condense via liquid-liquid phase separation (LLPS) into membraneless organelles with downstream effector proteins provides a further fundamental mechanism for signal regulation. Herein we highlight how this phenomenon contributes to cancer signalling and consider the potential impact of LLPS on outcomes for cancer patients.

The combined action of the intracellular regions regulates FGFR2 kinase activity.

Receptor tyrosine kinases (RTKs) are typically activated through a precise sequence of intracellular phosphorylation events starting with a tyrosine residue on the activation loop (A-loop) of the kinase domain (KD). From this point the mono-phosphorylated enzyme is active, but subject to stringent regulatory mechanisms which can vary dramatically across the different RTKs. In the absence of extracellular stimulation, fibroblast growth factor receptor 2 (FGFR2) exists in the mono-phosphorylated state in which catalytic activity is regulated to allow rapid response upon ligand binding, whilst restricting ligand-independent activation. Failure of this regulation is responsible for pathologic outcomes including cancer. Here we reveal the molecular mechanistic detail of KD control based on combinatorial interactions of the juxtamembrane (JM) and the C-terminal tail (CT) regions of the receptor. JM stabilizes the asymmetric dimeric KD required for substrate phosphorylation, whilst CT binding opposes dimerization, and down-regulates activity. Direct binding between JM and CT delays the recruitment of downstream effector proteins adding a further control step as the receptor proceeds to full activation. Our findings underscore the diversity in mechanisms of RTK oligomerisation and activation.

PLBD: protein-ligand binding database of thermodynamic and kinetic intrinsic parameters.

We introduce a protein-ligand binding database (PLBD) that presents thermodynamic and kinetic data of reversible protein interactions with small molecule compounds. The manually curated binding data are linked to protein-ligand crystal structures, enabling structure-thermodynamics correlations to be determined. The database contains over 5500 binding datasets of 556 sulfonamide compound interactions with the 12 catalytically active human carbonic anhydrase isozymes defined by fluorescent thermal shift assay, isothermal titration calorimetry, inhibition of enzymatic activity and surface plasmon resonance. In the PLBD, the intrinsic thermodynamic parameters of interactions are provided, which account for the binding-linked protonation reactions. In addition to the protein-ligand binding affinities, the database provides calorimetrically measured binding enthalpies, providing additional mechanistic understanding. The PLBD can be applied to investigations of protein-ligand recognition and could be integrated into small molecule drug design. Database URL https://plbd.org/.

Regulation of microRNA expression by the adaptor protein GRB2.

Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2). The N-terminal SH3 domain of GRB2 is recruited to the PAZ domain of AGO2 forming a ternary complex containing GRB2, AGO2 and DICER1. Using small-RNA sequencing we identified two groups of miRNAs which are regulated by the binding of GRB2. First, mature and precursor transcripts of mir-17~92 and mir-221 miRNAs are enhanced. Second, mature, but not precursor, let-7 family miRNAs are diminished suggesting that GRB2 directly affects loading of these miRNAs. Notably, the resulting loss of let-7 augments expression of oncogenic targets such as RAS. Thus, a new role for GRB2 is established with implications for cancer pathogenesis through regulation of miRNA biogenesis and oncogene expression.

Expansion microscopy reveals subdomains in C. elegans germ granules.

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid-liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule in C. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid-liquid phase separation.

Phase separation enhances probability of receptor signalling and drug targeting.

The probability of a given receptor tyrosine kinase (RTK) triggering a defined cellular outcome is low because of the promiscuous nature of signalling, the randomness of molecular diffusion through the cell, and the ongoing nonfunctional submembrane signalling activity or noise. Signal transduction is therefore a 'numbers game', where enough cell surface receptors and effector proteins must initially be engaged to guarantee formation of a functional signalling complex against a background of redundant events. The presence of intracellular liquid-liquid phase separation (LLPS) at the plasma membrane provides a mechanism through which the probabilistic nature of signalling can be weighted in favour of the required, discrete cellular outcome and mutual exclusivity in signal initiation.

The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation.

Activation of RAS is crucial in driving cellular outcomes including proliferation, differentiation, migration and apoptosis via the MAPK pathway. This is initiated on recruitment of Grb2, as part of a Grb2-Sos complex, to an up-regulated receptor tyrosine kinase (RTK), enabling subsequent interaction of Sos with the plasma membrane-localised RAS. Aberrant regulation at this convergence point for RTKs in MAPK signalling is a key driver of multiple cancers. Splicing of the GRB2 gene produces a deletion variant, Grb3-3, that is incapable of binding to RTKs. We show that, despite maintaining the ability to bind to Sos, the Grb3-3-Sos complex remains in the cytoplasm, unable to engage with RAS. Competition between Grb2 and Grb3-3 for binding to C-terminal proline-rich sequences on Sos modulates MAPK signalling. Additionally, we demonstrate that splicing is regulated by heterogenous nuclear riboproteins C1/C2, and that normal and malignant colon tissue show differential Grb3-3 expression.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Switching the Inhibitor-Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII.

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.

Grb2 binding induces phosphorylation-independent activation of Shp2.

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation.

Composition of receptor tyrosine kinase-mediated lipid micro-domains controlled by adaptor protein interaction.

Receptor tyrosine kinases (RTKs) are highly regulated, single pass transmembrane proteins, fundamental to cellular function and survival. Aberrancies in regulation lead to corruption of signal transduction and a range of pathological outcomes. Although control mechanisms associated with the receptors and their ligands are well understood, little is known with respect to the impact of lipid/lipid and lipid/protein interactions in the proximal plasma membrane environment. Given that the transmembrane regions of RTKs change in response to extracellular ligand binding, the lipid interactions have important consequences in influencing signal transduction. Fibroblast growth factor receptor 2 (FGFR2) is a highly regulated RTK, including under basal conditions. Binding of the adaptor protein, growth factor receptor-bound protein 2 (GRB2) to FGFR2 prevents full activation and recruitment of downstream signalling effector proteins in the absence of extracellular stimulation. Here we demonstrate that the FGFR2-GRB2 complex is sustained in a defined lipid environment. Dissociation of GRB2 from this complex due to ligand binding, or reduced GRB2 expression, facilitates the dispersion of FGFR2 into detergent-resistant membrane (DRM) micro-domains. This modification of the plasma membrane proximal to FGFR2 provides a further regulatory checkpoint which controls receptor degradation, recycling and recruitment of intracellular signalling proteins.

Drug Resistance in Glioma Cells Induced by a Mesenchymal-Amoeboid Migratory Switch.

Cancer cell invasion is a precondition for tumour metastasis and represents one of the most devastating characteristics of cancer. The development of drugs targeting cell migration, known as migrastatics, may improve the treatment of highly invasive tumours such as glioblastoma (GBM). In this study, investigations into the role of the cell adhesion protein Cellular communication network factor 1 (CCN1, also known as CYR61) in GBM cell migration uncovered a drug resistance mechanism adopted by cells when treated with the small molecule inhibitor CCG-1423. This inhibitor binds to importin α/ß inhibiting the nuclear translocation of the transcriptional co-activator MKL1, thus preventing downstream effects including migration. Despite this reported role as an inhibitor of cell migration, we found that CCG-1423 treatment did not inhibit GBM cell migration. However, we could observe cells now migrating by mesenchymal-amoeboid transition (MAT). Furthermore, we present evidence that CCN1 plays a critical role in the progression of GBM with increased expression in higher-grade tumours and matched blood samples. These findings support a potential role for CCN1 as a biomarker for the monitoring and potentially early prediction of GBM recurrence, therefore as such could help to improve treatment of and increase survival rates of this devastating disease.

Histone deacetylase inhibitors induce medulloblastoma cell death independent of HDACs recruited in REST repression complexes.

BACKGROUND: Repressor element 1-silencing transcription factor (REST) acts as a transcriptional repressor by recruiting several chromatin modifiers, including histone deacetylase (HDAC). Elevated REST expression in medulloblastoma has been associated with tumor progression nevertheless, the tumor shows high sensitivity to HDAC inhibitors (HDACi). However, the functional implications of REST and its requirement for HDACi-induced anti-cancer effects are not well understood. METHODS: In this study, the expression of REST was evaluated across the medulloblastoma subgroups and subtypes using published gene expression data. Further, the expression of REST was modulated using the CRISPR/Cas9 knockout and shRNA knockdown in the Daoy medulloblastoma cell line. RESULTS: The results of this study showed that the expression of REST is elevated in most medulloblastoma subgroups compared to the non-cancerous cerebellum. Blocking of REST expression resulted in increasing the expression of REST-regulated genes, a moderate decrease in the fraction of the cells in the S-phase, and reducing the cells' migration ability. However, REST deficiency did not lead to a marked decrease in the Daoy cell viability and sensitivity to HDACi. CONCLUSION: The findings of this study indicate that REST is not essential for sustaining the proliferation/viability of the Daoy cells. It also revealed that the anti-proliferative effect of HDACi is independent of REST expression.

A novel workflow for three-dimensional analysis of tumour cell migration.

The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal-amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

Targeting the Shc-EGFR interaction with indomethacin inhibits MAP kinase pathway signalling.

Receptor tyrosine kinase (RTK)-mediated hyperactivation of the MAPK/Erk pathway is responsible for a large number of pathogenic outcomes including many cancers. Considerable effort has been directed at targeting this pathway with varying degrees of long term therapeutic success. Under non-stimulated conditions Erk is bound to the adaptor protein Shc preventing aberrant signalling by sequestering Erk from activation by Mek. Activated RTK recruits Shc, via its phosphotyrosine binding (PTB) domain (ShcPTB), precipitating the release of Erk to engage in a signalling response. Here we describe a novel approach to inhibition of MAP kinase signal transduction through attempting to preserve the Shc-Erk complex under conditions of activated receptor. A library of existing drug molecules was computationally screened for hits that would bind to the ShcPTB and block its interaction with the RTKs EGFR and ErbB2. The primary hit from the screen was indomethacin, a non-steroidal anti-inflammatory drug. Validation of this molecule in vitro and in cellular efficacy studies in cancer cells provides proof of principle of the approach to pathway down-regulation and a potential optimizable lead compound.

Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function.

New blood vessel sprouting (angiogenesis) and vascular physiology are fundamental features of metazoan species but we do not fully understand how signal transduction pathways regulate diverse vascular responses. The vascular endothelial growth factor (VEGF) family bind membrane-bound receptor tyrosine kinases (VEGFRs), which trigger multiple signal transduction pathways and diverse cellular responses. We evaluated whether the MAP3K family member and proto-oncoprotein Tpl2 (MAP3K8) regulates basal and VEGF-A-stimulated signal transduction in endothelial cells. Notably, stimulation with exogenous VEGF-A increased Tpl2 mRNA levels and consequently de novo protein synthesis. Depletion of Tpl2 levels reveals a role in both basal and VEGF-A-stimulated endothelial cell responses, including endothelial-leukocyte interactions, monolayer permeability and new blood vessel formation. Under basal conditions, Tpl2 modulates a signal transduction cascade resulting in phosphorylation of a nuclear transcription factor (ATF-2) and altered endothelial gene expression, a pathway previously identified as crucial in VEGF-dependent vascular responses. Loss of Tpl2 expression or activity impairs signal transduction through Akt, eNOS and ATF-2, broadly impacting on endothelial function. Our study now provides a mechanism for Tpl2 as a central component of signal transduction pathways in the endothelium.

Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal Titration Calorimetry as Aid to Drug Design.

Isothermal titration calorimetry (ITC) is one of the main techniques to determine specific interactions between molecules dissolved in aqueous solution. This technique is commonly used in drug development programs when low-molecular-weight molecules are sought that bind tightly and specifically to a protein (disease target) molecule. The method allows a complete thermodynamic characterization of an interaction, i.e., ITC enables direct determination of the model-independent observed interaction change in enthalpy (ΔH) and a model-dependent observed interaction affinity (change in Gibbs free energy, ΔG) in a single experiment. The product of temperature and change in entropy (TΔS) can be obtained by the subtraction of ΔG from ΔH, and the change in heat capacity (ΔCp) can be determined as a slope of the temperature dependence of the binding ΔH.Despite the apparent value of ITC in characterization of interactions, it is often forgotten that many protein-ligand binding reactions are linked to protonation-deprotonation reactions or various conformational changes. In such cases, it is important to determine the linked-reaction contributions and obtain the intrinsic values of the changes in Gibbs energy (affinity), enthalpy, and entropy. These energy values can then be used in various SAR-type structure-thermodynamics and combined with structure-kinetics correlations in drug design, when searching for small molecules that would bind the protein target molecule. This manuscript provides a detailed protocol on how to determine the intrinsic values of protein-ligand binding thermodynamics by ITC.