Biology subjects

Byron, A.

Publications and source records attributed to Byron, A..

12 recordsLinked to original sources

bioRxiv2023

A novel ILK/STAT3 pathway controls plasticity in a neural stem cell model of glioblastoma.

Glioblastomas (GBM) are driven by malignant neural stem-like cells that display extensive heterogeneity and phenotypic plasticity, which drives tumour progression and therapeutic resistance. Here we show that the nodal extracellular matrix-cell adhesion protein integrin-linked kinase (ILK; a pseudokinase), is a key determinant of phenotypic plasticity and the mesenchymal-like, invasive cell state in mouse GBM stem cells. We found that a novel ILK-STAT3 signalling pathway is required for plasticity that enables the transition of GBM stem cells to an astrocyte-like state both in vitro and in vivo. GBM cells genetically depleted of ILK become predominantly stabilised in a transcriptionally-defined progenitor-like state that is characterised by lack of response to differentiation cues and constitutive proliferation. Loss of ILK or interference with STAT3 impairs differentiation potential, reducing phenotypic plasticity of tumour cell populations; additionally, ILK loss causes a mesenchymal- to epithelial-like morphological transition and suppression of malignancy-associated features. Our work defines ILK as a central regulator of multiple GBM phenotypes including phenotypic plasticity and mesenchymal state.

cancer biology↗

bioRxiv2022

Quantitative proteomics identifies tumour matrisome signatures in patients with non-small cell lung cancer

The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry, identifying 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue. We defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. These data reveal tumour matrisome signatures in human NSCLC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=19 SRC="FIGDIR/small/510064v1_ufig1.gif" ALT="Figure 1"> View larger version (8K): org.highwire.dtl.DTLVardef@b34045org.highwire.dtl.DTLVardef@40a5a9org.highwire.dtl.DTLVardef@3c150corg.highwire.dtl.DTLVardef@89c6f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Mena regulates the LINC complex to control actin-nuclear lamina associations, trans-nuclear membrane signalling and cancer gene expression

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes (IACs), play key roles in cancer progression and metastasis. We investigated systems-level changes in the integrin adhesome during metastatic progression of a patient-derived cutaneous squamous cell carcinoma (cSCC), and found that the actin regulatory protein Mena is enriched in IACs in metastatic cSCC cells. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope of metastatic cells. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We have uncovered an unexpected novel role for Mena at the nuclear membrane, where it controls the LINC complex, nuclear architecture, chromatin repositioning and cancer gene expression. This is the first description of an adhesion protein regulating gene transcription via direct signalling across the nuclear envelope. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/458340v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@14e2251org.highwire.dtl.DTLVardef@af341corg.highwire.dtl.DTLVardef@8624dforg.highwire.dtl.DTLVardef@deeb50_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Multicenter reverse-phase protein array data integration

Among the technologies available for protein biomarker discovery and validation, reverse-phase protein array (RPPA) benefits from unequalled sample throughput. Panels of high-quality antibodies enable the quantification by RPPA of protein abundance and posttranslational modifications in biological specimens with high precision and sensitivity. Incorporation of RPPA technology into clinical and drug development pipelines requires robust assays that generate reproducible results across multiple laboratories. We implemented the first international multicenter pilot study to investigate RPPA workflow variability. We characterized the proteomic responses of a series of breast cancer cells to two cancer drugs. This analysis quantified 86,832 sample spots, representing 108 biological samples, arrayed at three independent RPPA platforms. This unique integrated set of data is publicly available as a resource to the proteomic and cancer research communities to catalyse further analysis and investigation. We anticipate that this dataset will form a reference for the comparison of RPPA workflows and reagents, which can be expanded in the future, and will aid the identification of platform-robust treatment-marker antigens in breast cancer cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/458377v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@e2fb2forg.highwire.dtl.DTLVardef@1d8f425org.highwire.dtl.DTLVardef@84dd60org.highwire.dtl.DTLVardef@e1ccf7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Characterisation of a nucleo-adhesome

In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here defined a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions. In adding to nuclear FAKs known roles in regulating transcription, we now show that nuclear FAK regulates expression of many adhesion-related proteins that localise to the nucleus and that nuclear FAK binds to the adhesome component and nuclear protein Hic-5. FAK and Hic-C work together in the nucleus, co-regulating a subset of genes transcriptionally. We describe the first nucleo-adhesome using a squamous cancer cell model, and demonstrate the new principle that there are nuclear adhesion protein subcomplexes that cooperate to control transcription. O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/458428v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1c841ceorg.highwire.dtl.DTLVardef@586a75org.highwire.dtl.DTLVardef@b54439org.highwire.dtl.DTLVardef@921537_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery1-4. Here we show that translocation of lysosomes towards the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, are necessary for both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.

cell biology↗

bioRxiv2020

FAK regulates IL-33 expression via chromatin accessibility and c-Jun

Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 (Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression.

genomics↗

bioRxiv2020

A novel transcriptional signalling pathway mediated by the trafficking protein Ambra1 via scaffolding Atf2 complexes

Previously, we reported that Ambra1 is a core component of a cytoplasmic trafficking network, acting as a spatial rheostat to control active Src and FAK levels in addition to its critical roles in autophagy during neurogenesis. Here we identify a novel nuclear scaffolding function for Ambra1 that controls gene expression. Ambra1 binds to nuclear pore proteins, to other adaptor proteins like FAK and Akap8 in the nucleus, as well as to chromatin modifiers and transcriptional regulators such as Brg1, Cdk9 and the cAMP-regulated transcription factor Atf2. Ambra1 contributes to their association with chromatin and we identified genes whose transcription is regulated by Ambra1 complexes, likely via histone modifications and phospho-Atf2-dependent transcription. Therefore, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signalling in the nucleus; in particular, it recruits chromatin modifiers and transcriptional regulators to control expression of genes such as angpt1, tgfb2, tgfb3, itga8 and itgb7 that likely contribute to the role of Ambra1 in cancer cell invasion.

cell biology↗

bioRxiv2019

Integrative analysis of multi-platform reverse-phase protein array data for the pharmacodynamic assessment of response to targeted therapies

Reverse-phase protein array (RPPA) technology uses panels of high-specificity antibodies to measure proteins and protein post-translational modifications in cells and tissues. The approach offers sensitive and precise quantification of large numbers of samples and has thus found applications in the analysis of clinical and pre-clinical samples. For effective integration into drug development and clinical practice, robust assays with consistent results are essential. Leveraging a collaborative RPPA model, we set out to assess the variability between three different RPPA platforms using distinct instrument set-ups and workflows. Employing multiple RPPA-based approaches operated across distinct laboratories, we characterised a range of human breast cancer cells and their protein-level responses to two clinically relevant cancer drugs. We integrated multi-platform RPPA data and used unsupervised learning to identify protein expression and phosphorylation signatures that were not dependent on RPPA platform and analysis workflow. Our findings indicate that proteomic analyses of cancer cell lines using different RPPA platforms can identify concordant profiles of response to pharmacological inhibition, including when using different antibodies to measure the same target antigens. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of disease or response to therapy.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC=\"FIGDIR/small/769158v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (19K):\norg.highwire.dtl.DTLVardef@3b72aeorg.highwire.dtl.DTLVardef@e62fe4org.highwire.dtl.DTLVardef@5238c3org.highwire.dtl.DTLVardef@ca925_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

A Synergistic Anti-Cancer FAK and HDAC Inhibitor Combination Discovered by a Novel Chemical-Genetic High Content Phenotypic Screen

We mutated the Focal Adhesion Kinase (FAK) catalytic domain to inhibit binding of the chaperone, Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We re-expressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. HDAC inhibitors represented the major class of compounds that potently induced multi-parametric phenotypic changes when FAK was rendered kinase-defective, or inhibited pharmacologically in SCC cells. Indeed, combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a sub-set of cancer cell lines in vitro and efficiently inhibits tumor growth in vivo. Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and reverses FAK-associated nuclear YAP in sensitive cancer cell lines. Here we report the discovery of a new clinically actionable synergistic combination between FAK and HDAC inhibitors.\n\nSignificanceWe describe a chemical-genetic, high-content phenotypic screening approach to discover effective anti-cancer combinations of targeted therapeutics through which we identified a novel, synergistic and clinically actionable, combination of FAK and HDAC inhibitors.

cancer biology↗

bioRxiv2018

Eps8 is a convergence point integrating EGFR and integrin trafficking and crosstalk

Crosstalk between adhesion and growth factor receptors plays a critical role in tissue morphogenesis and repair, and aberrations contribute substantially to neoplastic disease. However, the mechanisms by which adhesion and growth factor receptor signalling are integrated, spatially and temporally, are unclear.\n\nWe used adhesion complex enrichment coupled with quantitative proteomic analysis to identify rapid changes to adhesion complex composition and signalling following growth factor stimulation. Bioinformatic network and ontological analyses revealed a substantial decrease in the abundance of adhesion regulatory proteins and co-ordinators of endocytosis within 5 minutes of EGF stimulation. Together these data suggested a mechanism of EGF-induced receptor endocytosis and adhesion complex turnover.\n\nCombinatorial interrogation of the networks allowed a global and dynamic view of adhesion and growth factor receptor crosstalk to be assembled. By interrogating network topology we identified Eps8 as a putative node integrating 5{beta}1 integrin and EGFR functions. Importantly, EGF stimulation promoted internalisation of both 5{beta}1 and EGFR. However, perturbation of Eps8 increased constitutive internalisation of 5{beta}1 and EGFR; suggesting that Eps8 constrains 5{beta}1 and EGFR endocytosis in the absence of EGF stimulation. Consistent with this, Eps8 regulated Rab5 activity and was required for maintenance of adhesion complex organisation and for EGF-dependent adhesion complex disassembly. Thus, by co-ordinating 5{beta}1 and EGFR trafficking mechanisms, Eps8 is able to control adhesion receptor and growth factor receptor bioavailability and cellular contractility.\n\nWe propose that during tissue morphogenesis and repair, Eps8 functions to spatially and temporally constrain endocytosis, and engagement, of 5{beta}1 and EGFR in order to precisely co-ordinate adhesion disassembly, cytoskeletal dynamics and cell migration.

cell biology↗

bioRxiv2017

Improved LC-MS chromatographic alignment increases the accuracy of label-free quantitative proteomics: Comparison of spectral counting versus ion intensity-based proteomic quantification strategies.

The ability to provide an unbiased qualitative and quantitative description of the global changes to proteins in a cell or an organism would permit the systems-wide study of complex biological systems. Label-free quantitative shotgun proteomic strategies (including LC-MS ion intensity quantification and spectral counting) are attractive because of their relatively low cost, ease of implementation, and the lack of multiplexing restrictions when comparing multiple samples. Owing to improvements in the resolution and sensitivity of mass spectrometers, and the availability of analytical software packages, protein quantification by LC-MS ion intensity has increased in popularity. Here, we have addressed the importance of chromatographic alignment on protein quantification, and then assessed how spectral counting compares to ion intensity-based proteomic quantification. Using a spiked-in protein strategy, we analysed two situations that commonly arise in the application of proteomics to cell biology: (i) samples with a small number of proteins of differential abundance in a larger non-changing background, and (ii) samples with a larger number of proteins of differential abundance. To perform these assessments on biologically relevant samples, we used isolated integrin adhesion complexes (IACs). Technical replicate analysis of isolated IACs resulted in a range of alignment scores using the Progenesis QI software package and demonstrated that higher LC-MS chromatographic alignment scores increased the precision of protein quantification. Furthermore, implementation of a simple sample batch-running strategy enabled good chromatographic alignment for hundreds of samples over multiple batches. Finally, we applied the sample batch-running strategy and compared quantification by LC-MS ion intensity to spectral counting and found that quantification by LC-MS ion intensity was more accurate and precise. In summary, these results demonstrate that chromatographic alignment is important for precise and accurate protein quantification based on LC-MS ion intensity and accordingly we present a simple sample re-ordering strategy to facilitate improved alignment. These findings are not only relevant to label-free quantification using Progenesis QI but may be useful to the wide range of MS-based quantification strategies that rely on chromatographic alignment.

bioinformatics↗