Biology subjects

Ewing, R. M.

Publications and source records attributed to Ewing, R. M..

9 recordsLinked to original sources

bioRxiv2026

Multi-omics analysis reveals integrin α3-dependent mechanisms of Zika virus oncolytic activity in pediatric neural tumor

Oncolytic virotherapy is an innovative approach to cancer treatment that uses replication-competent viruses to selectively target and destroy cancer cells while leaving healthy tissues largely unaffected. Zika virus (ZIKV), a neurotropic orthoflavivirus, has recently gained attention as a potential oncolytic agent due to its ability to infect neural-derived cells and suppress tumor growth in preclinical models. Although existing studies have examined ZIKVs oncolytic effects, the mechanisms underlying these effects remain largely unexplored. Additionally, the roles of individual ZIKV proteins and their interactions with host factors remain incompletely understood. Here, we used RNA sequencing, affinity purification-mass spectrometry, and functional assays to uncover previously unidentified mechanisms underlying ZIKVs oncolytic activity in pediatric neural tumors. We found that the ZIKV non-structural proteins NS4A and NS5 exert oncolytic effects, reducing tumorsphere size. ZIKV-host protein-protein interaction networks were characterized and showed that integrin 3 (gene: ITGA3), a mediator of cell-matrix adhesion, interacts with ZIKV NS2B and NS4A. Integrin 3 was further shown to be involved in ZIKV- and NS4A-induced tumorsphere size reduction, while ITGA3 knockdown and ZIKV infection additively inhibited 3D invasion. These findings provide critical mechanistic insights that could inform the rational design of ZIKV-based virotherapies and highlight opportunities for combination treatment strategies.

cancer biology↗

bioRxiv2024

Multiomics analysis reveals key immunogenic signatures induced by oncolytic Zika virus infection of paediatric brain tumour cells

Brain tumours disproportionately affect children and are the largest cause of paediatric cancer-related death. Despite decades of research, paediatric standard-of-care therapy still predominantly relies on surgery, radiotherapy, and systemic use of cytotoxic chemotherapeutic agents, all of which can result in debilitating acute and late effects. Novel therapies that engage the immune system, such as oncolytic viruses (OVs), hold great promise and are desperately needed. Zika virus (ZIKV) infects and destroys aggressive cells from paediatric medulloblastoma, atypical teratoid rhabdoid tumour (ATRT), diffuse midline glioma (DMG), ependymoma and neuroblastoma. Despite this, the molecular mechanisms underpinning this therapeutic response are grossly unknown. By profiling the transcriptome across a time-course, we comprehensively investigated the response of paediatric medulloblastoma and ATRT brain tumour cells to ZIKV infection at the transcriptome level for the first time. We observed conserved TNF signalling pathway and cytokine signalling-related signatures following ZIKV infection. We demonstrated that the canonical TNF-alpha signalling pathway is implicated in oncolysis by reducing the viability of ZIKV-infected brain tumour cells and is a likely contributor to the anti-tumoural immune response through TNF-alpha secretion. Our findings have highlighted TNF-alpha as a potential prognostic marker for oncolytic ZIKV virotherapy. Performing a 49-plex ELISA, we generated the most comprehensive ZIKV-infected cancer cell secretome to date. We demonstrated that ZIKV infection induces a clinically relevant and diverse pro-inflammatory brain tumour cell secretome, thus circumventing the need for transgene modification to boost efficacy. We assessed publicly available scRNA-Seq data to model how the ZIKV-induced secretome may (i) interact with medulloblastoma tumour microenvironment (TME) cells via paracrine signalling and (ii) polarise lymph node immune cells via endocrine signalling. Our modelling has provided significant insight into the cytokine response that orchestrates the diverse anti-tumoural immune response during oncolytic ZIKV infection of brain tumours. Our findings have significantly contributed to understanding the molecular mechanisms governing oncolytic ZIKV infection and will help pave the way towards ZIKV-based virotherapy.

cancer biology↗

bioRxiv2024

Knockdown proteomics reveals USP7 as a regulator of cell-cell adhesion in colorectal cancer via AJUBA

Ubiquitin-specific protease 7 (USP7) is implicated in many cancers including colorectal cancer in which it regulates cellular pathways such as Wnt signalling and the P53-MDM2 pathway. With the discovery of small-molecule inhibitors, USP7 has also become a promising target for cancer therapy, and therefore systematically identifying USP7 deubiquitinase interaction partners and substrates has become an important goal. In this study, we selected a colorectal cancer cell model that is highly dependent on USP7 and in which USP7 knockdown significantly inhibited colorectal cancer cell viability, colony formation, and cell-cell adhesion. We then used inducible knockdown of USP7 followed by LC-MS/MS to quantify USP7 dependent proteins. We identified the Ajuba LIM domain protein as an interacting partner of USP7 through co-IP, its substantially reduced protein levels in response to USP7 knockdown, and its sensitivity to the specific USP7 inhibitor FT671. The Ajuba protein has been shown to have oncogenic functions in colorectal and other tumours, including regulation of cell-cell adhesion. We show that both knockdown of USP7 or Ajuba results in a substantial reduction of cell-cell adhesion, with concomitant effects on other proteins associated with adherens junctions. Our findings underlie the role of USP7 in colorectal cancer through its protein interaction networks and show that the Ajuba protein is a component of USP7 protein networks present in colorectal cancer.

systems biology↗

bioRxiv2024

Identification of natural Zika virus peptides presented on the surface of paediatric brain tumour cells by HLA classI

Despite decades of research, survival from brain cancer has scarcely improved and is drastically lower than that of other cancers. Novel therapies, such as immunotherapy, hold great promise for treating brain tumours and are desperately needed. Zika virus (ZIKV) infects and kills aggressive cancer cells with stem-like properties (CSCs) from both paediatric and adult brain tumours. Whilst T cell recruitment into ZIKV-infected brain tumours is becoming well documented, the specific mechanisms through which they are activated are poorly understood. We address this by employing a combined LC-MS/MS global proteome and immunopeptidome approach to describe, for the first time, human leukocyte antigen (HLA) presentation of ZIKV peptides on the surface of infected brain tumour cells. We first show that HLA class I (HLA-I) antigen processing & presentation is the most highly enriched immune response pathway in the global proteome of aggressive paediatric USP7-ATRT brain tumour cells following ZIKV infection. We identify USP7-ATRT cells as a good immunopeptidome model due to their homozygous of the globally most common HLA-A allotype (A*02:01). We predict the majority of the 19 ZIKV peptides that we identify here to strongly bind and be presented by HLA-A*02:01. We show that immunopeptide presentation corresponds with cellular ZIKV protein abundance, with ten peptides arising from the most abundant viral protein; non-structural protein 3 (NS3). Specifically, we show the ZIKV NS3 helicase domain to be a rich source of peptides. Finally, we verify that the identified ZIKV peptides do not mimic predicted peptides of the human proteome. The ZIKV peptides we identify here are potential candidates for developing novel epitope-specific brain tumour immunotherapies, and our findings provide potential insight into the efficacious cytotoxic T cell response that oncolytic ZIKV virotherapy can induce against brain tumours. Author SummaryViruses can attack cancer through two mechanisms: 1) infecting and killing the cancer cell and 2) activating the immune system against the tumour. Zika virus (ZIKV) uses both mechanisms to fight brain cancer. Here, we employ a powerful proteomic technique to identify fragments of viral proteins (peptides) presented by cell surface receptors on brain cancer cells infected with ZIKV. In the human body, immune system cells such as T cells recognise and become activated in response to these viral peptides and subsequently attack the infected patient tumour. We identify 19 ZIKV peptides, three of which have been shown previously to elicit T cell responses, four identified elsewhere, and twelve are novel. Our work helps delineate a component of how ZIKV acts as an immunotherapy, the T cell-specific immune response that the virus raises to promote clearance of brain tumours. The significance of our study is that the ZIKV peptides we identify may lead to the development of a novel brain tumour immunotherapy.

cancer biology↗

bioRxiv2022

Integrative transcriptomic and proteomic meta-analysis of Zika viral infection reveals potential mechanisms for oncolytic therapy in neuroblastoma

BACKGROUNDPaediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) is neurotropic and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma. METHODSHere, we perform an extensive meta-analysis of ZIKV infection studies to identify molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We summarise the neuroblastoma cell lines and ZIKV strains utilised and re-evaluate the infection data to deduce the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating transcriptomics, interaction proteomics, dependency factor and compound datasets we show the involvement of multiple host systems during ZIKV infection. RESULTSWe identify that most paediatric neuroblastoma cell lines are highly susceptible to ZIKV infection and that the PRVABC59 ZIKV strain is the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV is dependent on SREBP-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV nonstructural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon. CONCLUSIONSOur work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials. KEYPOINTSO_LIThe Zika virus may provide the basis for an oncolytic virotherapy against Neuroblastoma C_LIO_LIMost paediatric neuroblastoma cell lines are susceptible to Zika viral infection C_LIO_LIWe identified molecular mechanisms that may induce the oncolytic response in Neuroblastoma C_LI Contribution to the fieldThe ability to both induce direct oncolysis and provoke an anti-tumoral immune response makes oncolytic virotherapy an attractive candidate to combat aggressive and heterogenous cancers, such as high-risk neuroblastoma. To progress oncolytic virotherapy to clinical trial it is essential to understand the host mechanisms the virus manipulates to kill cancer cells, alongside any pathology as a consequence of infection of normal cells. Here, we show that ZIKV efficiently infects and induces oncolysis of paediatric neuroblastoma cells and propose a potential TNF pathway-driven immune response. ZIKVs specificity for infection of nervous system cancer cells, while rarely causing nervous system-related pathology in young children, addresses many of its safety concerns. The inclusion of more effective and less toxic novel therapies, such as a potential ZIKV-based therapeutic, in multimodal treatment regimens will pave the way for improving patient long-term health and overall survival.

cancer biology↗

bioRxiv2021

Predicting Relative Protein Abundance via Sequence-Based Information

Understanding the complex interactions between transcriptome and proteome is essential in uncovering cellular mechanisms both in health and disease contexts. The limited correlations between corresponding transcript and protein abundance suggest that regulatory processes tightly govern information flow surrounding transcription and translation, and beyond. In this study we adopt an approach which expands the feature scope that models the human proteome: we develop machine learning models that incorporate sequence-derived features (SDFs), sometimes in conjunction with corresponding mRNA levels. We develop a large resource of sequence-derived features which cover a significant proportion of the H. sapiens proteome, demonstrate which of these features are significant in prediction on multiple cell lines, and suggest insights into which biological processes can be explained using these features. We reveal that (a) SDFs are significantly better at protein abundance prediction across multiple cell lines both in steady-state and dynamic contexts, (b) that SDFs can cover the domain of translation with relative efficiency but struggle with cell-line specific pathways and (c) provide a resource which can be plugged into many subsequent protein-centric analyses.

bioinformatics↗

bioRxiv2021

Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells

Glycogen-specific kinase (GSK3{beta}) is an integral regulator of the Wnt signalling pathway as well as many other diverse signalling pathways and processes. Dys-regulation of GSK3{beta} is implicated in many different pathologies, including neurodegenerative disorders as well as many different tumour types. In the context of tumour development, GSK3{beta} has been shown to play both oncogenic and tumour suppressor roles, depending upon tissue, signalling environment or disease progression. Although multiple substrates of the GSK3{beta} kinase have been identified, the wider protein networks within which GSK3{beta} participates are not well known, and the consequences of these interactions not well understood. In this study, LC-MS/MS expression analysis was performed using knockout GSK3{beta} colorectal cancer cells and isogenic controls in colorectal cancer cell lines carrying dominant stabilizing mutations of {beta}-Catenin. Consistent with the role GSK3{beta}, we found that {beta}-Catenin levels and canonical Wnt activity are unaffected by knockout of GSK3{beta} and therefore use this knockout cell model to identify other processes in which GSK3{beta} is implicated. Quantitative proteomic analysis revealed perturbation of proteins involved in cell-cell adhesion, and we characterize the phenotype and altered proteomic profiles associated with this. We also characterize the perturbation of metabolic pathways resulting from GSK3{beta} knockout and identify defects in glycogen metabolism. In summary, using a precision colorectal cancer cell-line knockout model with constitutively activated {beta}-Catenin we are able to identify several of the diverse pathways and processes associated with GSK3{beta} function.

cancer biology↗

bioRxiv2019

Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dys-regulation of Epithelial-Mesenchymal Transition and subcellular re-localization of Beta-Catenin

BackgroundDNA methyltransferase I is the primary eukaryotic DNA methyltransferase engaged in maintenance of CpG DNA methylation patterns across the genome. Alteration of CpG methylation patterns and levels is a frequent and significant occurrence across many cancers, and targeted inhibition of Dnmt1 has become an approach of choice for select malignancies. There has been significant interest both in the methyltransferase activity as well as methylation-independent functions of Dnmt1. A previously generated hypomorphic allele of Dnmt1 in HCT116 colorectal cancer cells has become an important tool for understanding Dnmt1 function and how CpG methylation patterns are modulated across the genome. Colorectal cancer cells with the Dnmt1 hypomorphic allele carry a homozygous deletion of exons 3 to 5 of Dnmt1, resulting in greatly reduced Dnmt1 protein expression whilst still exhibiting a limited functional activity and methyltransferase ability. Although this cell model of reduced Dnmt1 levels and function have been used to study the downstream effects on the epigenome and transcriptome, the broader effects of the Dnmt1 hypomorph on the proteome and wider cell signalling are largely unknown. ResultsIn this study, we used quantitative proteomic analysis of nuclear-enriched samples of HCT116 Dnmt1 hypomorph cells to identify signalling pathways and processes dysregulated in the hypomorph cells as compared to wild-type HCT116 cells. Unexpectedly, we observed a clear signature of increased expression of Epithelial-to-Mesenchymal (EMT) in Dnmt1 hypomorph cells. We also observed reduced expression and sub-cellular re-localization of Beta-Catenin in Dnmt1 hypomorph cells. Expression of wild-type Dnmt1 in hypomorph cells or knock-down of wild-type Dnmt1 did not recapitulate or rescue the observed protein profiles in Dnmt1 hypomorph cells suggesting that hypomorphic Dnmt1 causes changes not solely attributable ConclusionsIn summary we present the first comprehensive proteomic analysis of the widely studied Dnmt1 hypomorph colorectal cancer cells and identify redistribution of Dnmt1 and its interaction partner Beta-Catenin as well as the dysregulation of EMT related processes and signalling pathways related to the development of a cancer stem cell phenotype.

cancer biology↗

bioRxiv2019

A Topological Data Analysis Network Model of Asthma Based on Blood Gene Expression Profiles

Stratified medicine requires discretisation of disease populations for targeted treatments. We have developed and applied a discrete Morse theory clustering algorithm to a Topological Data Analysis (TDA) network model of 498 gene expression profiles of peripheral blood from asthma and healthy participants. The Morse clustering algorithm defined nine clusters, BC1-9, representing molecular phenotypes with discrete phenotypes including Type-1, 2 & 17 cytokine inflammatory pathways. The TDA network model and clusters were also characterised by activity of glucocorticoid receptor signalling associated with different expression profiles of glucocorticoid receptor (GR), according to microarray probesets targeted to the start or end of the GR mRNAs 3 UTR; suggesting differential GR mRNA processing as a possible driver of asthma phenotypes including steroid insensitivity.

molecular biology↗