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Supplementary proteomics data

Supplementary Figures 1-7. Supplementary Figure 1.Cell-cycle profiles of HEK293T cells following treatment with Wnt3a. Supplementary Figure 2. Protein lysate samples used in the immunoprecipitation or immunoblots analyses were treated with Nuclease. Supplementary Figure 3. Dnmt1 (native antibody) immuno-precipitates β-catenin in parent HCT116 cells. Supplementary Figure 4. DNMT1 protein is stabilized by treatment with Wnt3a in HCT116 but not in CTNNB1KO-HCT116 cells. Supplementary Figure 5. Degradation profiles for β-catenin and Dnmt1 in HCT116, DNMT1KO-HCT116 or CTNNB1KO-HCT116 cells following cycloheximide treatment. Supplementary Figure 6. Abundance of β-catenin and Dnmt1 in DNMT1KO-HCT116 (A) or CTNNB1KO-HCT116 (B) cells treated with MG-132 proteasome inhibitor. Supplementary Figure 7. RT-PCR analysis of H19 expression in HCT116 and CTNNB1KO-HCT116 cells shows increase H19 transcript levels in the KO cells.

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.KEYPOINTSThe Zika virus may provide the basis for an oncolytic virotherapy against NeuroblastomaMost paediatric neuroblastoma cell lines are susceptible to Zika viral infectionWe identified molecular mechanisms that may induce the oncolytic response in NeuroblastomaContribution 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. ZIKV’s 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.

The apoptosis-stimulating protein of p53 (ASPP) family of proteins can regulate apoptosis by interacting with the p53 family and have been identified to play an important role in cancer progression. Previously, we have demonstrated that ASPP2 downregulation can promote invasion and migration by controlling β-catenin- dependent regulation of ZEB1, however, the role of ASPP1 in colorectal cancer (CRC) remains unclear. We analyzed data from The Cancer Genome Atlas (TCGA) and coupled this to in vitro experiments in CRC cell lines as well as to experimental pulmonary metastasis in vivo. Tissue microarrays of CRC patients with information of clinical-pathological parameters were also used to investigate the expression and function of ASPP1 in CRC. Here, we report that loss of ASPP1 is capable of enhancing migration and invasion in CRC, both in vivo and in vitro. We demonstrate that depletion of ASPP1 could activate expression of Snail2 via the NF-κB pathway and in turn, induce EMT; and this process is further exacerbated in RAS-mutated CRC. ASPP1 could be a prognostic factor in CRC, and the use of NF-κB inhibitors may provide new strategies for therapy against metastasis in ASPP1-depleted CRC patients.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFβ increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

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.

Motivation Protein–protein interactions (PPIs) play a key role in diverse biological processes but only a small subset of the interactions has been experimentally identified. Additionally, high-throughput experimental techniques that detect PPIs are known to suffer various limitations, such as exaggerated false positives and negatives rates. The semantic similarity derived from the Gene Ontology (GO) annotation is regarded as one of the most powerful indicators for protein interactions. However, while computational approaches for prediction of PPIs have gained popularity in recent years, most methods fail to capture the specificity of GO terms. Results We propose TransformerGO, a model that is capable of capturing the semantic similarity between GO sets dynamically using an attention mechanism. We generate dense graph embeddings for GO terms using an algorithmic framework for learning continuous representations of nodes in networks called node2vec. TransformerGO learns deep semantic relations between annotated terms and can distinguish between negative and positive interactions with high accuracy. TransformerGO outperforms classic semantic similarity measures on gold standard PPI datasets and state-of-the-art machine-learning-based approaches on large datasets from Saccharomyces cerevisiae and Homo sapiens. We show how the neural attention mechanism embedded in the transformer architecture detects relevant functional terms when predicting interactions. Availability and implementation https://github.com/Ieremie/TransformerGO. Supplementary information Supplementary data are available at Bioinformatics online.

Despite the extensive study of MYCN-amplified neuroblastomas, there is a significant unmet clinical need in MYCN non-amplified neuroblastomas. In particular, the extent of heterogeneity within the MYCN non-amplified population is unknown. Here, we investigate whether transcriptional subtyping of MYCN non-amplified neuroblastomas can identify molecular subtypes with discrete prognosis and therapeutic vulnerabilities. Using tumour expression data and ConsensusClusterPlus, we demonstrate that MYCN non-amplified neuroblastomas are heterogeneous and can be classified into 3 subgroups based on their transcriptional signatures. Within these groups, subgroup 2 has the worst prognosis and this group shows a "MYCN" signature that is potentially induced by the overexpression of Aurora Kinase A (AURKA); whilst subgroup 3 is characterised by an "inflamed" gene signature. The clinical implications of this subtype classification are significant, as each subtype demonstrates unique prognosis and vulnerability to investigational therapies. We propose that matching baseline tumour subtype to therapy may enhance precision prognosis and therapy stratification for patients with MYCN non-amplified neuroblastomas.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we show that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of oxygen status (pseudohypoxia). Whilst TGFβ increased rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting ‘bone-type’ cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knock down of Factor Inhibiting HIF (FIH) or oxidative stress caused pseudohypoxic HIF activation in normal fibroblasts. In contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of IPF lung mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

BACKGROUND: The consequences of COVID-19 in those who recover from acute infection requiring hospitalisation have yet to be clearly defined. We aimed to describe the temporal trends in respiratory outcomes over 12 months in patients hospitalised for severe COVID-19 and to investigate the associated risk factors. METHODS: In this prospective, longitudinal, cohort study, patients admitted to hospital for severe COVID-19 who did not require mechanical ventilation were prospectively followed up at 3 months, 6 months, 9 months, and 12 months after discharge from Renmin Hospital of Wuhan University, Wuhan, China. Patients with a history of hypertension; diabetes; cardiovascular disease; cancer; and chronic lung disease, including asthma or chronic obstructive pulmonary disease; or a history of smoking documented at time of hospital admission were excluded at time of electronic case-note review. Patients who required intubation and mechanical ventilation were excluded given the potential for the consequences of mechanical ventilation itself to influence the factors under investigation. During the follow-up visits, patients were interviewed and underwent physical examination, routine blood test, pulmonary function tests (ie, diffusing capacity of the lungs for carbon monoxide [DLCO]; forced expiratory flow between 25% and 75% of forced vital capacity [FVC]; functional residual capacity; FVC; FEV1; residual volume; total lung capacity; and vital capacity), chest high-resolution CT (HRCT), and 6-min walk distance test, as well as assessment using a modified Medical Research Council dyspnoea scale (mMRC). FINDINGS: Between Feb 1, and March 31, 2020, of 135 eligible patients, 83 (61%) patients participated in this study. The median age of participants was 60 years (IQR 52-66). Temporal improvement in pulmonary physiology and exercise capacity was observed in most patients; however, persistent physiological and radiographic abnormalities remained in some patients with COVID-19 at 12 months after discharge. We found a significant reduction in DLCO over the study period, with a median of 77% of predicted (IQR 67-87) at 3 months, 76% of predicted (68-90) at 6 months, and 88% of predicted (78-101) at 12 months after discharge. At 12 months after discharge, radiological changes persisted in 20 (24%) patients. Multivariate logistic regression showed increasing odds of impaired DLCO associated with female sex (odds ratio 8·61 [95% CI 2·83-26·2; p=0·0002) and radiological abnormalities were associated with peak HRCT pneumonia scores during hospitalisation (1·36 [1·13-1·62]; p=0·0009). INTERPRETATION: In most patients who recovered from severe COVID-19, dyspnoea scores and exercise capacity improved over time; however, in a subgroup of patients at 12 months we found evidence of persistent physiological and radiographic change. A unified pathway for the respiratory follow-up of patients with COVID-19 is required. FUNDING: National Natural Science Foundation of China, UK Medical Research Council, and National Institute for Health Research Southampton Biomedical Research Centre. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer that lacks the oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, making it difficult to target therapeutically. Targeting synthetic lethality is an alternative approach for cancer treatment. TNBC shows frequent loss of phosphatase and tensin homologue (PTEN) expression, which is associated with poor prognosis and treatment response. To identify PTEN synthetic lethal interactions, TCGA analysis coupled with a whole-genome siRNA screen in isogenic PTEN-negative and -positive cells were performed. Among the candidate genes essential for the survival of PTEN-inactive TNBC cells, WDHD1 (WD repeat and high-mobility group box DNA-binding protein 1) expression was increased in the low vs. high PTEN TNBC samples. It was also the top hit in the siRNA screen and its knockdown significantly inhibited cell viability in PTEN-negative cells, which was further validated in 2D and 3D cultures. Mechanistically, WDHD1 is important to mediate a high demand of protein translation in PTEN-inactive TNBC. Finally, the importance of WDHD1 in TNBC was confirmed in patient samples obtained from the TCGA and tissue microarrays with clinic-pathological information. Taken together, as an essential gene for the survival of PTEN-inactive TNBC cells, WDHD1 could be a potential biomarker or a therapeutic target for TNBC.

Glycogen-specific kinase (GSK3β) is an integral regulator of the Wnt signalling pathway as well as many other diverse signalling pathways and processes. Dys-regulation of GSK3β is implicated in many different pathologies, including neurodegenerative disorders as well as many different tumour types. In the context of tumour development, GSK3β has been shown to play both oncogenic and tumour suppressor roles, depending upon tissue, signalling environment or disease progression. Although multiple substrates of the GSK3β kinase have been identified, the wider protein networks within which GSK3β 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 GSK3β colorectal cancer cells and isogenic controls in colorectal cancer cell lines carrying dominant stabilizing mutations of β-Catenin. Consistent with the role GSK3β, we found that β-Catenin levels and canonical Wnt activity are unaffected by knockout of GSK3β and therefore use this knockout cell model to identify other processes in which GSK3β 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 GSK3β knockout and identify defects in glycogen metabolism. In summary, using a precision colorectal cancer cell-line knockout model with constitutively activated β-Catenin we are able to identify several of the diverse pathways and processes associated with GSK3β function. p { margin-bottom: 0.25cm; direction: ltr; color: #00000a; line-height: 120%; text-align: left; orphans: 2; widows: 2 }p.western { font-family: "Times New Roman", serif }p.cjk { font-family: "PMingLiU", "????"; so-language: zh-CN }p.ctl { font-family: "Times New Roman"; font-size: 10pt }a:link { color: #0000ff }

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer that lacks the oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, making it difficult to target therapeutically. Targeting synthetic lethality is an alternative approach for cancer treatment. TNBC shows frequent loss of phosphatase and tensin homologue (PTEN) expression, which is associated with poor prognosis and treatment response. To identify PTEN synthetic lethal interactions, TCGA analysis coupled with a whole-genome siRNA screen in isogenic PTEN-negative and -positive cells were performed. Among the candidate genes essential for the survival of PTEN-inactive TNBC cells, WDHD1 (WD repeat and high-mobility group box DNA-binding protein 1) expression was increased in the low vs. high PTEN TNBC samples. It was also the top hit in the siRNA screen and its knockdown significantly inhibited cell viability in PTEN-negative cells, which was further validated in 2D and 3D cultures. Mechanistically, WDHD1 is important to mediate a high demand of protein translation in PTEN-inactive TNBC. Finally, the importance of WDHD1 in TNBC was confirmed in patient samples obtained from the TCGA and tissue microarrays with clinic-pathological information. Taken together, as an essential gene for the survival of PTEN-inactive TNBC cells, WDHD1 could be a potential biomarker or a therapeutic target for TNBC.

Ubiquitin-specific protease (USP7), also known as Herpesvirus-associated ubiquitin-specific protease (HAUSP), is a deubiquitinase. There has been significant recent attention on USP7 following the discovery that USP7 is a key regulator of the p53-MDM2 pathway. The USP7 protein is 130 kDa in size and has multiple domains which bind to a diverse set of proteins. These interactions mediate key developmental and homeostatic processes including the cell cycle, immune response, and modulation of transcription factor and epigenetic regulator activity and localization. USP7 also promotes carcinogenesis through aberrant activation of the Wnt signalling pathway and stabilization of HIF-1α. These findings have shown that USP7 may induce tumour progression and be a therapeutic target. Together with interest in developing USP7 as a target, several studies have defined new protein interactions and the regulatory networks within which USP7 functions. In this review, we focus on the protein interactions of USP7 that are most important for its cancer-associated roles.

DNA methylation is a critical epigenetic modification that is established and maintained across the genome by DNA methyltransferase enzymes (Dnmts). Altered patterns of DNA methylation are a frequent occurrence in many tumor genomes, and inhibitors of Dnmts have become important epigenetic drugs. Azacitidine is a cytidine analog that is incorporated into DNA and induces the specific inhibition and proteasomal-mediated degradation of Dnmts. The downstream effects of azacitidine on CpG methylation and on gene transcription have been widely studied in many systems, but how azacitidine impacts the proteome is not well-understood. In addition, with its specific ability to induce the rapid degradation of Dnmts (in particular, the primary maintenance DNA methyltransferase, Dnmt1), it may be employed as a specific chemical knockdown for investigating the Dnmt1-associated functional or physical interactome. In this study, we use quantitative proteomics to analyze the degradation profile of proteins in the nuclear proteome of cells treated with azacitidine. We identify specific proteins as well as multiple pathways and processes that are impacted by azacitidine. The Dnmt1 interaction partner, Uhrf1, exhibits significant azacitidine-induced degradation, and this azacitidine-induced degradation is independent of the levels of Dnmt1 protein. We identify multiple other chromatin- and epigenetic-associated factors, including the bromodomain-containing transcriptional regulator, Brd2. We show that azacitidine induces highly specific perturbations of the Dnmt1-associated proteome, and while interaction partners such as Uhrf1 are sensitive to azacitidine, others such as the Dnmt1 interaction partner and stability regulator, Usp7, are not. In summary, we have conducted the first comprehensive proteomic analysis of the azacitidine-sensitive nuclear proteome, and we show how 5-azacitidine can be used as a specific probe to explore Dnmt- and chromatin-related protein networks.

Background: COVID-19 is “public enemy number one” and has placed an enormous burden on health authorities across the world. Given the wide clinical spectrum of COVID-19, understanding the factors that can predict disease severity will be essential since this will help frontline clinical staff to stratify patients with increased confidence.Purpose: To investigate the diagnostic value of the temporal radiographic changes, and the relationship to disease severity and viral clearance in COVID-19 patients.Methods: In this retrospective cohort study, we included 99 patients admitted to the Renmin Hospital of Wuhan University, with laboratory confirmed moderate or severe COVID-19. Temporal radiographic changes and viral clearance were explored using appropriate statistical methods.Results: Radiographic features from HRCT scans included ground-glass opacity, consolidation, air bronchogram, nodular opacities and pleural effusion. The HRCT scores (peak) during disease course in COVID-19 patients with severe pneumonia (median: 24.5) were higher compared to those with pneumonia (median: 10) (p=3.56×10-12), with more frequency of consolidation (p=0.025) and air bronchogram (p=7.50×10-6). The median values of days when the peak HRCT scores were reached in pneumonia or severe pneumonia patients were 12 vs. 14, respectively (p=0.048). Log-rank test and Spearman's Rank-Order correlation suggested temporal radiographic changes as a valuable predictor for viral clearance. In addition, follow up CT scans from 11 pneumonia patients showed full recovery.Conclusion: Given the values of HRCT scores for both disease severity and viral clearance, a standardised HRCT score system for COVID-19 is highly demanded.

Hyperbaric oxygen (HBO) is widely applied to treat several hypoxia-related diseases. Previous studies have focused on the immediate effect of HBO-exposure induced oxidative stress on the lungs, but knowledge regarding the chronic effects from repetitive HBO exposure is limited, especially at the gene expression level. We found that repetitive HBO exposure did not alter the morphology of murine lungs. However, by deconvolution of RNA-seq from those mice lungs using CIBERSORTx and the expression profile matrices of 8 mesenchymal cell subtypes obtained from bleomycin-treated mouse lungs, we identify several mesenchymal cell subtype changes. These include increases in Col13a1 matrix fibroblasts, mesenchymal progenitors and mesothelial cell populations and decreases in lipofibroblasts, endothelial and Pdgfrb high cell populations. Our data suggest that repetitive HBO exposure may affect biological processes in the lungs such as response to wounding, extracellular matrix, vasculature development and immune response.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a key step in the transformation of epithelial cells into migratory and invasive tumour cells. Intricate positive and negative regulatory processes regulate EMT. Many oncogenic signalling pathways can induce EMT, but the specific mechanisms of how this occurs, and how this process is controlled are not fully understood.METHODS: RNA-Seq analysis, computational analysis of protein networks and large-scale cancer genomics datasets were used to identify ELF3 as a negative regulator of the expression of EMT markers. Western blotting coupled to siRNA as well as analysis of tumour/normal colorectal cancer panels was used to investigate the expression and function of ELF3.RESULTS: RNA-Seq analysis of colorectal cancer cells expressing mutant and wild-type β-catenin and analysis of colorectal cancer cells expressing inducible mutant RAS showed that ELF3 expression is reduced in response to oncogenic signalling and antagonizes Wnt and RAS oncogenic signalling pathways. Analysis of gene-expression patterns across The Cancer Genome Atlas (TCGA) and protein localization in colorectal cancer tumour panels showed that ELF3 expression is anti-correlated with β-catenin and markers of EMT and correlates with better clinical prognosis.CONCLUSIONS: ELF3 is a negative regulator of the EMT transcription factor (EMT-TF) ZEB1 through its function as an antagonist of oncogenic signalling.

Glycogen-specific kinase (GSK3β) is an integral regulator of the Wnt signalling pathway as well as many other diverse signalling pathways and processes. Dys-regulation of GSK3β is implicated in many different pathologies, including neurodegenerative disorders as well as many different tumour types. In the context of tumour development, GSK3β has been shown to play both oncogenic and tumour suppressor roles, depending upon tissue, signalling environment or disease progression. Although multiple substrates of the GSK3β kinase have been identified, the wider protein networks within which GSK3β 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 GSK3β colorectal cancer cells and isogenic controls in colorectal cancer cell lines carrying dominant stabilizing mutations of β-catenin. Consistent with the role of GSK3β, we found that β-catenin levels and canonical Wnt activity are unaffected by knockout of GSK3β and therefore used this knockout cell model to identify other processes in which GSK3β is implicated. Quantitative proteomic analysis revealed perturbation of proteins involved in cell-cell adhesion, and we characterized the phenotype and altered proteomic profiles associated with this. We also characterized the perturbation of metabolic pathways resulting from GSK3β knockout and identified defects in glycogen metabolism. In summary, using a precision colorectal cancer cell-line knockout model with constitutively activated β-catenin we identified several of the diverse pathways and processes associated with GSK3β function.