Characterisation of ACBD3 and PI4Kβ expression in breast cancer and the effects of ACBD3 overexpression

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel UniversityTargeted breast cancer treatments are essential for increasing chemotherapy effectiveness whilst simultaneously reducing side effects and are the focus of a whole generation of drug development in cancer and elsewhere. The ACBD3 gene encodes an essential structural tether protein of the same name that has an unusually large number of cellular roles, diverse binding partners, and few redundancies. Chromosome 1q is frequently amplified in breast cancer and the ACBD3 locus (1q42.12) was previously found to be amplified in multiple breast cell lines and primary breast tumours. Previous research found that ACBD3 mRNA was upregulated in breast tumour tissue matched against adjacent normal tissue and that ACBD3 overexpression promoted cancer stem cell renewal and activated the Wnt/β-Catenin signalling pathway in breast cancer cell lines. Due to the broad functions of ACBD3 and its contextual role in cells it was hypothesised that ACBD3 expression may have other affects in breast cancer. ACBD3 was overexpressed at the mRNA and protein level in breast cancer patient tumours compared to normal tissue and mRNA expression over the median value was detrimental for breast cancer patient survival, relapse free survival and distant metastasis free survival. IHC staining of breast cancer and normal breast tissue cores found that ACBD3 was highly expressed in epithelial ductal cells. ACBD3 mRNA and protein expression was higher in a panel of breast cancer cell lines compared to a normal like breast cell line and ER+ cell lines had the highest protein expression of ACBD3. ACBD3 mRNA and protein expression was upregulated in a previously engineered T47D everolimus chemotherapy resistant cell, the T47D breast cancer cell line was transfected with eGFP-ACBD3 but this did not affect everolimus resistance. ACBD3 overexpression did increase cell growth and there were also a number of expression changes to oncoproteins. A GOLD domain deletion mutant of ACBD3 was constructed and this led to more oncoprotein expression changes when expressed in the T47D cell line. Transcriptional and translational regulation are sensitive to cell density which has implications for all ex vivo study of ACBD3 and several compounds have been found that augment ACBD3 expression. ACBD3 was hypothesised to be a marker of progression in breast cancer and may promote a Luminal B pathology over Luminal A. its overexpression increased growth in a Luminal A cell line, increased expression of proteins associated with inflammation and secretion and reduced immunogenic protein expression. Luminal B patients had the largest reduction in relapse free survival when ACBD3 mRNA expression was high. ACBD3 expression appears to be a biomarker for breast cancer patient outcomes and may have some validity in predicting response to therapy and was also associated with ER+ and signalling. New mechanisms by which ACBD3 might cause inflammation were determined in addition to known roles for ACBD3 in redox stress and in iron import. ACBD3 also reduced immunogenic proteins when overexpressed. ACBD3 is certainly associated with worse outcomes and with progression in breast cancer and ACBD3 dependent pathways should be considered as a target for treatment in the future. The consensus of these results agree that ACBD3 expression in breast cancer is associated with characteristics of stemness and that ACBD3 may decrease immune system detection in addition to Wnt signalling.Breast Cancer Hope and Brunel University Londo

Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein

CHK1 is a protein kinase that functions downstream of activated ATR to phosphorylate multiple targets as part of intra-S and G2/M DNA damage checkpoints. Its role in allowing cells to survive replicative stress has made it an important target for anti-cancer drug discovery. Activation of CHK1 by ATR depends on their mutual interaction with CLASPIN, a natively unstructured protein that interacts with CHK1 through a cluster of phosphorylation sites in its C-terminal half. We have now determined the crystal structure of the kinase domain of CHK1 bound to a high-affinity motif from CLASPIN. Our data show that CLASPIN engages a conserved site on CHK1 adjacent to the substrate-binding cleft, involved in phosphate sensing in other kinases. The CLASPIN motif is not phosphorylated by CHK1, nor does it affect phosphorylation of a CDC25 substrate peptide, suggesting that it functions purely as a scaffold for CHK1 activation by ATR

Longitudinal proteomic profiling of dialysis patients with COVID-19 reveals markers of severity and predictors of death

Funder: The Sidharth Burman endowmentEnd-stage kidney disease (ESKD) patients are at high risk of severe COVID-19. We measured 436 circulating proteins in serial blood samples from hospitalised and non-hospitalised ESKD patients with COVID-19 (n = 256 samples from 55 patients). Comparison to 51 non-infected patients revealed 221 differentially expressed proteins, with consistent results in a separate subcohort of 46 COVID-19 patients. Two hundred and three proteins were associated with clinical severity, including IL6, markers of monocyte recruitment (e.g. CCL2, CCL7), neutrophil activation (e.g. proteinase-3), and epithelial injury (e.g. KRT19). Machine-learning identified predictors of severity including IL18BP, CTSD, GDF15, and KRT19. Survival analysis with joint models revealed 69 predictors of death. Longitudinal modelling with linear mixed models uncovered 32 proteins displaying different temporal profiles in severe versus non-severe disease, including integrins and adhesion molecules. These data implicate epithelial damage, innate immune activation, and leucocyte–endothelial interactions in the pathology of severe COVID-19 and provide a resource for identifying drug targets

The widespread nature of Pack-TYPE transposons reveals their importance for plant genome evolution

Pack-TYPE transposable elements (TEs) are a group of non-autonomous DNA transposons found in plants. These elements can efficiently capture and shuffle coding DNA across the host genome, accelerating the evolution of genes. Despite their relevance for plant genome plasticity, the detection and study of Pack-TYPE TEs are challenging due to the high similarity these elements have with genes. Here, we produced an automated annotation pipeline designed to study Pack-TYPE elements and used it to successfully annotate and analyse more than 10,000 new Pack-TYPE TEs in the rice and maize genomes. Our analysis indicates that Pack-TYPE TEs are an abundant and heterogeneous group of elements. We found that these elements are associated with all main superfamilies of Class II DNA transposons in plants and likely share a similar mechanism to capture new chromosomal DNA sequences. Furthermore, we report examples of the direct contribution of these TEs to coding genes, suggesting a generalised and extensive role of Pack-TYPE TEs in plant genome evolution

Longitudinal plasma proteomics reveals biomarkers of alveolar-capillary barrier disruption in critically ill COVID-19 patients

Abstract The pathobiology of respiratory failure in COVID-19 consists of a complex interplay between viral cytopathic effects and a dysregulated host immune response. In critically ill patients, imatinib treatment demonstrated potential for reducing invasive ventilation duration and mortality. Here, we perform longitudinal profiling of 6385 plasma proteins in 318 hospitalised patients to investigate the biological processes involved in critical COVID-19, and assess the effects of imatinib treatment. Nine proteins measured at hospital admission accurately predict critical illness development. Next to dysregulation of inflammation, critical illness is characterised by pathways involving cellular adhesion, extracellular matrix turnover and tissue remodelling. Imatinib treatment attenuates protein perturbations associated with inflammation and extracellular matrix turnover. These proteomic alterations are contextualised using external pulmonary RNA-sequencing data of deceased COVID-19 patients and imatinib-treated Syrian hamsters. Together, we show that alveolar capillary barrier disruption in critical COVID-19 is reflected in the plasma proteome, and is attenuated with imatinib treatment. This study comprises a secondary analysis of both clinical data and plasma samples derived from a clinical trial that was registered with the EU Clinical Trials Register (EudraCT 2020–001236–10, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001236-10/NL ) and Netherlands Trial Register (NL8491, https://www.trialregister.nl/trial/8491 )

ACBD3 Bioinformatic Analysis and Protein Expression in Breast Cancer Cells

ACBD3 overexpression has previously been found to correlate with worse prognosis for breast cancer patients and, as an incredibly diverse protein in both function and cellular localisation, ACBD3 may have a larger role in breast cancer than previously thought. This study further investigated ACBD3′s role in breast cancer. Bioinformatic databases were queried to characterise ACBD3 expression and mutation in breast cancer and to investigate how overexpression affects breast cancer patient outcomes. Immunohistochemistry was carried out to examine ACBD3 location within cells and tissue structures. ACBD3 was more highly expressed in breast cancer than in any other cancer or matched normal tissue, and expression over the median level resulted in reduced relapse-free, overall, and distant metastasis-free survival for breast cancer patients as a whole, with some differences observed between subtypes. IHC analysis found that ACBD3 levels varied based on hormone receptor status, indicating that ACBD3 could be a candidate biomarker for poor patient prognosis in breast cancer and may possibly be a biomarker for ER signal reprogramming of precancerous breast tissue

Mendelian randomisation identifies alternative splicing of the FAS death receptor as a mediator of severe COVID-19

Severe COVID-19 is characterised by immunopathology and epithelial injury. Proteomic studies have identified circulating proteins that are biomarkers of severe COVID-19, but cannot distinguish correlation from causation. To address this, we performed Mendelian randomisation (MR) to identify proteins that mediate severe COVID-19. Using protein quantitative trait loci (pQTL) data from the SCALLOP consortium, involving meta-analysis of up to 26,494 individuals, and COVID-19 genome-wide association data from the Host Genetics Initiative, we performed MR for 157 COVID-19 severity protein biomarkers. We identified significant MR results for five proteins: FAS, TNFRSF10A, CCL2, EPHB4 and LGALS9. Further evaluation of these candidates using sensitivity analyses and colocalization testing provided strong evidence to implicate the apoptosis-associated cytokine receptor FAS as a causal mediator of severe COVID-19. This effect was specific to severe disease. Using RNA-seq data from 4,778 individuals, we demonstrate that the pQTL at the FAS locus results from genetically influenced alternate splicing causing skipping of exon 6. We show that the risk allele for very severe COVID-19 increases the proportion of transcripts lacking exon 6, and thereby increases soluble FAS. Soluble FAS acts as a decoy receptor for FAS-ligand, inhibiting apoptosis induced through membrane-bound FAS. In summary, we demonstrate a novel genetic mechanism that contributes to risk of severe of COVID-19, highlighting a pathway that may be a promising therapeutic target