Biochemistry and Cell Biology - Masters by Research Theses

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Novel RNA-based biomarkers for ovarian cancer – uncovering how LINC01132 is regulated by the tumour suppressor protein, p53
(University College Cork, 2023) Hartigan, Shaun; Dean, Kellie; McKenna, Sharon L.; Irish Research Council
Ovarian cancer is one of the deadliest female cancers worldwide. Most cases are detected in advanced stages, as it is difficult to diagnose ovarian cancer early due to nonspecific symptoms. Presently, there are no sensitive biomarkers to identify early-stage disease. Over 60% of ovarian cancer cases have a mutation in p53, a tumour suppressor transcription factor which counteracts cell stress and oncogenic signals. Most mutations occur within p53’s DNA-binding domain, a vital region which facilitates its anchorage to target gene promoters. Previous data found numerous long-non-coding RNAs (lncRNAs) are differentially expressed in ovarian cancer cells with mutant TP53, compared to those with the wildtype gene. Here, we show that three p53 mutants (R175H, I195T and R248Q) commonly found in ovarian cancer patients, were unable to activate firefly luciferase expression from a synthetic p53-responsive promoter, reflecting the impact of p53 core domain mutations on its ability to bind target promoters and transactivate gene expression. In silico genome-wide ChIP-seq analysis of differentially expressed lncRNAs identified three (MEG3, LINC01132, and LINC2960) containing regions within -1 kb of their transcription start sites, showing interaction with p53. Another four (EMX20S, PRICKLE2-DT, LINC00887 and LINC02610 contained p53-interacting regions within -5 kb. Division of the region up to -6,995 bp of the LINC01132 transcription start site into a distal, middle, and proximal segment, and subsequent cloning upstream of firefly luciferase, allowed us to assess p53 activity at the LINC01132 promoter. Western blot analysis could not detect luciferase expression from either segment under wildtype p53 overexpression, despite the proximal segment containing six p53-interacting sites. To determine the true response of wildtype and mutant p53 binding to the LINC01132 promoter, future quantitative reverse-transcriptase PCR and luciferase assays should be conducted, given their higher sensitivity compared to Western blot analysis. To improve patient prognosis in ovarian cancer, there is vital necessity to discover specific biomarkers, which can diagnose and monitor disease progression. LncRNAs can be detected in blood, so linking expression of differentially expressed lncRNAs to the mutational status of p53 in ovarian cancer, and studying how these change with the therapies, is novel, previously unexplored, and may aid in biomarker discovery.
Interrogating regulated intramembrane proteolysis of IGF-1R, and its role in cell migration
(University College Cork, 2023) O'Donoghue, Jordan Christopher; McCarthy, Justin V.; O'Connor, Rosemary; University College Cork
The IGF-1R has long been implicated in numerous malignant characteristics of cancers including enhanced proliferation, insensitivity to apoptosis, chemoresistance, increased migratory and invasive capacity, epithelial-to-mesenchymal transition, and metabolic augmentation. IGF-1R, much like other receptor tyrosine kinases, has been characterised as a substrate for gamma-secretase-mediated regulated intramembrane proteolysis (RIP). This proteolytic pathway begins with ectodomain shedding, mediated by sheddases, which produces a soluble fragment in the extracellular space. Following ectodomain shedding, a membrane anchored C-terminal fragment (CTF) is produced which is then cleaved by gamma-secretase at the internal juxtamembrane region to produce a soluble intracellular domain (ICD). Classically, RIP was thought to be a means by which cells would terminate receptor signalling, however contemporary research indicates a more nuanced role. It is now understood that the fragments generated through RIP can retain signalling of the full-form receptors and, in some instances, may acquire novel functionality. Although the RIP of IGF-1R has been outlined, the underlying dynamics of this pathway and the consequences of such remain to be elucidated. With this in mind, we endeavoured to identify the regulatory mechanisms underlying IGF-1R RIP and subsequently aimed to identify potential functions of the fragments produced. Firstly, we generated a GFP-tagged K1003 IGF-1R point mutant, termed a kinase dead mutant, which lacks the ability to catalyse trans-autophosphorylation of the IGF-1R kinase domains thus preventing downstream signal transduction. Trans-autophosphorylation is a critical step in the activation of receptor tyrosine kinases during which the kinase domain of one intracellular domain catalyses the phosphorylation of tyrosine residues within the other intracellular domain. With this tool, we validated the cleavage of IGF-1R and the production of its CTF via transient transfection of HEK293T cells with plasmids expressing either wild-type or K1003R kinase dead mutant forms of IGF-1R C-terminally tagged with GFP. We subsequently identified receptor kinase activation as a potential driver of IGF-1R RIP and determined that ligand stimulation significantly catalyses the accumulation of IGF-1R CTF. This was accomplished via transient transfection of HEK293T cells with plasmids expressing the aforementioned IGF-1R forms. Following this, we elucidated clathrin-mediated endocytosis as a probable regulatory pre-requisite step for IGF-1R RIP by gamma-secretase. Furthermore, our data indicates that ectodomain shedding of IGF-1R is likely catalysed in a metalloprotease dependent fashion, consistent with other receptor tyrosine kinases. We also confirmed that the CTF can undergo nuclear translocation in a clathrin-mediated endocytosis dependent manner, consistent with the translocation of the holoreceptor. Utilising wound healing assays, conducted in Hela cells, our data indicates that gamma-secretase inhibition by DAPT (a small molecule gamma-secretase inhibitor) alone does not antagonise IGF-1-induced cell migration. Similarly, the administration of Pitstop alone does not impinge upon IGF-1 induced cell migration. Interestingly, the administration of batimistat (a broad spectrum metalloprotease inhibitor) significantly suppresses cell migration in the presence of IGF-1. Collectively, our data provides a framework for the construction of the IGF-1R RIP cascade. Following ligand-binding it appears that IGF-1R undergoes metalloprotease-dependent ectodomain shedding leading to loss of the extracellular alpha-chains. The CTF, which remains in the plasma membrane, then undergoes clathrin-mediated endocytosis and packaging into early endosomes. Following internalisation, the CTF is cleaved by gamma-secretase to generate a soluble ICD. Our data also demonstrates that IGF-1R CTF undergoes nuclear translocation in a clathrin-mediated endocytosis dependent fashion. Lastly, data gathered from our wound healing assays indicates that IGF-1R ectodomain shedding is likely a required event for the efficient IGF-1-induced migration of cells, possibly through the proteolytic augmentation of focal adhesion complexes containing IGF-1R.
Investigating the granule forming properties of SMAUG1 and the consequence of interaction with the 14-3-3 protein family
(University College Cork, 2023-03-01) Fehilly, John Denis; Dean, Kellie
SMAUG1 is an intrinsically disordered RNA binding protein that forms granules in cells containing RNA. SMAUG1 has been linked to several diseases such as Alzheimers, muscular dystrophy, and cancer. SMAUG1 represents a potentially distinct class of RNA granules and thus is a very interesting target for study. This work furthers our understanding of the SMAUG1 protein by showing that it undergoes rapid recovery following photobleaching indicative of liquid-liquid phase separated granules. Here we also report partial colocalization of SMAUG1 with P-body component Enhancer Of MRNA Decapping 4 (EDC4) and stress granule component Ras GTPase-activating protein-binding protein 1 (G3BP1). Previous work from the Dean lab identified interaction between the 14-3-3 family of proteins and SMAUG1. Here this interaction is validated via co-immunoprecipitation western blot analysis. Further to this potential 14-3-3 binding motifs within SMAUG were mutated and shown to be functional for 14-3-3 binding. Finally, the consequences of 14-3-3 interaction with SMAUG1 granules were assessed. 14-3-3 binding mutant form of SMAUG1 showed a higher propensity for granule formation within cells. Mutant forms of SMAUG1 also showed impaired recovery following photobleaching. This taken together suggests that the 14-3-3 family proteins are negative regulators of SMAUG1 granule formation.
Investigation of mechanisms underpinning cytokine-induced cell death of colon cancer cells
(University College Cork, 2023) Linehan, Eva; Nally, Ken; Lee, Ciaran
Inflammatory cytokines induce regulated cell death as part of a robust immune response. Many unique forms of regulated cell death have been described that are critically dependent on specific signal transduction molecules or cellular events. Often, molecules or events canonically associated with cell death signalling pathways, while contributing factors, are not essential for cell death. Given the extensive emerging crosstalk and plasticity between modes of cell death, determining dependency is crucial for categorising cell death and for downstream clinical applications. IFN-γ and TNF-α are inflammatory, Th1-type cytokines elevated in immune-mediated inflammatory diseases and infectious diseases such as COVID-19, that synergistically induce cell death in diverse cell types. Recently IFN-γ-induced JAK/STAT signalling has emerged as an important pathway for triggering cell death, but the downstream mechanisms underpinning JAK/STAT-mediated cell death are incompletely understood. Here, IFN-γ+TNF-α-induced synergistic cell death was confirmed to be dependent on JAK1, JAK2, STAT1, and partially dependent on CASP8, using knockout colon cancer cell lines. IFN-γ+TNF-α-treated cells displayed biochemical hallmarks of multiple cell death pathways. Use of JAK1/2 inhibitors highlighted the unique kinetics of IFN-γ+TNF-α-induced cell death, suggesting that an accumulation of intracellular event/s, reaching a threshold or point of no return, may be responsible for cell death. The inconsistent effects of p300/CBP inhibitors on IFN-γ+TNF-α-mediated cell death suggest that STAT1-dependent transcription is not the main effector of cell death downstream of JAK1 and JAK2. The STAT1-dependency observed may just be due to its effects on JAK2 expression in cytokine-treated cells over time. Flow cytometry assays were used to investigate the kinetics of several biomarkers of mitochondrial and cellular stress in IFN-γ+TNF-α-treated cells, compared to cells undergoing canonical cell death pathways. Loss of mitochondrial integrity coincided with the crucial turning point observed in JAK1/2 inhibitor chase experiments, followed by severe increase in superoxide levels. Though these events cannot be confirmed to be the main drivers of IFN-γ+TNF-α-induced cell death, they are hallmarks of death and may be contributing factors. IFN-γ+TNF-α-induced cell death occurs by a non-canonical and redundant cell death mechanism that is dependent on JAK activity and potentially involves induction of components of multiple cell death pathways.
Generation of a CRISPR-Cas9 mediated knock-in reporter for the GRIA3 candidate gene for schizophrenia
(University College Cork, 2022-09-27) Breen, Lisa; McCarthy, Tommie V.
Glutamatergic neurotransmission impairment is considered a major feature of the neurobiology of Schizophrenia (SZ) and implicates genes in this pathway as potential candidates for the condition. A study on α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor genes found strong evidence of association between the Glutamate Ionotropic Receptor AMPA Type Subunit 3 (GRIA3) gene and SZ. Similarly, a recent report has identified a number of genes, including GRIA3, with ultra-rare disabling variants that promote SZ. The association of a rare disabling GRIA3 variant with SZ indicates that reduced expression of the gene predisposes people to SZ and suggests that increasing the expression GRIA3 could be a potential therapeutic avenue for treatment of the condition. The aim of this thesis was to establish a cell model enabling rapid analysis of GRIA3 expression. Such a model would be of high value and in addition to facilitating expression studies on GRIA3, would enable screening for new drugs that increase GRIA3 expression which could have therapeutic potential. This project aimed to modify the cell line using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR associated protein 9 (Cas9) homology directed repair (HDR) approach so that a donor reporter vector containing the Gaussia secreted luciferase gene and linked green fluorescent protein (GFP) or neomycin resistance gene (Neo) gene would be integrated directly under the control of the endogenous promoter of the GRIA3 gene, in a manner that retains intact expression of the GRIA3 protein. This donor reporter vector was successfully constructed and has significant general use as it facilitates cloning of any pair of homology arms and the insertion of a reporter cassette into any target gene via CRISPR-Cas9 HDR. Flanking GRIA3 homology arms were inserted 5’ and 3’ of the reporter cassette for CRISPR-Cas9 HDR mediated insertion into the GRIA3 locus in human U87 glioblastoma cells. Luciferase activity was monitored post-transfection and was present at low levels suggesting successful HDR events. However, the presence of the donor cassette could not be demonstrated at the GRIA3 locus. It was not possible to distinguish if the luciferase activity resulted from read through of the donor plasmid or if a low number of targeted integration events had occurred. Further work involving isolation of individual clones of the targeted U87 cells and checking for the presence of the donor at the GRIA3 locus will be necessary to resolve this question. Overall, this reporter system should be of high value for targeting other loci and can be improved further by modifications to ensure luciferase is only active when inserted into the targeted locus.

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