Browsing Cancer Biology at UCC by Issue Date
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- ItemIn silico-aided design, build and test of synthetic proteins(University College Cork, 2019-12-20) Yallapragada, V. V. B.; Tangney, MarkSince the discovery of proteins in 1838, the field of protein engineering and our understanding of proteins have improved exponentially. Synthetic proteins have found applications in various biomedical, food and material-based settings. This rise in synthetic proteins was complemented with the parallel expansion in the availability of in silico tools for protein modelling. The complexity in the composition and design of synthetic proteins requires careful in silico validation to screen for potential pitfalls in the design. In silico tools for protein modelling and design have been used extensively to computationally validate the structure and functioning of the synthetic proteins prior to wet-lab testing. In this thesis, the workflow of design-model-build-test of synthetic proteins with novel applications in imaging is described. The in silico-aided design, screening and the in vitro testing of synthetic proteins targeting S. aureus surface antigen Clumping factor A are discussed in Chapter 2. In this chapter, a suitable candidate worthy of examining in a future in vivo setting was identified. During the in silico-aided screening, the complexity of data obtained from various in silico tools posed new challenges. This was termed as ‘the in silico myriad problem’. In Chapter 3, a mathematical strategy (Function2Form bridge) was tested to address the in silico myriad problem, by combining the scores of different design parameters pertaining to the synthetic protein being analysed into a single easily interpreted output describing overall performance. The strategy comprises 1. A mathematical strategy combining data from a myriad of in silico tools into an Overall Performance-score (a singular score informing on a user-defined overall performance); 2. The F2F-Plot, a graphical means of informing the wet-lab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F bridge was implemented during the design process of all the synthetic proteins in Chapter 4 and Chapter 5. The synthetic protein design strategy used in Chapter 2 was implemented to design synthetic proteins targeting cancer cells, and to assess their potential as in vivo imaging agents in Chapter 4. For both MUC1 and ClfA targeted proteins, in vivo luminescence imaging studies involving systemic intravenous administration of proteins, validated synthetic protein specific accumulation at target cell locations within mice as evidenced by localised luminescence. Dose response studies indicated that luminescence output was both target cell and administered protein quantity related. In Chapter 5, a self-assembling protein ‘cage’ was designed, built and tested in vitro. An accompanying novel fluorescence-based protein-protein interaction reporting strategy was introduced, involving incorporation of cysteine residues at the interaction interface of monomeric proteins of the self-assembling protein cage. In silico tools were used to ensure the conformational and functional stability. FlAsH EDT2 (fluorescin arsenical hairpin binder-ethanedithiol) mediated fluorescence was used to confirm the self-assembly. This demonstrates the level of accuracy and detail that can be incorporated into synthetic protein design using in silico tools. In Chapter 6, the scope of introducing miniaturised optical devices to aid biological experimentation was explored. A novel handheld device for monitoring continuous bacterial growth, with prospects of measuring biofluorescence was developed. The device was tested using different bacterial strains and showed accuracy levels similar to a standard benchtop spectrophotometer. This thesis demonstrates the use of computational methods and various in silico tools for protein design. Modern day biomedical science demands novel concepts with deployable technology to assist their translation into user-based settings. In this thesis, various interdisciplinary concepts have been applied to deliver on a holistic end-goal.
- ItemRegulation of insulin-like growth factor receptor activity and its therapeutic targeting in cancer(University College Cork, 2020-04-08) Rieger, Leonie; O'Connor, Rosemary; Science Foundation IrelandInsulin-like Growth Factor-1 Receptor signalling is essential for cell growth, and also promotes migration, survival and cellular transformation, thus, linking it to cancer progression. IGF-1R activity and signalling in cancer cells is regulated by the C terminal tail of the receptor. In particular the Tyr1250/1251, as their mutation to phenylalanine (FF) profoundly impairs IGF-1 signalling and the crosstalk between Integrin and IGF 1R signalling that facilitates a migratory phenotype in cancer cells. This thesis investigated the mechanisms of adhesion-modulated IGF-1R signalling, the function of Tyr1250/1251, and whether these mechanisms might influence the efficacy of IGF-1R-targeted drugs. Following the identification of FER kinase as a mediator of sensitivity to IGF 1R kinase inhibition, we found that ectopic expression of FER enhanced IGF-1R and SHC/MAPK pathway activation in an IGF-1R kinase independent manner. FER also specifically phosphorylated the IGF-1R on Tyr1250/1251. FER activity was cell adhesion-dependent and FER expression correlated with a migratory cancer phenotype. FER inhibition profoundly decreased IGF-1R and SHC signalling and reduced cell migration in breast cancer cells. The mechanisms and consequences of IGF-1R phosphorylation on Tyr1250/1251 phosphorylation were investigated for their contribution to IGF-1R function. It was established that IGF-1R, FER and FAK kinase activity may all contribute to Tyr1250/1251 phosphorylation and that cell adhesion is required for this phosphorylation. The consequence of Tyr1250/1251 phosphorylation was further investigated using a phosphomimetic Y1250E/Y1251E (EE) and the non-phosphorylatable FF IGF-1R mutant. These studies determined that IGF 1R phosphorylation on Tyr1250/1251 IGF-1R enhanced IGF-1R internalisation and proteasomal degradation. Moreover, the WT and EE receptors became rapidly internalised in response to IGF-1 ligation and accumulated in the Golgi apparatus, while the FF mutant remained at the plasma membrane or sites of cell adhesion. Importantly, Golgi associated IGF 1R signalling correlated with a migratory cancer cell phenotype, and disruption of the Golgi apparatus impaired IGF-1-promoted SHC phosphorylation and cell migration. In migratory cells, the formation of new focal adhesion points caused a transient release of the IGF-1R from the Golgi-apparatus to the membrane and lower IGF-1R Tyr1250/1251 phosphorylation. Thus, phosphorylation on Tyr1250/1251 enables IGF-1R signalling from the Golgi apparatus, and Golgi-derived IGF-1R signalling can support an aggressive cancer phenotype. Overall, this study identifies FER kinase and Tyr1250/1251 phosphorylation as adhesion-dependent mechanisms that modulate IGF-1R signalling and localisation in migratory cancer cells. The fact that the IGF-1R may be activated by other kinases and is predominantly located in the Golgi in migratory cells may explain the poor clinical efficacy of monoclonal antibodies and kinase inhibitors that specifically target the surface IGF-1R.
- ItemEvaluation of selective γ-secretase inhibitors as novel modulators of TNF-α-mediated apoptosis(University College Cork, 2021-05-04) O'Brien, Ciara; McCarthy, Justin V.; Science Foundation IrelandThe presenilin proteins (presenilin 1 and presenilin 2) were identified in mutagenesis screens causing the early onset forms of familial Alzheimer’s disease (FAD) in 1995. Subsequently characterized as the catalytic subunits of the γ-secretase protease complexes, the presenilins are responsible for the cleavage of the amyloid precursor protein (APP) and generation of amyloid beta (Aβ). To date, γ-secretase proteases have over 120 substrates which demonstrate contribution to a diverse range of cellular processes and signalling events. However, recent findings have revealed several γ-secretase-independent presenilin functions, including calcium signalling, autophagy and apoptosis. We and others have previously reported members of the TNF receptor super-family as substrates for γ-secretase proteolysis, and that presenilin-deficient cells have increased resistance to TNFα-induced apoptosis. In this study, we sought to determine whether loss of presenilin expression or loss of γ-secretase protease activity is associated with increased resistance to TNFα-induced apoptosis. Utilizing the cleavage of caspase 3 and Poly ADP ribose polymerase (PARP) in target cells as a readout, we tested the anti-apoptotic characteristics of a panel of well characterized γ-secretase inhibitors. In this study, we show that loss of presenilin expression is associated with increased resistance to TNFα-induced apoptosis, and that loss of γ-secretase protease activity does not affect sensitivity to TNFα-induced apoptosis. These observations suggest a γ-secretase-independent role of presenilins in the regulation of TNFα-induced apoptosis.
- ItemTargeting transcription-regulating cyclin dependent kinase 12 for the treatment of breast cancer(University College Cork, 2022-11-27) Burns, Martha; Krajewska, MalgorzataCyclin-dependent Kinase 12 (CDK12) with its binding partner Cyclin K regulates transcription through phosphorylation of RNA Polymerase II (RNA Pol II) at serine 2. Previous work showed that inhibition of CDK12 leads to decreased expression of DNA damage response (DDR) genes and sensitizes cancer cells to DNA damage-inducing agents. However, the exact mechanism by which this is achieved remains unclear. This study aimed to investigate CDK12 as a therapeutic target in breast cancer and to identify new drug combinations involving the inhibition of CDK12 and clinically relevant inhibitors of DNA repair. We demonstrated that CDK12 inhibitor SR-4835 is cytotoxic in MCF-7 breast cancer cells at a low nanomolar concentration. The observed toxicity was associated with G2-M cell cycle arrest, increased apoptosis, and DNA damage. We showed that CDK12 inhibition had a minor effect on the phosphorylation of RNA Pol II at serine 2 indicating that global transcription was not affected. Interestingly, we observed that CDK12 inhibition resulted in decreased expression of chromodomain helicase DNA binding protein 2 (CHD2), a potentially new target of CDK12. Next, we tested the combination of CDK12 inhibition with inhibitors of DNA repair including inhibitors of PARP (olaparib) and CHK1 (AZD7762) using a colony formation assay. The preliminary results indicate that combining CDK12 and CHK1 inhibition will likely have greater therapeutic potential than the combination of CDK12 and PARP inhibition. Future studies are required to establish the exact role of CDK12 in transcription and DDR as well as to investigate further the potential of combining CDK12 and CHK1 inhibition in breast cancer.
- ItemIGF-1 signalling controls mitochondrial morphology and basal mitophagy in cancer(University College Cork, 2023-01-06) Murray, Joss; O'Connor, Rosemary; Science Foundation IrelandInsulin-Like Growth Factor 1 (IGF-1) signalling is known to support oncogenic transformation and the promotion of cancer development. A growing body of evidence has outlined the protective effect IGF-1 signalling has on the mitochondria, however this has been relatively underexplored in cancer. Therefore, this thesis aims to elucidate the mechanisms by which IGF-1 promotes mitochondrial protection in cancer. Previously, we determined that the mitophagy receptor BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is induced by IGF-1 to support mitochondrial turnover and protection. Here, we analysed a publicly available gene expression dataset of breast cancer cells stimulated by IGF-1. Gene ontology classification revealed a signature of genes induced and repressed by IGF-1 involved with mitochondrial functions. Further analysis to classify genes by biological process suggested that genes involved with apoptosis and suppression of mitochondrial metabolism were most enriched in the gene groups regulated by IGF-1. To further interrogate mitochondrial dynamics downstream of IGF-1 signalling, we assessed mitochondrial morphology. Lack of IGF-1R promoted mitochondrial fusion, while IGF-1 stimulation promoted mitochondrial fragmentation. Mitochondrial fragmentation was associated with increased mitochondrial transport to the leading edge of invasive breast cancer cells. Pharmacological inhibition of mitochondrial fission inhibited the migration of cells expressing the IGF-1R but was ineffective at moderating migration of cells lacking the IGF-1R. Finally, we interrogated the function of BNIP3 downstream of IGF-1 stimulation. While BNIP3 is induced, IGF-1 stimulation suppressed mitophagy. However, BNIP3 turnover was higher in basal cell culture conditions than in nutrient deprived conditions, suggesting that BNIP3-mediates basal mitophagy in cancer cells. Indeed, IGF-1R knockout reduced the basal turnover of BNIP3 implying that IGF-1 regulates basal mitophagy via BNIP3. In totality, this thesis presents evidence that IGF-1 signalling promotes mitochondrial protection by regulating genes involved with redox homeostasis while tempering mitochondrial metabolism. Mitochondrial fragmentation is induced by IGF-1 and can also regulate cancer cell migration, while also supporting basal mitophagy mediated by BNIP3. These findings demonstrate that targeting IGF-1 signalling in cancer could impair mitochondrial protection mechanisms, which offers an avenue for novel therapeutic opportunities.