29 results on '"Caitriona Holohan"'
Search Results
2. Supplementary Figure 2 from FLIP: A Targetable Mediator of Resistance to Radiation in Non–Small Cell Lung Cancer
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Daniel B. Longley, Kevin M. Prise, Karl T. Butterworth, Gerard G. Hanna, Timothy Harrison, Patrick G. Johnston, Caitriona Holohan, Nyree Crawford, Catherine Higgins, Joanna Majkut, Catherine Fenning, Izabela Stasik, Luke Humphreys, Conor A. Bradley, Zsuzsanna Nemeth, and Kylie A. McLaughlin
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Caspase-8 dependent apoptosis induced by IR
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- 2023
3. Supplementary Figure 3 from FLIP: A Targetable Mediator of Resistance to Radiation in Non–Small Cell Lung Cancer
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Daniel B. Longley, Kevin M. Prise, Karl T. Butterworth, Gerard G. Hanna, Timothy Harrison, Patrick G. Johnston, Caitriona Holohan, Nyree Crawford, Catherine Higgins, Joanna Majkut, Catherine Fenning, Izabela Stasik, Luke Humphreys, Conor A. Bradley, Zsuzsanna Nemeth, and Kylie A. McLaughlin
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IR-induced changes in cell surface expression of death receptors
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- 2023
4. Supplementary Figure 1 from FLIP: A Targetable Mediator of Resistance to Radiation in Non–Small Cell Lung Cancer
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Daniel B. Longley, Kevin M. Prise, Karl T. Butterworth, Gerard G. Hanna, Timothy Harrison, Patrick G. Johnston, Caitriona Holohan, Nyree Crawford, Catherine Higgins, Joanna Majkut, Catherine Fenning, Izabela Stasik, Luke Humphreys, Conor A. Bradley, Zsuzsanna Nemeth, and Kylie A. McLaughlin
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Response of H460 and A549 cells to IR
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- 2023
5. Supplementary Figure 4 from FLIP: A Targetable Mediator of Resistance to Radiation in Non–Small Cell Lung Cancer
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Daniel B. Longley, Kevin M. Prise, Karl T. Butterworth, Gerard G. Hanna, Timothy Harrison, Patrick G. Johnston, Caitriona Holohan, Nyree Crawford, Catherine Higgins, Joanna Majkut, Catherine Fenning, Izabela Stasik, Luke Humphreys, Conor A. Bradley, Zsuzsanna Nemeth, and Kylie A. McLaughlin
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Combination of SAHA with IR in H460 and A549 cells using different treatment schedules
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- 2023
6. The pseudo-caspase FLIP(L) regulates cell fate following p53 activation
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Kirsty M. McLaughlin, Tamas Sessler, Peter Gallagher, Emma M. Kerr, Wendy L. Allen, Alexander J. McIntyre, Gemma M.A. Gregg, Vicky M. Coyle, Gerard P. Quinn, Melissa Labonte-Wilson, Nyree Crawford, Daniel B. Longley, Jamie Z. Roberts, Andrea Lees, Fiammetta Falcone, Mark Wappett, Patrick G. Johnston, Christopher McCann, Katherine McAllister, Caitriona Holohan, Laurence J. Egan, Aideen E. Ryan, Philip D Dunne, and Simon S. McDade
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Pyridines ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Antineoplastic Agents ,Apoptosis ,Cell fate determination ,Models, Biological ,Piperazines ,TNF-Related Apoptosis-Inducing Ligand ,chemistry.chemical_compound ,SDG 3 - Good Health and Well-being ,Cell Line, Tumor ,Puma ,Humans ,Gene silencing ,Protein Kinase Inhibitors ,Caspase ,Caspase 8 ,Multidisciplinary ,biology ,Entinostat ,Cell Cycle ,Imidazoles ,Acetylation ,Drug Synergism ,Proto-Oncogene Proteins c-mdm2 ,Biological Sciences ,biology.organism_classification ,Cell biology ,Receptors, TNF-Related Apoptosis-Inducing Ligand ,Gene Expression Regulation ,chemistry ,Flip ,Benzamides ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 ,Protein Binding - Abstract
p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.
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- 2020
7. Pevonedistat (MLN4924): mechanism of cell death induction and therapeutic potential in colorectal cancer
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Jennifer Ferris, Alex J. McIntyre, Margarita Espona-Fiedler, Victoria Coyle, Daniel B. Longley, Jamie Z. Roberts, Nyree Crawford, Simon S. McDade, Mark Wappett, Claudia Hamilton, and Caitriona Holohan
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0301 basic medicine ,Cancer Research ,Programmed cell death ,Colorectal cancer ,medicine.medical_treatment ,Immunology ,Mitochondrion ,lcsh:RC254-282 ,Article ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Mediator ,Medicine ,lcsh:QH573-671 ,Chemotherapy ,business.industry ,lcsh:Cytology ,Cell Biology ,medicine.disease ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Colon cancer ,030104 developmental biology ,Pevonedistat ,Flip ,Apoptosis ,030220 oncology & carcinogenesis ,Cancer research ,business - Abstract
Pevonedistat (MLN4924), a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit (NAE1), has demonstrated significant therapeutic potential in several malignancies. Although multiple mechanisms-of-action have been identified, how MLN4924 induces cell death and its potential as a combinatorial agent with standard-of-care (SoC) chemotherapy in colorectal cancer (CRC) remains largely undefined. In an effort to understand MLN4924-induced cell death in CRC, we identified p53 as an important mediator of the apoptotic response to MLN4924. We also identified roles for the extrinsic (TRAIL-R2/caspase-8) and intrinsic (BAX/BAK) apoptotic pathways in mediating the apoptotic effects of MLN4924 in CRC cells, as well as a role for BID, which modulates a cross-talk between these pathways. Depletion of the anti-apoptotic protein FLIP, which we identify as a novel mediator of resistance to MLN4924, enhanced apoptosis in a p53-, TRAIL-R2/DR5-, and caspase-8-dependent manner. Notably, TRAIL-R2 was involved in potentiating the apoptotic response to MLN4924 in the absence of FLIP, in a ligand-independent manner. Moreoever, when paired with SoC chemotherapies, MLN4924 demonstrated synergy with the irinotecan metabolite SN38. The cell death induced by MLN4924/SN38 combination was dependent on activation of mitochondria through BAX/BAK, but in a p53-independent manner, an important observation given the high frequency of TP53 mutation(s) in advanced CRC. These results uncover mechanisms of cell death induced by MLN4924 and suggest that this second-generation proteostasis-disrupting agent may have its most widespread activity in CRC, in combination with irinotecan-containing treatment regimens.
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- 2020
8. The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L)
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Joel S. Riley, Hajrah Khawaja, Jennifer Fox, Joanna Majkut, Caitriona Holohan, Luke M Humphreys, Jennifer Ferris, Margarita Espona-Fiedler, Daniel B. Longley, Jamie Z. Roberts, Catherine A. Higgins, Paul N. Moynagh, Nyree Crawford, Emma Evergren, Simon S. McDade, and Tamas Sessler
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0303 health sciences ,biology ,Chemistry ,Cell Biology ,Ubiquitin ligase ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Pevonedistat ,Ubiquitin ,Flip ,030220 oncology & carcinogenesis ,Ubiquitin ligase complex ,embryonic structures ,biology.protein ,SKP2 ,Neddylation ,FADD ,Molecular Biology ,030304 developmental biology - Abstract
TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2’s pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1’s DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1’s recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)’s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.
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- 2020
9. The SCF
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Jamie Z, Roberts, Caitriona, Holohan, Tamas, Sessler, Jennifer, Fox, Nyree, Crawford, Joel S, Riley, Hajrah, Khawaja, Joanna, Majkut, Emma, Evergren, Luke M, Humphreys, Jennifer, Ferris, Catherine, Higgins, Margarita, Espona-Fiedler, Paul, Moynagh, Simon S, McDade, and Daniel B, Longley
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Caspase 8 ,Death Domain Receptor Signaling Adaptor Proteins ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Apoptosis ,Cyclopentanes ,Cullin Proteins ,TNF Receptor-Associated Factor 2 ,Article ,TNF-Related Apoptosis-Inducing Ligand ,Receptors, TNF-Related Apoptosis-Inducing Ligand ,Pyrimidines ,Cell Line, Tumor ,Protein Interaction Mapping ,Proteolysis ,Humans ,S-Phase Kinase-Associated Proteins ,Protein Binding ,Signal Transduction ,Cancer - Abstract
TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2’s pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1’s DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1’s recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)’s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.
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- 2019
10. FLIP(L) determines p53 induced life or death
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Vicky M. Coyle, Simon S. McDade, Laurence J. Egan, Aideen E. Ryan, Katherine McAllister, Phillip D. Dunne, Andrea Lees, Peter Gallagher, Christopher McCann, Gemma M.A. Gregg, Tamas Sessler, Daniel B. Longley, Jamie Z. Roberts, Wendy L. Allen, Nyree Crawford, Emma M. Kerr, Mark Wappett, Patrick G. Johnston, Kirsty M. McLaughlin, Melissa Labonte-Wilson, Alexander J. McIntyre, Caitriona Holohan, Fiammetta Falcone, and Gerard P. Quinn
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0303 health sciences ,Programmed cell death ,Cell cycle checkpoint ,Chemistry ,Regulator ,Cell fate determination ,Cell cycle ,3. Good health ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Flip ,Apoptosis ,030220 oncology & carcinogenesis ,030304 developmental biology - Abstract
How p53 differentially activates cell cycle arrest versus cell death remains poorly understood. Here, we demonstrate that upregulation of canonical pro-apoptotic p53 target genes in colon cancer cells imposes a critical dependence on the long splice form of the caspase-8 regulator FLIP (FLIP(L)), which we identify as a direct p53 transcriptional target. Inhibiting FLIP(L) expression with siRNA or Class-I HDAC inhibitors promotes apoptosis in response to p53 activation by the MDM2 inhibitor Nutlin-3A, which otherwise predominantly induces cell-cycle arrest. When FLIP(L) upregulation is inhibited, apoptosis is induced in response to p53 activation via a novel ligand-independent TRAIL-R2/caspase-8 complex, which, by activating BID, induces mitochondrial-mediated apoptosis. Notably, FLIP(L) depletion inhibits p53-induced expression of the cell cycle regulator p21 and enhances p53-mediated upregulation of PUMA, with the latter activating mitochondrial-mediated apoptosis in FLIP(L)-depleted, Nutlin-3A-treated cells lacking TRAIL-R2/caspase-8. Thus, we report two previously undescribed, novel FLIP(L)-dependent mechanisms that determine cell fate following p53 activation.
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- 2019
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11. Bcl-xL as a poor prognostic biomarker and predictor of response to adjuvant chemotherapy specifically in BRAF-mutant stage II and III colon cancer
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Helen G. Coleman, Alan Gilmore, Stephen McQuaid, Ronan T. Gray, Federica Di Nicolantonio, Philip D Dunne, Sabine Tejpar, Darragh G. McArt, Matthew Alderdice, Patrick G. Johnston, Daniel B. Longley, Mark Lawler, Victoria Bingham, D. Klingbiel, Peter Bankhead, Jacqueline James, Caitriona Holohan, Amy M.B. McCorry, and Maurice B Loughrey
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Oncology ,medicine.medical_specialty ,education.field_of_study ,molecular stratification ,Bcl-xL ,Colorectal cancer ,Proportional hazards model ,business.industry ,Population ,Disease ,relapse risk ,medicine.disease ,Colon cancer ,Gene expression profiling ,Molecular stratification ,Relapse risk ,colon cancer ,Internal medicine ,Genotype ,Gene expression ,Cohort ,medicine ,gene expression profiling ,Immunohistochemistry ,education ,business ,Research Paper - Abstract
BackgroundBRAF mutation occurs in 8-15% of colon cancers (CC), and is associated with poor prognosis in metastatic disease. Compared to wild-type BRAF (BRAFWT) disease, stage II/III CC patients with BRAF mutant (BRAFMT) tumors have shorter overall survival after relapse; however, time-to-relapse is not significantly different. The aim of this investigation was to identify, and validate, novel predictors of relapse of stage II/III BRAFMT CC.Patients and methodsWe used gene expression data from a cohort of 460 patients (GSE39582) to perform a supervised classification analysis based on risk-of-relapse within BRAFMT stage II/III CC, to identify transcriptomic biomarkers associated with prognosis within this genotype. These findings were validated using immunohistochemistry in an independent population-based cohort of Stage II/III CC (n=691), applying Cox proportional hazards analysis to determine associations with survival.ResultsHigh gene expression levels of Bcl-xL, a key regulator of apoptosis, were associated with increased risk of relapse, specifically in BRAFMT tumors (HR=8.3, 95% CI 1.7-41.7), but not KRASMT/BRAFWT or KRASWT/BRAFWT tumors. High Bcl-xL protein expression in BRAFMT, untreated, stage II/III CC was confirmed to be associated with an increased risk of death in an independent cohort (HR=12.13, 95% CI 2.49-59.13). Additionally, BRAFMT tumors with high levels of Bcl-xL protein expression appeared to benefit from adjuvant chemotherapy (P for interaction =0.006), indicating the potential predictive value of Bcl-xL expression in this setting.ConclusionsThese findings provide evidence that Bcl-xL gene and/or protein expression identifies a poor prognostic subgroup of BRAFMT stage II/III CC patients, who may benefit from adjuvant chemotherapy.Key MessageUsing a combination of computational biology discovery and immunohistochemistry validation in independent patient cohorts, we show that high expression of the apoptosis regulator Bcl-xL is associated with disease relapse specifically within BRAF mutant stage II/III colon cancer.This data could enable tailored disease management to reduce relapse rates in the most aggressive subtype.
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- 2018
12. Abstract 2409: FLIP(L) determines colon cancer cell fate following p53 activation
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P.G. Johnston, Vicky M. Coye, Emma M. Kerr, Peter Gallagher, Phillip D. Dunne, Fiammetta Falcone, Caitriona Holohan, Simon S. McDade, Alexander J. McIntyre, Andrea Lees, Laurence J. Egan, Gemma M.A. Gregg, Aideen E. Ryan, Nyree Crawford, Wendy L. Allen, Chris McCann, Thomas Sessler, Katherine McAllister, Daniel B. Longley, Jamie Z. Roberts, Melissa Labonte-Wilson, Gerard P. Quinn, Mark Wappett, and Kirsty M. McLaughlin
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Cancer Research ,Oncology ,Flip ,Chemistry ,Cancer research ,Colon cancer cell - Abstract
Background: How p53 differentially activates cell cycle arrest versus cell death remains poorly understood. Understanding the mechanisms governing the switch from p53-induced cell-fates is important for optimising efficacy of p53-activating therapies, such as DNA damaging chemotherapy and radiotherapy. HDAC inhibitors (HDACi) have potential to enhance p53 induced cell death through enhancing p53 activation and altering regulation of other cell death regulatory proteins. Methods: We utilised a panel of matched p53 wild-type and deficient colorectal cancer cell line models to the potential for HDACi to augment cell death induced by direct and indirect p53 activating agents. A number of molecular (Western blot, RT-PCR), phenotypic (cell death) and functional genomic (RNA-seq, CRISPR, ChIP-seq) analyses were used to investigate the importance of p53 and its downstream transcriptional programs. Results: Here we report that upregulation of canonical pro-apoptotic p53 target genes in colon cancer cells imposes critical dependence on the long splice form of the caspase-8 regulator FLIP (FLIP(L)), which we identify as a direct p53 transcriptional target. Inhibiting FLIP(L) expression with siRNA or Class-I HDAC inhibitors promotes apoptosis in response to p53 activation by the MDM2 inhibitor Nutlin-3A, which otherwise predominantly induces cell-cycle arrest. When FLIP(L) upregulation is inhibited, apoptosis is induced in response to p53 activation via a ligand-independent TRAIL-R2/caspase-8 complex, which is distinct from the ligand-dependent DISC. Notably, FLIP(L) depletion inhibits p53-induced expression of the cell cycle regulator p21 and enhances p53-mediated upregulation of PUMA, with the latter activating mitochondrial-mediated apoptosis in FLIP(L)-depleted, Nutlin-3A-treated cells lacking TRAIL-R2/caspase-8. Conclusion: Acute p53-mediated transcriptional upregulation of FLIP(L) plays an unexpected nodal role in determining cell fate following p53 activation. This is mediated through two previously undescribed mechanisms, preventing apoptosis by a ligand-independent TRAIL-R2 complex and by suppressing expression of pro-apoptotic PUMA. Which, importantly imposes a critical dependence on FLIP(L) which can be overcome through combinations with class-I HDAC inhibitors such as Entinostat. Citation Format: Andrea Lees, Alexander J. McIntyre J. McIntyre, Nyree T. Crawford, Fiammetta Falcone, Chris McCann, Gerard P. Quinn, Jamie Z. Roberts, Thomas Sessler, Peter F. Gallagher, Gemma M. Gregg, Katherine McAllister, Kirsty M. McLaughlin, Wendy L. Allen, Caitriona Holohan, Laurence J. Egan, Aideen E. Ryan, Melissa Labonte-Wilson, Phillip D. Dunne, Mark Wappett, Vicky M. Coye, Patrick G. Johnston, Emma M. Kerr, Daniel B. Longley, Simon S. McDade. FLIP(L) determines colon cancer cell fate following p53 activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2409.
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- 2020
13. Cytoplasmic FLIP(S) and nuclear FLIP(L) mediate resistance of castrate-resistant prostate cancer to apoptosis induced by IAP antagonists
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Christopher, McCann, Nyree, Crawford, Joanna, Majkut, Caitriona, Holohan, Chris W D, Armstrong, Pamela J, Maxwell, Chee Wee, Ong, Melissa J, LaBonte, Simon S, McDade, David J, Waugh, and Daniel B, Longley
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Cell Nucleus ,Male ,Caspase 8 ,Cytoplasm ,Mice, Inbred BALB C ,THP-1 Cells ,Tumor Necrosis Factor-alpha ,CASP8 and FADD-Like Apoptosis Regulating Protein ,NF-kappa B ,Apoptosis ,Mice, SCID ,urologic and male genital diseases ,Histone Deacetylases ,Article ,Inhibitor of Apoptosis Proteins ,Histone Deacetylase Inhibitors ,Mice ,Prostatic Neoplasms, Castration-Resistant ,Drug Resistance, Neoplasm ,Cell Line, Tumor ,PC-3 Cells ,Animals ,Humans - Abstract
Expression of tumor necrosis factor-α (TNFα) in the serum of prostate cancer patients is associated with poorer outcome and progression to castrate-resistant (CRPC) disease. TNFα promotes the activity of NFκB, which regulates a number of anti-apoptotic and proinflammatory genes, including those encoding the inhibitor of apoptosis proteins (IAPs); however, in the presence of IAP antagonists, TNFα can induce cell death. In the presence of recombinant or macrophage-derived TNFα, we found that IAP antagonists triggered degradation of cIAP1 and induced formation of Complex-IIb, consisting of caspase-8, FADD and RIPK1 in CRPC models; however, no, or modest levels of apoptosis were induced. This resistance was found to be mediated by both the long (L) and short (S) splice forms of the caspase-8 inhibitor, FLIP, another NFκB-regulated protein frequently overexpressed in CRPC. By decreasing FLIP expression at the post-transcriptional level in PC3 and DU145 cells (but not VCaP), the Class-I histone deacetylase (HDAC) inhibitor Entinostat promoted IAP antagonist-induced cell death in these models in a manner dependent on RIPK1, FADD and Caspase-8. Of note, Entinostat primarily targeted the nuclear rather than cytoplasmic pool of FLIP(L). While the cytoplasmic pool of FLIP(L) was highly stable, the nuclear pool was more labile and regulated by the Class-I HDAC target Ku70, which we have previously shown regulates FLIP stability. The efficacy of IAP antagonist (TL32711) and Entinostat combination and their effects on cIAP1 and FLIP respectively were confirmed in vivo, highlighting the therapeutic potential for targeting IAPs and FLIP in proinflammatory CRPC.
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- 2018
14. Cancer drug resistance: an evolving paradigm
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Caitriona Holohan, Daniel B. Longley, Sandra Van Schaeybroeck, and Patrick G. Johnston
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Drug ,Screening techniques ,Epithelial-Mesenchymal Transition ,DNA Repair ,General Mathematics ,media_common.quotation_subject ,Cancer drugs ,Drug target ,Antineoplastic Agents ,Apoptosis ,Drug resistance ,Pharmacology ,Bioinformatics ,Models, Biological ,Autophagy ,Tumor Microenvironment ,Humans ,Medicine ,Gene Silencing ,Molecular Targeted Therapy ,Biotransformation ,Predictive biomarker ,media_common ,business.industry ,Applied Mathematics ,Biological Transport ,Adaptation, Physiological ,Neoplasm Proteins ,Gene Expression Regulation, Neoplastic ,Drug Resistance, Neoplasm ,Drug Design ,Mutation ,Neoplastic Stem Cells ,Molecular targets ,ATP-Binding Cassette Transporters ,Apoptosis Regulatory Proteins ,business ,Patient stratification ,DNA Damage - Abstract
Resistance to chemotherapy and molecularly targeted therapies is a major problem facing current cancer research. The mechanisms of resistance to 'classical' cytotoxic chemotherapeutics and to therapies that are designed to be selective for specific molecular targets share many features, such as alterations in the drug target, activation of prosurvival pathways and ineffective induction of cell death. With the increasing arsenal of anticancer agents, improving preclinical models and the advent of powerful high-throughput screening techniques, there are now unprecedented opportunities to understand and overcome drug resistance through the clinical assessment of rational therapeutic drug combinations and the use of predictive biomarkers to enable patient stratification.
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- 2013
15. FLIP: A Targetable Mediator of Resistance to Radiation in Non-Small Cell Lung Cancer
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Zsuzsanna Németh, Izabela Stasik, Catherine Fenning, Kevin M. Prise, Conor A Bradley, Kylie A. McLaughlin, Gerard G Hanna, Joanna Majkut, Catherine A. Higgins, Caitriona Holohan, Nyree Crawford, Luke M Humphreys, Patrick G. Johnston, Karl T. Butterworth, Tim Harrison, and Daniel B. Longley
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0301 basic medicine ,Cancer Research ,Programmed cell death ,Lung Neoplasms ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Antineoplastic Agents ,Apoptosis ,Radiation Tolerance ,03 medical and health sciences ,chemistry.chemical_compound ,SDG 3 - Good Health and Well-being ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,medicine ,Humans ,FADD ,RNA, Small Interfering ,Vorinostat ,biology ,Entinostat ,B131 ,A100 ,3. Good health ,Cell biology ,Gene Expression Regulation, Neoplastic ,Histone Deacetylase Inhibitors ,030104 developmental biology ,Oncology ,chemistry ,Flip ,Cancer cell ,biology.protein ,RNA Interference ,Signal transduction ,Signal Transduction ,medicine.drug - Abstract
Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non–small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor FLIP is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were downregulated by RNAi, IR-induced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the antitumor activity of IR in vivo. Therefore, FLIP downregulation induced by HDAC inhibitors is a potential clinical strategy to radiosensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR. Mol Cancer Ther; 15(10); 2432–41. ©2016 AACR.
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- 2016
16. Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis
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David Haigh, Miriam Sgobba, Kirsty McLaughlin, Rosemary O'Connor, Emma M. Kerr, Cathy Fenning, Joel S. Riley, P.G. Johnston, Izabela Stasik, Daniel B. Longley, Joanna Majkut, Keara Redmond, Caitriona Holohan, Sharron Dolan, Patrick A. Kiely, M. Crudden, Kirsty M. McLaughlin, and S. Van Schaeybroeck
- Subjects
CASP8 and FADD-Like Apoptosis Regulating Protein ,Down-Regulation ,Apoptosis ,Biology ,Histone Deacetylase 6 ,Hydroxamic Acids ,Transfection ,Caspase 8 ,Histone Deacetylases ,Mice ,Ubiquitin ,medicine ,Animals ,Humans ,Amino Acid Sequence ,RNA, Small Interfering ,Ku Autoantigen ,Molecular Biology ,Vorinostat ,Original Paper ,Mice, Inbred BALB C ,Acetylation ,Antigens, Nuclear ,Cell Biology ,HDAC6 ,HCT116 Cells ,DNA-Binding Proteins ,Histone Deacetylase Inhibitors ,Proteasome ,Flip ,Cancer research ,biology.protein ,Female ,Histone deacetylase ,HT29 Cells ,Protein Processing, Post-Translational ,medicine.drug - Abstract
FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.
- Published
- 2012
17. Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing
- Author
-
Timothy R. Wilson, Dean A. Fennell, Kelly M. Redmond, Nyree Crawford, Patrick G. Johnston, Kirsty McLaughlin, Daniel B. Longley, Kathy Gately, Caitriona Holohan, Kenneth J. O'Byrne, and C. Le-Clorrenec
- Subjects
Programmed cell death ,Lung Neoplasms ,Transplantation, Heterologous ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Mice, Nude ,Antineoplastic Agents ,Apoptosis ,Biology ,Caspase 8 ,TNF-Related Apoptosis-Inducing Ligand ,Mice ,RNA interference ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Gene silencing ,RNA, Small Interfering ,Molecular Biology ,Cisplatin ,Enzyme Precursors ,Cell growth ,Cell Biology ,Flow Cytometry ,respiratory tract diseases ,Receptors, TNF-Related Apoptosis-Inducing Ligand ,Flip ,Cancer research ,Female ,RNA Interference ,BH3 Interacting Domain Death Agonist Protein ,medicine.drug - Abstract
We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.
- Published
- 2009
18. Cytokine-induced β-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL
- Author
-
Thomas Ritter, Timothy O'Brien, Caitriona Holohan, Afshin Samali, and Eva Szegezdi
- Subjects
bcl-x-l ,endoplasmic-reticulum stress ,Programmed cell death ,Necrosis ,Bcl-XL ,nitric-oxide synthase ,kappa-b ,bcl-X Protein ,Nitric Oxide Synthase Type II ,Apoptosis ,Images in Cellular / Molecular Medicine ,Bcl-xL ,Nitric Oxide ,outer-membrane ,Models, Biological ,cytochrome-c release ,Adenoviridae ,Nitric oxide ,Islets of Langerhans ,chemistry.chemical_compound ,death ,Cell Line, Tumor ,medicine ,Animals ,induction ,Caspase ,omi/htra2 protease ,Membrane Potential, Mitochondrial ,biology ,caspase activation ,Cytochrome c ,Cell Biology ,cytokines ,Mitochondria ,Rats ,Cell biology ,Enzyme Activation ,iNOS ,Nitric oxide synthase ,Type 1 diabetes ,Microscopy, Fluorescence ,caspases ,chemistry ,biology.protein ,Molecular Medicine ,Insulinoma ,medicine.symptom ,diabetes-mellitus - Abstract
Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1 beta (IL-1 beta), interferon-gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1 beta and IFN gamma (IL-1 beta/IFN gamma)-induced apoptotic cell death. TNF alpha neither induced beta-cell death nor did it potentiate the effects of IL-1 beta, IFN gamma or IL-1 beta/IFN gamma . The cytotoxic effect of IL-1 beta/IFN gamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1 beta/IFN gamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1 beta/IFN gamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -3. Transduction of cells with Ad-Bcl-X-L blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X-L as a potential target to combat beta-cell apoptosis.
- Published
- 2008
19. DED or alive: assembly and regulation of the death effector domain complexes
- Author
-
A Malik, Joel S. Riley, Daniel B. Longley, and Caitriona Holohan
- Subjects
Models, Molecular ,Cancer Research ,Programmed cell death ,Fas-Associated Death Domain Protein ,Necroptosis ,Immunology ,Antineoplastic Agents ,Apoptosis ,Review ,Q1 ,Mice ,Cellular and Molecular Neuroscience ,SDG 3 - Good Health and Well-being ,Neoplasms ,Autophagy ,Animals ,Humans ,Protein Interaction Domains and Motifs ,FADD ,Adaptor Proteins, Signal Transducing ,Caspase 8 ,Clinical Trials as Topic ,biology ,Effector ,Cell Biology ,Acquired immune system ,Cell biology ,Gene Expression Regulation, Neoplastic ,biology.protein ,Death effector domain ,Signal transduction ,Protein Binding ,Signal Transduction - Abstract
Death effector domains (DEDs) are protein–protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.
- Published
- 2015
20. Differential affinity of FLIP and procaspase 8 for FADD’s DED binding surfaces regulates DISC assembly
- Author
-
Daniel B. Longley, M. Sgobba, Keara Redmond, Joel S. Riley, Joanna Majkut, Caitriona Holohan, Dean A. Fennell, Shozeb Haider, David Haigh, Patrick G. Johnston, S. Van Schaeybroeck, Andrew Logan, Izabela Stasik, Nyree Crawford, Emma M. Kerr, and Catherine A. Higgins
- Subjects
Immunoprecipitation ,FLIP ,Fas-Associated Death Domain Protein ,C130 ,Blotting, Western ,CASP8 and FADD-Like Apoptosis Regulating Protein ,General Physics and Astronomy ,Plasma protein binding ,Caspase 8 ,General Biochemistry, Genetics and Molecular Biology ,Article ,Humans ,FADD ,DR5 ,Receptor ,Multidisciplinary ,biology ,C760 ,apoptosis ,General Chemistry ,DISC ,HCT116 Cells ,C700 ,Cell biology ,Blot ,Flip ,biology.protein ,Chromatography, Gel ,Death effector domain ,Protein Binding - Abstract
Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.
- Published
- 2014
21. Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer
- Author
-
Joel S. Riley, Ryan Hutchinson, Nyree Crawford, Manuel Salto-Tellez, Caitriona Holohan, Kenneth J. O'Byrne, Darragh G. McArt, Patrick G. Johnston, Ian M. Paul, Elaine W. Kay, Daniel B. Longley, Kathy Gately, Robert Cummins, Dean A. Fennell, Peter W. Hamilton, S. Van Schaeybroeck, and Izabela Stasik
- Subjects
Cancer Research ,C131 ,Cell Survival ,FLIP ,Blotting, Western ,Immunology ,Cell ,CASP8 and FADD-Like Apoptosis Regulating Protein ,TRAIL ,In Vitro Techniques ,Biology ,Caspase 8 ,Q1 ,caspase-8 ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,SDG 3 - Good Health and Well-being ,HDAC inhibitor ,Carcinoma, Non-Small-Cell Lung ,medicine ,Humans ,Lung cancer ,non-small cell lung cancer ,Retrospective Studies ,Cisplatin ,Entinostat ,B131 ,B132 ,A100 ,Cell Biology ,Flow Cytometry ,medicine.disease ,Molecular biology ,respiratory tract diseases ,medicine.anatomical_structure ,chemistry ,Flip ,Cell culture ,Apoptosis ,Cancer research ,Original Article ,medicine.drug - Abstract
Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.
- Published
- 2013
22. Prognostic and therapeutic relevance of c-FLIP in acute myeloid leukaemia
- Author
-
Patrick G. Johnston, Daniel B. Longley, Jodie Hay, Mary Frances McMullin, Ken I. Mills, Caitriona Holohan, Donal P. McLornan, Kirsty McLaughlin, Robert Kerrin Hills, and Alan Kenneth Burnett
- Subjects
Adult ,Male ,Adolescent ,medicine.drug_class ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Down-Regulation ,Apoptosis ,Kaplan-Meier Estimate ,Biology ,Histone Deacetylase 6 ,CFLAR Gene ,Histone Deacetylases ,Leukemia, Myelomonocytic, Acute ,CFLAR ,TNF-Related Apoptosis-Inducing Ligand ,Young Adult ,Downregulation and upregulation ,Cell Line, Tumor ,medicine ,Humans ,RNA, Messenger ,RNA, Neoplasm ,RNA, Small Interfering ,Vorinostat ,Aged ,Gene Expression Regulation, Leukemic ,Histone deacetylase inhibitor ,Hematology ,Middle Aged ,Prognosis ,Molecular biology ,Recombinant Proteins ,Neoplasm Proteins ,Histone Deacetylase Inhibitors ,Alternative Splicing ,Leukemia, Myeloid, Acute ,Drug Resistance, Neoplasm ,Female ,RNA Interference ,Histone deacetylase ,medicine.drug ,K562 cells - Abstract
Chemoresistance is a major contributor to the aggressiveness of AML and is often due to insufficient apoptosis. The CFLAR gene is expressed as long and short splice forms encoding the anti-apoptotic proteins c-FLIP(L) and c-FLIP(S) (CFLAR(L) and CFLAR(S) , respectively) that play important roles in drug resistance. In univariate analyses of CFLAR mRNA expression in adult AML patients, those individuals with higher than median mRNA expression of the long splice form CFLAR(L) (but not the short splice form) had significantly lower 3 year overall survival (P = 0·04) compared to those with low expression. In cell line studies, simultaneous down-regulation of c-FLIP(L) and c-FLIP(S) proteins using siRNA induced apoptosis in U937 and NB-4 AML cells, but not K562 or OCI-AML3 cells. However, dual c-FLIP(L/S) downregulation sensitized all four cell lines to apoptosis induced by recombinant tumour necrosis factor-related apoptosis-inducing ligand (rTRAIL). Moreover, specific downregulation of c-FLIP(L) was found to recapitulate the phenotypic effects of dual c-FLIP(L/S) downregulation. The histone deacetylase (HDAC)1/2/3/6 inhibitor Vorinostat was found to potently down-regulate c-FLIP(L) expression by transcriptional and post-transcriptional mechanisms and to sensitize AML cells to rTRAIL. Further analyses using more selective HDAC inhibitors revealed that HDAC6 inhibition was not required for c-FLIP(L) down-regulation. These results suggest that c-FLIP(L) may have clinical relevance both as a prognostic biomarker and potential therapeutic target for HDAC inhibitors in AML although this requires further study.
- Published
- 2012
23. Cytokine-Induced β-Cell Stress and Death in Type 1 Diabetes Mellitus
- Author
-
Afshin Samali, Lisa Vincenz, Timothy O'Brien, Richard Jäger, Caitriona Holohan, and Eva Szegezdi
- Subjects
0303 health sciences ,medicine.medical_specialty ,Type 1 diabetes ,business.industry ,medicine.medical_treatment ,030209 endocrinology & metabolism ,medicine.disease ,03 medical and health sciences ,Cell stress ,0302 clinical medicine ,Cytokine ,Endocrinology ,Internal medicine ,medicine ,business ,030304 developmental biology - Published
- 2011
24. Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent
- Author
-
Caitriona Holohan, Steven G. Gray, Izabela Stasik, Jane L. Hurwitz, V. Courtney Broaddus, Daniel B. Longley, Sara Busacca, Kenneth J. O'Byrne, Emma M. Kerr, Kelly M. Redmond, Dario Barbone, Dean A. Fennell, Patrick G. Johnston, and Kirsty M. McLaughlin
- Subjects
Male ,Mesothelioma ,Cancer Research ,Programmed cell death ,Pleural Neoplasms ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Apoptosis ,Caspase 8 ,Hydroxamic Acids ,Cell Line, Tumor ,Spheroids, Cellular ,medicine ,Humans ,Clonogenic assay ,Vorinostat ,Caspase ,Cisplatin ,biology ,Molecular biology ,Caspase Inhibitors ,DNA-Binding Proteins ,Histone Deacetylase Inhibitors ,DNA Repair Enzymes ,Oncology ,Flip ,Cancer research ,biology.protein ,RNA Interference ,Apoptosis Regulatory Proteins ,medicine.drug - Abstract
Introduction Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease.
- Published
- 2011
25. SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma
- Author
-
Nyree Crawford, David Waugh, Daniel B. Longley, George Ward, Izabela Stasik, Gianni Chessari, Patrick G. Johnston, Dean A. Fennell, Joanna Majkut, Caitriona Holohan, and Mike McGrath
- Subjects
SMAC mimetics ,Mesothelioma ,Cancer Research ,C131 ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Poly (ADP-Ribose) Polymerase-1 ,Hydroxamic Acids ,Inhibitor of Apoptosis Proteins ,HDAC inhibitors ,B290 ,Caspase 8 ,Vorinostat ,Caspase 3 ,B131 ,Histone deacetylase inhibitor ,Drug Synergism ,mesothelioma ,Receptor-Interacting Protein Serine-Threonine Kinases ,Original Article ,Poly(ADP-ribose) Polymerases ,medicine.drug ,Programmed cell death ,FLIP ,medicine.drug_class ,Pleural Neoplasms ,Immunology ,Down-Regulation ,Antineoplastic Agents ,Biology ,Cellular and Molecular Neuroscience ,SDG 3 - Good Health and Well-being ,Cell Line, Tumor ,medicine ,Humans ,Tumor microenvironment ,Molecular Mimicry ,A100 ,Cell Biology ,SAHA/Vorinostat ,Enzyme Activation ,Histone Deacetylase Inhibitors ,Drug Resistance, Neoplasm ,Apoptosis ,Flip ,Cancer research ,Cisplatin ,Drug Screening Assays, Antitumor - Abstract
Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFa) signaling for their activity. As circulating TNFa expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFa. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFa promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.
- Published
- 2013
26. 160 Ku70 Acetylation as a Determinant of Chemosensitivity in Colorectal Cancer
- Author
-
P.G. Johnston, Caitriona Holohan, and Daniel B. Longley
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,Ku70 ,business.industry ,Colorectal cancer ,medicine.disease ,Acetylation ,Internal medicine ,Cancer research ,medicine ,business ,A determinant - Published
- 2012
27. 80 Rational Targeting of Inhibitor of Apoptosis Proteins (IAPs) for Effective Therapy of Malignant Pleural Mesothelioma
- Author
-
David Waugh, Nyree Crawford, Caitriona Holohan, and Daniel B. Longley
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,Pleural mesothelioma ,business.industry ,Internal medicine ,medicine ,business ,Inhibitor of apoptosis - Published
- 2012
28. 93 Mapping the Interaction Between FLIP and FADD
- Author
-
David Haigh, Daniel B. Longley, M. Sgobba, Caitriona Holohan, and J. Majkut
- Subjects
Cancer Research ,biology ,Chemistry ,Mutant ,Mutagenesis (molecular biology technique) ,Signal transducing adaptor protein ,urologic and male genital diseases ,Cell biology ,Blot ,Oncology ,Flip ,Death-inducing signaling complex ,biology.protein ,FADD ,biological phenomena, cell phenomena, and immunity ,Site-directed mutagenesis - Abstract
Introduction: cFLIP is a major anti-apoptotic protein that blocks the apoptotic pathway mediated by death receptors. It is overexpressed in many cancers resulting in chemoresistance and limiting the effectiveness of commonly used anticancer therapies. Both the long (FLIPL) and the short (FLIPS) splice forms compete with procaspase 8 for binding with the adaptor protein FADD at the death inducing signaling complex (DISC), which is formed after stimulation of death receptors by their ligands. Materials and Methods:Molecular modeling of the FLIP–FADD interaction was performed using published structures viral FLIP (MC159) and human FADD. Site directed mutagenesis of was performed to study the importance of specific residues of FLIP. Protein–protein interactions were studied by GST-pulldown assay. DISC recruitment of FLIP mutants was investigated in a DISC IP assay. Western blotting was performed to assess protein levels. Results: Modelling identified several key residues which appeared to be important for the cFLIP–FADD interaction. Site directed mutagenesis was performed against these key residues, and a panel of cFLIP and FADD mutants was generated. These mutants were then screened for their ability to interact with either FADD or cFLIP using GST-pulldown assays. A functional assay for DISC recruitment was also carried out to confirm which of these mutations resulted in impaired recruitment. This analysis established the critical amino acids which mediate DED interactions between cFLIP and FADD. Conclusions: Iterative rounds of computer modelling and mutagenesis have characterised the molecular features of the interaction between the pro-apoptotic adaptor molecule FADD and the anti-apoptotic protein FLIP.
- Published
- 2012
29. Corrigendum to 'Characterization of a serine protease-mediated cell death program activated in human leukemia cells' [Exp. Cell Res. 312 (2006) 27–39]
- Author
-
Caitriona Holohan, Alicia Torriglia, B.W. Lee, A.R. O'Connell, and C. Stenson-Cox
- Subjects
Serine protease ,Programmed cell death ,Human leukemia ,medicine.anatomical_structure ,biology ,Cell ,biology.protein ,medicine ,Cell Biology ,Molecular biology ,Cell biology - Published
- 2006
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