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1.
Cell Death Differ ; 29(2): 272-284, 2022 02.
Article in English | MEDLINE | ID: mdl-34912054

ABSTRACT

Cell death pathways have evolved to maintain tissue homoeostasis and eliminate potentially harmful cells from within an organism, such as cells with damaged DNA that could lead to cancer. Apoptosis, known to eliminate cells in a predominantly non-inflammatory manner, is controlled by two main branches, the intrinsic and extrinsic apoptotic pathways. While the intrinsic pathway is regulated by the Bcl-2 family members, the extrinsic pathway is controlled by the Death receptors, members of the tumour necrosis factor (TNF) receptor superfamily. Death receptors can also activate a pro-inflammatory type of cell death, necroptosis, when Caspase-8 is inhibited. Apoptotic pathways are known to be tightly regulated by post-translational modifications, especially by ubiquitination. This review discusses research on ubiquitination-mediated regulation of apoptotic signalling. Additionally, the emerging importance of ubiquitination in regulating necroptosis is discussed.


Subject(s)
Apoptosis , Necroptosis , Apoptosis/genetics , DNA Damage/genetics , Receptors, Death Domain/genetics , Receptors, Death Domain/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction/genetics , Ubiquitination/genetics , Ubiquitination/physiology
2.
Cell Death Dis ; 11(10): 930, 2020 10 29.
Article in English | MEDLINE | ID: mdl-33122623

ABSTRACT

RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.


Subject(s)
Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , GTP Phosphohydrolases/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , ral GTP-Binding Proteins/metabolism , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzimidazoles/administration & dosage , Colorectal Neoplasms/genetics , Humans , Mutation , Proto-Oncogene Proteins p21(ras)/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Recombinant Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand/administration & dosage , Transfection , ral GTP-Binding Proteins/antagonists & inhibitors , ral GTP-Binding Proteins/biosynthesis , ral GTP-Binding Proteins/genetics
3.
Proc Natl Acad Sci U S A ; 117(30): 17808-17819, 2020 07 28.
Article in English | MEDLINE | ID: mdl-32661168

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.


Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Acetylation , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Benzamides/pharmacology , Caspase 8/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Drug Synergism , Gene Expression Regulation , Humans , Imidazoles/metabolism , Models, Biological , Piperazines/metabolism , Protein Binding , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-mdm2/metabolism , Pyridines/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Suppressor Protein p53/genetics
4.
Cell Death Discov ; 6: 61, 2020.
Article in English | MEDLINE | ID: mdl-32714568

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.

5.
Cell Death Differ ; 27(9): 2726-2741, 2020 09.
Article in English | MEDLINE | ID: mdl-32313199

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.


Subject(s)
Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Apoptosis/drug effects , Caspase 8/metabolism , Cell Line, Tumor , Cullin Proteins/metabolism , Cyclopentanes/pharmacology , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Humans , Protein Binding/drug effects , Protein Interaction Mapping , Proteolysis/drug effects , Pyrimidines/pharmacology , Signal Transduction/drug effects , TNF Receptor-Associated Factor 2/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology
6.
EMBO Rep ; 21(3): e49254, 2020 03 04.
Article in English | MEDLINE | ID: mdl-32009295

ABSTRACT

The long FLIP splice form FLIP(L) can act as both an inhibitor and promoter of caspase-8 at death-inducing signalling complexes (DISCs) formed by death receptors such as TRAIL-R2 and related intracellular complexes such as the ripoptosome. Herein, we describe a revised DISC assembly model that explains how FLIP(L) can have these opposite effects by defining the stoichiometry (with respect to caspase-8) at which it converts from being anti- to pro-apoptotic at the DISC. We also show that in the complete absence of FLIP(L), procaspase-8 activation at the TRAIL-R2 DISC has significantly slower kinetics, although ultimately the extent of apoptosis is significantly greater. This revised model of DISC assembly also explains why FLIP's recruitment to the TRAIL-R2 DISC is impaired in the absence of caspase-8 despite showing that it can interact with the DISC adaptor protein FADD and why the short FLIP splice form FLIP(S) is the more potent inhibitor of DISC-mediated apoptosis.


Subject(s)
Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein , Apoptosis/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Caspase 8/genetics , Caspase 8/metabolism , Humans , Receptors, TNF-Related Apoptosis-Inducing Ligand , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand/genetics
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