RESUMEN
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.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteínas de Unión al GTP ral/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bencimidazoles/administración & dosificación , Neoplasias Colorrectales/genética , Humanos , Mutación , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Transfección , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/biosíntesis , Proteínas de Unión al GTP ral/genéticaRESUMEN
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.
Asunto(s)
Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Ciclopentanos/farmacología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Humanos , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Proteolisis/efectos de los fármacos , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
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.
Asunto(s)
Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 8/genética , Caspasa 8/metabolismo , Humanos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Núcleo Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Animales , Caspasa 8/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , FN-kappa B/metabolismo , Células PC-3 , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.