RESUMO
BACKGROUND: Recent experimental studies of neuroinflammation in glaucoma pointed to cFLIP as a molecular switch for cell fate decisions, mainly regulating cell type-specific caspase-8 functions in cell death and inflammation. This study aimed to determine the importance of cFLIP for regulating astroglia-driven neuroinflammation in experimental glaucoma by analyzing the outcomes of astroglia-targeted transgenic deletion of cFLIP or cFLIPL. METHODS: Glaucoma was modeled by anterior chamber microbead injections to induce ocular hypertension in mouse lines with or without conditional deletion of cFLIP or cFLIPL in astroglia. Morphological analysis of astroglia responses assessed quantitative parameters in retinal whole mounts immunolabeled for GFAP and inflammatory molecules or assayed for TUNEL. The molecular analysis included 36-plexed immunoassays of the retina and optic nerve cytokines and chemokines, NanoString-based profiling of inflammation-related gene expression, and Western blot analysis of selected proteins in freshly isolated samples of astroglia. RESULTS: Immunoassays and immunolabeling of retina and optic nerve tissues presented reduced production of various proinflammatory cytokines, including TNFα, in GFAP/cFLIP and GFAP/cFLIPL relative to controls at 12 weeks of ocular hypertension with no detectable alteration in TUNEL. Besides presenting a similar trend of the proinflammatory versus anti-inflammatory molecules displayed by immunoassays, NanoString-based molecular profiling detected downregulated NF-κB/RelA and upregulated RelB expression of astroglia in ocular hypertensive samples of GFAP/cFLIP compared to ocular hypertensive controls. Analysis of protein expression also revealed decreased phospho-RelA and increased phospho-RelB in parallel with an increase in caspase-8 cleavage products. CONCLUSIONS: A prominent response limiting neuroinflammation in ocular hypertensive eyes with cFLIP-deletion in astroglia values the role of cFLIP in the molecular regulation of glia-driven neuroinflammation during glaucomatous neurodegeneration. The molecular responses accompanying the lessening of neurodegenerative inflammation also seem to maintain astroglia survival despite increased caspase-8 cleavage with cFLIP deletion. A transcriptional autoregulatory response, dampening RelA but boosting RelB for selective expression of NF-κB target genes, might reinforce cell survival in cFLIP-deleted astroglia.
Assuntos
Astrócitos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Glaucoma , Doenças Neuroinflamatórias , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Camundongos , Astrócitos/metabolismo , Astrócitos/patologia , Glaucoma/metabolismo , Glaucoma/patologia , Glaucoma/genética , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Camundongos Transgênicos , Modelos Animais de Doenças , Citocinas/metabolismo , Retina/metabolismo , Retina/patologia , Camundongos Endogâmicos C57BL , Nervo Óptico/patologia , Nervo Óptico/metabolismo , Proteína Glial Fibrilar Ácida/metabolismoRESUMO
Apoptosis, or programmed cell death, maintains tissue homeostasis by eliminating damaged or unnecessary cells. However, cells can evade this process, contributing to conditions such as cancer. Escape mechanisms include anoikis, mitochondrial DNA depletion, cellular FLICE inhibitory protein (c-FLIP), endosomal sorting complexes required for transport (ESCRT), mitotic slippage, anastasis, and blebbishield formation. Anoikis, triggered by cell detachment from the extracellular matrix, is pivotal in cancer research due to its role in cellular survival and metastasis. Mitochondrial DNA depletion, associated with cellular dysfunction and diseases such as breast and prostate cancer, links to apoptosis resistance. The c-FLIP protein family, notably CFLAR, regulates cell death processes as a truncated caspase-8 form. The ESCRT complex aids apoptosis evasion by repairing intracellular damage through increased Ca2+ levels. Antimitotic agents induce mitotic arrest in cancer treatment but can lead to mitotic slippage and tetraploid cell formation. Anastasis allows cells to resist apoptosis induced by various triggers. Blebbishield formation suppresses apoptosis indirectly in cancer stem cells by transforming apoptotic cells into blebbishields. In conclusion, the future of apoptosis research offers exciting possibilities for innovative therapeutic approaches, enhanced diagnostic tools, and a deeper understanding of the complex biological processes that govern cell fate. Collaborative efforts across disciplines, including molecular biology, genetics, immunology, and bioinformatics, will be essential to realize these prospects and improve patient outcomes in diverse disease contexts.
Assuntos
Apoptose , Neoplasias , Humanos , Neoplasias/patologia , Neoplasias/genética , Neoplasias/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genéticaRESUMO
Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Caspase 8 , Proteína de Domínio de Morte Associada a Fas , Humanos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Células HEK293 , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de SinaisRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it kills cancer cells while sparing normal cells. However, many cancers, including pancreatic ductal adenocarcinoma (PDAC), exhibit intrinsic or acquired resistance to TRAIL, and the molecular mechanisms underlying TRAIL resistance in cancers, particularly in PDAC, remain unclear. In this study, we demonstrated that glutamine (Gln) endows PDAC cells with resistance to TRAIL through KDM4C-mediated epigenetic regulation of cFLIP. Inhibition of glutaminolysis significantly reduced the cFLIP level, leading to TRAIL-mediated formation of death-inducing signaling complexes. Overexpression of cFLIP dramatically rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Alpha-Ketoglutarate (aKG) supplementation significantly reversed the decrease in the cFLIP level induced by glutaminolysis inhibition and rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Knockdown of glutamic-oxaloacetic transaminase 2, which facilitates the conversion of oxaloacetate and glutamate into aspartate and aKG, decreased aKG production and the cFLIP level and activated TRAIL-induced apoptosis. AKG-mediated epigenetic regulation was necessary for maintaining a high level of cFLIP. Glutaminolysis inhibition increased the abundance of H3K9me3 in the cFLIP promoter, indicating that Gln-derived aKG production is important for Jumonji-domain histone demethylase (JHDM)-mediated cFLIP regulation. The JHDM KDM4C regulated cFLIP expression by binding to its promoter, and KDM4C knockdown sensitized PDAC cells to TRAIL-induced apoptosis. The present findings suggest that Gln-derived aKG production is required for KDM4C-mediated epigenetic regulation of cFLIP, which leads to resistance to TRAIL.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glutamina , Histona Desmetilases com o Domínio Jumonji , Neoplasias Pancreáticas , Ligante Indutor de Apoptose Relacionado a TNF , Humanos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Glutamina/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ácidos Cetoglutáricos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Aspartato Aminotransferase Citoplasmática/metabolismo , Aspartato Aminotransferase Citoplasmática/genética , Animais , Regiões Promotoras GenéticasRESUMO
TRAIL and FasL are potent inducers of apoptosis but can also promote inflammation through assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) complexes, wherein caspase-8 acts as a scaffold to drive FADD/RIPK1-mediated nuclear factor κB (NF-κB) activation. cFLIP is also recruited to FADDosomes and restricts caspase-8 activity and apoptosis, but whether cFLIP also regulates death receptor-initiated inflammation is unclear. Here, we show that silencing or deletion of cFLIP leads to robustly enhanced Fas-, TRAIL-, or TLR3-induced inflammatory cytokine production, which can be uncoupled from the effects of cFLIP on caspase-8 activation and apoptosis. Mechanistically, cFLIPL suppresses Fas- or TRAIL-initiated NF-κB activation through inhibiting the assembly of caspase-8/FADD/RIPK1 FADDosome complexes, due to the low affinity of cFLIPL for FADD. Consequently, increased cFLIPL occupancy of FADDosomes diminishes recruitment of FADD/RIPK1 to caspase-8, thereby suppressing NF-κB activation and inflammatory cytokine production downstream. Thus, cFLIP acts as a dual suppressor of apoptosis and inflammation via distinct modes of action.
Assuntos
Proteínas Reguladoras de Apoptose , NF-kappa B , Humanos , NF-kappa B/metabolismo , Caspase 8/metabolismo , Proteínas Reguladoras de Apoptose/farmacologia , Apoptose , Inflamação , Citocinas/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteína de Domínio de Morte Associada a Fas/metabolismoRESUMO
Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.
Assuntos
Catequina , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamento farmacológico , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Apoptose , Catequina/farmacologia , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Tubeimoside-1 (TBMS-1), a traditional Chinese medicinal herb, is commonly used as an anti-cancer agent. In this study, we aimed to investigate its effect on the sensitization of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Our results revealed that even though monotherapy using TBMS-1 or TRAIL at sublethal concentrations did not affect cancer cell death, combination therapy using TBMS-1 and TRAIL increased apoptotic cell death. Mechanistically, TBMS-1 destabilized c-FLIP expression by downregulating STAMBPL1, a deubiquitinase (DUB). Specifically, when STAMBPL1 and c-FLIP bound together, STAMBPL1 deubiquitylated c-FLIP. Moreover, STAMBPL1 knockdown markedly increased sensitivity to TRAIL by destabilizing c-FLIP. These findings were further confirmed in vivo using a xenograft model based on the observation that combined treatment with TBMS-1 and TRAIL decreased tumor volume and downregulated STAMBPL1 and c-FLIP expression levels. Overall, our study revealed that STAMBPL1 is essential for c-FLIP stabilization, and that STAMBPL1 depletion enhances TRAIL-mediated apoptosis via c-FLIP downregulation.
Assuntos
Apoptose , Ligante Indutor de Apoptose Relacionado a TNF , Humanos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Ligantes , Peptídeo Hidrolases/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , AnimaisRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival, although KSHV encodes a viral homolog of this protein (vFLIP). Cellular and viral FLIP proteins have several functions, including, most importantly, the inhibition of pro-apoptotic caspase 8 and modulation of NF-κB signaling. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, we first performed rescue experiments with human or viral FLIP proteins known to affect FLIP target pathways differently. The long and short isoforms of cFLIP and molluscum contagiosum virus MC159L, which are all strong caspase 8 inhibitors, efficiently rescued the loss of endogenous cFLIP activity in PEL cells. KSHV vFLIP was unable to fully rescue the loss of endogenous cFLIP and is therefore functionally distinct. Next, we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout. Results from these screens and our validation experiments implicate the canonical cFLIP target caspase 8 and TRAIL receptor 1 (TRAIL-R1 or TNFRSF10A) in promoting constitutive death signaling in PEL cells. However, this process was independent of TRAIL receptor 2 or TRAIL, the latter of which is not detectable in PEL cell cultures. The requirement for cFLIP is also overcome by inactivation of the ER/Golgi resident chondroitin sulfate proteoglycan synthesis and UFMylation pathways, Jagunal homolog 1 (JAGN1) or CXCR4. UFMylation and JAGN1, but not chondroitin sulfate proteoglycan synthesis or CXCR4, contribute to TRAIL-R1 expression. In sum, our work shows that cFLIP is required in PEL cells to inhibit ligand-independent TRAIL-R1 cell death signaling downstream of a complex set of ER/Golgi-associated processes that have not previously been implicated in cFLIP or TRAIL-R1 function.
Assuntos
Apoptose , Herpesvirus Humano 8 , Humanos , Apoptose/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular , Herpesvirus Humano 8/metabolismo , Ligantes , Proteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Sulfatos/metabolismoRESUMO
AIMS: Further investigation on the mechanism of action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in NSCLC would shed light on the understanding of TRAIL resistance and provide new clues for the counter-strategy. BACKGROUND: Cellular FLICE-inhibitory protein (c-FLIP) is a critical inhibitor of TRAIL-induced apoptosis. Our previous study suggested that glycogen synthase kinase 3ß (GSK3ß) positively regulated c-FLIP expression in human lung adenocarcinoma cells. Meanwhile, other studies reported that c-FLIP was degraded by HECT-type E3 ligase ITCH (Itchy E3 Ubiquitin Protein Ligase) via the proteasome pathway. OBJECTIVE: We will explore whether ITCH is involved in the expression regulation of c-FLIP positively controlled by GSK3ß during the treatment of TRAIL. METHODS: Human lung adenocarcinoma cells were used to stably overexpress and knockdown GSK3ß. Quantitative real-time PCR (qRT-PCR) assay was used to test the expressional level of mRNA of genes. Western blot analysis was employed to detect the expression of proteins at the protein level. siRNA of ITCH was used to knock down its expression. TRAIL treatment was used to cause apoptosis. RESULTS: In the present study, we have confirmed the degradation of c-FLIP by ITCH protein and the downregulation of ITCH expression by GSK3ß in lung adenocarcinoma cells. Moreover, ITCH silencing reversed the downregulation of c-FLIP protein caused by GSK3ß-knockdown in the cells. Accordingly, TRAIL-induced apoptosis facilitated by GSK3ß knockdown was blocked by the combined interference of ITCH. CONCLUSION: These results suggested that GSK3ß/ITCH axis regulated the stability of c-FLIP and influenced TRAIL-induced apoptosis. Taken together, our study revealed a GSK3ß/ITCH/c-FLIP axis, which counteracts TRAIL-induced apoptosis in human lung adenocarcinoma cells.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Ligantes , Glicogênio Sintase Quinase 3 beta/metabolismo , Linhagem Celular Tumoral , Apoptose , Ubiquitina-Proteína Ligases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
PURPOSE: Endoplasmic reticulum stress (ERS) plays a crucial role in myocardial ischemia-reperfusion injury (MIRI). Cellular FLICE-inhibitory protein (cFLIP) is an essential regulator of apoptosis and plays a major role in regulating ERS. The present study aimed to investigate the effects of long isoform cFLIP (cFLIPL) on endogenous apoptosis and the mechanism of ERS in MIRI. METHODS: The cFLIPL recombinant adenovirus vector was used to infect H9c2 cells and Sprague-Dawley (SD) rats. After infection for 72 h, ischemia was induced for 30 min, and reperfusion was then performed for 2 h to establish the MIRI model in SD rats. H9c2 cells were hypoxic for 4 h and then reoxygenated for 12 h to simulate ischemia/reperfusion (I/R) injury. Model parameters were evaluated by assessing cardiomyocyte viability, cell death (apoptosis), and ERS-related protein expression. In addition, tunicamycin (TM), an ERS agonist, was also added to the medium for pretreatment. Coimmunoprecipitation (Co-IP) of cFLIPL and p38 MAPK protein was performed. RESULTS: cFLIPL expression was decreased in I/R injury and hypoxia/reoxygenation (H/R) injury, and cFLIPL overexpression reduced myocardial infarction in vivo and increased the viability of H9c2 cells in vitro. I/R and H/R upregulated the protein expression of GRP78, IRE-1, and PERK to induce ERS and apoptosis. Interestingly, overexpression of cFLIPL significantly inhibited ERS and subsequent apoptosis, which was reversed by an agonist of ERS. Moreover, Co-IP showed that cFLIPL attenuated ERS and was associated with inhibiting the activation of p38 protein. CONCLUSION: The expression of cFLIPL is significantly downregulated in MIRI, and it is accompanied by excessive ERS and apoptosis. Upregulated cFLIPL suppresses ERS to reduce myocardial apoptosis, which is associated with inhibiting the activity of p38 MAPK. Therefore, cFLIPL may be a potential intervention target for MIRI.
Assuntos
Traumatismo por Reperfusão Miocárdica , Ratos , Animais , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos Sprague-Dawley , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/farmacologia , Estresse do Retículo Endoplasmático , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/farmacologiaRESUMO
Cellular FLICE-like inhibitory protein (c-FLIP) regulates extrinsic apoptosis by controlling procaspase-8 activation through its tandem N-terminal death effector domains (DEDs). Here, we present the expression and purification of c-FLIP tandem DEDs (tDED) from Escherichia coli. We observed that the c-FLIPtDED maintains monomeric form under near-physiological pH condition in vitro. Our results also reveal a significant correlation between the pH conditions and the structure of c-FLIPtDED (F114A). The described methods and results would be helpful for follow-up study on the structural and functional of c-FLIP.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Domínio Efetor de Morte , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , SeguimentosRESUMO
The forkhead box M1 (FoxM1) protein, a transcription factor, plays critical roles in regulating tumor growth and drug resistance, while cellular FLICE-inhibitory protein (c-FLIP), an anti-apoptotic regulator, is involved in the ubiquitin-proteasome pathway. In this study, we investigated the effects of c-FLIP on the expression and ubiquitination levels of FoxM1 along with drug susceptibility in non-small-cell lung cancer (NSCLC) cells. We first showed that the expression levels of FoxM1 and c-FLIP were increased and positively correlated (R2 = 0.1106, P < 0.0001) in 90 NSCLC samples. The survival data from prognostic analysis demonstrated that high expression of c-FLIP and/or FoxM1 was related to poor prognosis in NSCLC patients and that the combination of FoxM1 and c-FLIP could be a more precise prognostic biomarker than either alone. Then, we explored the functions of c-FLIP/FoxM1 in drug resistance in NSCLC cell lines and a xenograft mouse model in vivo. We showed that c-FLIP stabilized FoxM1 by inhibiting its ubiquitination, thus upregulated the expression of FoxM1 at post-transcriptional level. In addition, a positive feedback loop composed of FoxM1, ß-catenin and p65 also participated in c-FLIP-FoxM1 axis. We revealed that c-FLIP promoted the resistance of NSCLC cells to thiostrepton and osimertinib by upregulating FoxM1. Taken together, these results reveal a new mechanism by which c-FLIP regulates FoxM1 and the function of this interaction in the development of thiostrepton and osimertinib resistance. This study provides experimental evidence for the potential therapeutic benefit of targeting the c-FLIP-FoxM1 axis for lung cancer treatment.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Proteína Forkhead Box M1 , Animais , Humanos , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Tioestreptona/farmacologia , Tioestreptona/uso terapêutico , Tioestreptona/metabolismo , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
Hepatocellular carcinoma (HCC) is an aggressive malignancy, and its effective treatment has been hampered by drug resistance. Extracellular vesicle (EV) delivery of TNF-related apoptosis-inducing ligand (TRAIL) (EV-T) was demonstrated to be superior to recombinant TRAIL (rTRAIL) for cancer treatment previously. And AZD5582, a potent antagonist of inhibitors of apoptosis proteins (IAPs) can potentiate apoptosis-based cancer therapies. However, the combination of EV-T and AZD5582 has never been examined for their possible apoptosis inducing synergism in cancers. In this study, we proposed and tested the combination of EV-T and AZD5582 as a potential novel therapy for effective treatment of HCC. Two HCC lines Huh7 and HepG2 that are both resistant to rTRAIL were examined. The results confirmed that AZD5582 and EV-T are synergistic for apoptosis induction in some cancer lines including Huh7 and HepG2 while sparing normal cells. More importantly, this study revealed that TRAIL sensitization by AZD5582 is mediated through the concomitant suppression of anti-apoptotic factors including cFLIP, MCL-1, and IAPs (XIAP, Survivin and cIAP-1). Particularly the downregulation of cFLIP and IAP's appeared to be essential and necessary for the synergism between AZD5582 and TRAIL. In vivo, we first time demonstrated that the combined therapy with low doses of AZD5582 and EV-Ts triggered drastically enhanced apoptosis leading to the complete eradication of Huh7 tumor development without any apparent adverse side effects examined. We thus have unraveled the important molecular mechanism underlying TRAIL sensitization by AZD5582, rationalizing the next development of a combination therapy with AZD5582 and EV-T for HCC treatment. KEY MESSAGES: It confirmed the TRAIL sensitization by AZD5582, a potent antagonist of IAPs in hepatocarcinoma. It revealed that the sensitization is via the concomitant suppression of antiapoptotic factors including cFLIP, MCL-1, and IAPs. The downregulation of cFLIP and IAPs like Survivin appeared to be essential and necessary for the synergism between AZD5582 and nanosomal TRAIL. In vivo the combined therapy with AZD5582 and nanosomal TRAIL led to complete eradication of hepatocarcinoma tumors. This study has rationalized the next development of a combination therapy with AZD5582 and nanosomal TRAIL for cancer treatment.
Assuntos
Carcinoma Hepatocelular , Vesículas Extracelulares , Neoplasias Hepáticas , Alcinos , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Vesículas Extracelulares/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Oligopeptídeos , Survivina/metabolismo , Survivina/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
During viral evolution and adaptation, many viruses have utilized host cellular factors and machinery as their partners. HBx, as a multifunctional viral protein encoded by the hepatitis B virus (HBV), promotes HBV replication and greatly contributes to the development of HBV-associated hepatocellular carcinoma (HCC). HBx interacts with several host factors in order to regulate HBV replication and evolve carcinogenesis. The cellular FADD-like IL-1ß-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) is a major factor that functions in a variety of cellular pathways and specifically in apoptosis. It has been shown that the interaction between HBx and c-FLIP determines HBV fate. In this review, we provide a comprehensive and detailed overview of the interplay between c-FLIP and HBV in various environmental circumstances. We describe strategies adapted by HBV to establish its chronic infection. We also summarize the conventional roles of c-FLIP and highlight the functional outcome of the interaction between c-FLIP and HBV or other viruses in viral replication and the innate immune system.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno , Sistema Imunitário/metabolismo , Replicação Viral , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Carcinoma Hepatocelular/virologia , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/virologia , Proteínas Virais Reguladoras e AcessóriasRESUMO
Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIPL expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIPL knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell's demise in the unfavorable conditions of the tumor microenvironment.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Neoplasias do Colo , Estresse do Retículo Endoplasmático , Apoptose/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Regulação para Baixo/genética , Estresse do Retículo Endoplasmático/genética , Humanos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Microambiente TumoralRESUMO
Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteases Específicas de Ubiquitina , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/biossíntese , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The extrinsic pathway is mediated by death receptors (DRs), including CD95 (APO-1/Fas) or TRAILR-1/2. Defects in apoptosis regulation lead to cancer and other malignancies. The master regulator of the DR networks is the cellular FLICE inhibitory protein (c-FLIP). In addition to its key role in apoptosis, c-FLIP may exert other cellular functions, including control of necroptosis, pyroptosis, nuclear factor κB (NF-κB) activation, and tumorigenesis. To gain further insight into the molecular mechanisms of c-FLIP action in cancer networks, we focus on the structure, isoforms, interactions, and post-translational modifications of c-FLIP. We also discuss various avenues to target c-FLIP in cancer cells for therapeutic benefit.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Neoplasias , Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transdução de Sinais , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models, whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to suppress MEDI3039-induced cell death. High-throughput drug screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics analysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell lines and patient-derived xenografts identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination screening of TRAIL-R2, caspase-8, FADD, and BID knockout models with 60 compounds with varying mechanisms of action identified two inhibitor of apoptosis proteins (IAP) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8:MCL-1 as potential predictive biomarkers for second-generation TRAIL-R2 agonists and loss of key effectors such as FADD and caspase-8 as likely drivers of clinical resistance in solid tumors.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2 , Ligante Indutor de Apoptose Relacionado a TNF , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Linhagem Celular Tumoral , Genômica , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
BACKGROUND: The tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL, an apoptosis-inducing cytokine, has attracted much attention in the treatment of cancer for its selective toxicity to malignant rather than normal cells. However, the apoptosis-inducing ability of TRAIL is weaker than expected primarily due to cancer cell resistance. As one of the dietary flavonoids, kaempferol, has been shown to be antiproliferative and might have a protective effect against TRAIL resistance, particularly for hematologic malignancies. METHODS AND RESULTS: Here, we studied the potential of kaempferol to enhance the TRAIL-induced cytotoxicity and apoptosis in human chronic myelogenous leukemia (CML) cell line K-562, as well as the expression of specific genes with impact on TRAIL signal regulation. Analysis of flowcytometry data showed that treatment with kaempferol did enhance sensitivity of CML cells to pro-apoptotic effects of anti-TRAIL antibody. Although the gene expression levels were heterogeneous, cFLIP, cIAP1 and cIAP2 expression were generally downregulated where co-treatment of kaempferol and TRAIL was employed and these effects appeared to be dose-dependent. We further demonstrated that the expression of death receptors 4 and 5 tended to increase subsequent to the combination treatment. CONCLUSIONS: Consequently, it is reasonable to conclude that sensitization of chronic leukemia cells to TRAIL by kaempferol in vitro should be considered as a way of focusing clinical attention on leukemia therapy.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Quempferóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína 3 com Repetições IAP de Baculovírus/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/genética , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cellular FLICE-like inhibitory protein (cFLIP) is a member of the Death Domain superfamily with pivotal roles in many cellular processes and disease states, including cancer and autoimmune disorders. In the context of the death-inducing signaling complex (DISC), cFLIP isoforms regulate extrinsic apoptosis by controlling procaspase-8 activation. The function of cFLIP is mediated through a series of protein-protein interactions, engaging the two N-terminal death effector domains (DEDs). Here, we solve the structure of an engineered DED1 domain of cFLIP using solution nuclear magnetic resonance (NMR) and we define the interaction with FADD and calmodulin, protein-protein interactions that regulate the function of cFLIP in the DISC. cFLIP DED1 assumes a canonical DED fold characterized by six α helices and is able to bind calmodulin and FADD through two separate interfaces. Our results clearly demonstrate the role of DED1 in the cFLIP/FADD association and contribute to the understanding of the assembly of DISC filaments.