RESUMEN
Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.
Asunto(s)
Apoptosis , Señalización del Calcio , Retículo Endoplásmico/metabolismo , Proteína Ligando Fas/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Línea Celular , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Femenino , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Patients with high-risk neuroblastoma (NBL) tumors have a high mortality rate. Consequently, there is an urgent need for the development of new treatments for this condition. Targeting death receptor signaling has been proposed as an alternative to standard chemo- and radio-therapies in various tumors. In NBL, this therapeutic strategy has been largely disregarded, possibly because ~50-70% of all human NBLs are characterized by caspase-8 silencing. However, the expression of caspase-8 is detected in a significant group of NBL patients, and they could therefore benefit from treatments that induce cell death through death receptor activation. Given that cytokines, such as TNFα, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNFα and FasL in NBL. METHODS: For the purpose of the study we used a set of eight NBL cell lines. Here we explore the cell death induced by TNFα, FasL, cisplatin, and etoposide, or a combination thereof by Hoechst staining and calcein viability assay. Further assessment of the signaling pathways involved was performed by caspase activity assays and Western blot experiments. Characterization of Fas expression levels was achieved by qRT-PCR, cell surface biotinylation assays, and cytometry. RESULTS: We have found that TNFα is able to increase FasL-induced cell death by a mechanism that involves the NF-κB-mediated induction of the Fas receptor. Moreover, TNFα sensitized NBL cells to DNA-damaging agents (i.e. cisplatin and etoposide) that induce the expression of FasL. Priming to FasL-, cisplatin-, and etoposide-induced cell death could only be achieved in NBLs that display TNFα-induced upregulation of Fas. Further analysis denotes that the high degree of heterogeneity between NBLs is also manifested in Fas expression and modulation thereof by TNFα. CONCLUSIONS: In summary, our findings reveal that TNFα sensitizes NBL cells to FasL-induced cell death by NF-κB-mediated upregulation of Fas and unveil a new mechanism through which TNFα enhances the efficacy of currently used NBL treatments, cisplatin and etoposide.
Asunto(s)
Resistencia a Antineoplásicos , Proteína Ligando Fas/farmacología , Regulación Neoplásica de la Expresión Génica , FN-kappa B/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/genética , Caspasa 8/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Activación Enzimática/efectos de los fármacos , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Transcripción GenéticaRESUMEN
Polypyrimidine tract binding protein (PTB) regulates pre-mRNA splicing, having special relevance for determining gene expression in the differentiating muscle. We have previously shown that PTB protein abundance is progressively reduced during heart development without reduction of its own transcript. Simultaneous reduction of histone deacetylase (HDAC) expression prompted us to investigate the potential link between these events. HDAC5-deficient mice have reduced cardiac PTB protein abundance, and HDAC inhibition in myocytes causes a reduction in endogenous expression of cellular FLICE-like inhibitory protein (cFLIP) and caspase-dependent cleavage of PTB. In agreement with this, cardiac PTB expression is abnormally high in mice with cardiac-specific executioner caspase deficiency, and cFLIP overexpression prevents PTB cleavage in vitro. Caspase-dependent cleavage triggers further fragmentation of PTB, and these fragments accumulate in the presence of proteasome inhibitors. Experimental modification of the above processes in vivo and in vitro results in coherent changes in the alternative splicing of genes encoding tropomyosin-1 (TPM1), tropomyosin-2 (TPM2) and myocyte enhancer factor-2 (MEF2). Thus, we report a pathway connecting HDAC, cFLIP and caspases regulating the progressive disappearance of PTB, which enables the expression of the adult variants of proteins involved in the regulation of contraction and transcription during cardiac muscle development.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasas/metabolismo , Histona Desacetilasas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Animales , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Caspasas/genética , Electroforesis en Gel de Poliacrilamida , Histona Desacetilasas/genética , Ratones , Ratones Noqueados , Miocardio/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tropomiosina/genética , Tropomiosina/metabolismoRESUMEN
The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/farmacología , Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fármacos Neuroprotectores , Ubiquitinación/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Ligada a X/fisiología , Receptor fas/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Citocromos c/metabolismo , Femenino , Inmunoprecipitación , Proteínas Inhibidoras de la Apoptosis/genética , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Mutagénesis Sitio-Dirigida , Células PC12 , Plásmidos/genética , Unión Proteica , Conformación Proteica , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína X Asociada a bcl-2/metabolismo , Receptor fas/genéticaRESUMEN
TNFα can promote either cell survival or cell death. The activation of NF-κB plays a central role in cell survival while its inhibition makes TNFα-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-κB (super-repressor (SR)-IκBα) sensitizes HeLa cells towards TNFα-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFα-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-κB. Then, the activation of the NF-κB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFα-induced cell death, meaning that TNFα is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IκBα-overexpressing cells. This indicates that TNFα-induced apoptosis in SR-IκBα-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFα-induced apoptosis after Bcl-xL knockdown despite that NF-κB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFα-triggered NF-κB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-κB-dependent highly chemoresistant proliferative malignancies.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2 , Factor de Necrosis Tumoral alfa/metabolismo , Proteína bcl-X , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Células HeLa , Humanos , Proteínas I-kappa B/farmacología , Mitocondrias , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.
Asunto(s)
Melanoma , Animales , Línea Celular , Humanos , Melanoma/patología , Ratones , Metástasis de la NeoplasiaRESUMEN
Melanoma is a highly prevalent cancer with an increasing incidence worldwide and high metastatic potential. Brain metastasis is a major complication of the disease, as more than 50% of metastatic melanoma patients eventually develop intracranial disease. MicroRNAs (miRNAs) have been found to play an important role in the tumorigenicity of different cancers and have potential as markers of disease outcome. Identification of relevant miRNAs has generally stemmed from miRNA profiling studies of cells or tissues, but these approaches may have missed miRNAs with relevant functions that are expressed in subfractions of cancer cells. We performed an unbiased in vivo screen to identify miRNAs with potential functions as metastasis suppressors using a lentiviral library of miRNA decoys. Notably, we found that a significant fraction of melanomas that metastasized to the brain carried a decoy for miR-124a, a miRNA that is highly expressed in the brain/neurons. Additional loss- and gain-of-function in vivo validation studies confirmed miR-124a as a suppressor of melanoma metastasis and particularly of brain metastasis. miR-124a overexpression did not inhibit tumor growth in vivo, underscoring that miR-124a specifically controls processes required for melanoma metastatic growth, such as seeding and growth post-extravasation. Finally, we provide proof of principle of this miRNA as a promising therapeutic agent by showing its ability to impair metastatic growth of melanoma cells seeded in distal organs. Our efforts shed light on miR-124a as an antimetastatic agent, which could be leveraged therapeutically to impair metastatic growth and improve patient survival.
RESUMEN
The contribution of epigenetic dysregulation to metastasis remains understudied. Through a meta-analysis of gene expression datasets followed by a mini-screen, we identified Plant Homeodomain Finger protein 8 (PHF8), a histone demethylase of the Jumonji C protein family, as a previously unidentified prometastatic gene in melanoma. Loss- and gain-of-function approaches demonstrate that PHF8 promotes cell invasion without affecting proliferation in vitro and increases dissemination but not subcutaneous tumor growth in vivo, thus supporting its specific contribution to the acquisition of metastatic potential. PHF8 requires its histone demethylase activity to enhance melanoma cell invasion. Transcriptomic and epigenomic analyses revealed that PHF8 orchestrates a molecular program that directly controls the TGFß signaling pathway and, as a consequence, melanoma invasion and metastasis. Our findings bring a mechanistic understanding of epigenetic regulation of metastatic fitness in cancer, which may pave the way for improved therapeutic interventions.
Asunto(s)
Histona Demetilasas , Melanoma , Proliferación Celular , Epigénesis Genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Melanoma/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.
Asunto(s)
Encéfalo/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuritas/fisiología , Neurogénesis/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Muerte Celular/fisiología , Diferenciación Celular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Neuronas Motoras/fisiología , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso , Neuroglía/metabolismo , Células PC12 , Ratas , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptores de Factores de Crecimiento , Receptores de Factor de Crecimiento Nervioso/metabolismo , Ganglio Cervical Superior/embriología , Ganglio Cervical Superior/crecimiento & desarrollo , Ganglio Cervical Superior/fisiologíaRESUMEN
Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Calpaína/metabolismo , Necrosis/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Alquilantes/farmacología , Alquilación/efectos de los fármacos , Animales , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Metilnitronitrosoguanidina/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Transporte de Proteínas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
The long isoform of Fas apoptosis inhibitory molecule (FAIM-L) is a neuron-specific death receptor antagonist that modulates apoptotic cell death and mechanisms of neuronal plasticity. FAIM-L exerts its antiapoptotic action by binding to X-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases, which are the main effectors of apoptosis. XIAP levels are regulated by the ubiquitin-proteasome pathway. FAIM-L interaction with XIAP prevents the ubiquitination and degradation of the latter, thereby allowing it to inhibit caspase activation. This interaction also modulates non-apoptotic functions of caspases, such as the endocytosis of AMPA receptor (AMPAR) in hippocampal long-term depression (LTD). The molecular mechanism of action exerted by FAIM-L is unclear since the consensus binding motifs are still unknown. Here, we performed a two-hybrid screening to discover novel FAIM-L-interacting proteins. We found a functional interaction of SIVA-1 with FAIM-L. SIVA-1 is a proapoptotic protein that has the capacity to interact with XIAP. We describe how SIVA-1 regulates FAIM-L function by disrupting the interaction of FAIM-L with XIAP, thereby promoting XIAP ubiquitination, caspase-3 activation and neuronal death. Furthermore, we report that SIVA-1 plays a role in receptor internalization in synapses. SIVA-1 is upregulated upon chemical LTD induction, and it modulates AMPAR internalization via non-apoptotic activation of caspases. In summary, our findings uncover SIVA-1 as new functional partner of FAIM-L and demonstrate its role as a regulator of caspase activity in synaptic function.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas Inhibidoras de la Apoptosis/metabolismo , Plasticidad Neuronal , Animales , Proteínas Reguladoras de la Apoptosis/genética , Caspasa 3/metabolismo , Células Cultivadas , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ratones , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células PC12 , Unión Proteica , Ratas , Receptores AMPA/metabolismo , UbiquitinaciónRESUMEN
Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover important tumor biology and/or yield promising therapeutic insights. Here, we investigated the role of circular RNAs (circRNA) in metastasis, using melanoma as a model aggressive tumor. We identified silencing of cerebellar degeneration-related 1 antisense (CDR1as), a regulator of miR-7, as a hallmark of melanoma progression. CDR1as depletion results from epigenetic silencing of LINC00632, its originating long non-coding RNA (lncRNA) and promotes invasion in vitro and metastasis in vivo through a miR-7-independent, IGF2BP3-mediated mechanism. Moreover, CDR1as levels reflect cellular states associated with distinct therapeutic responses. Our study reveals functional, prognostic, and predictive roles for CDR1as and expose circRNAs as key players in metastasis.
Asunto(s)
Autoantígenos/genética , Epigénesis Genética , Silenciador del Gen , Melanoma/patología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Humanos , MicroARNs/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Pronóstico , ARN sin Sentido/genética , ARN Circular/genética , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.
Asunto(s)
Carcinogénesis/patología , Melanocitos/patología , Melanoma/patología , Células Madre Neoplásicas/patología , Animales , Carcinogénesis/metabolismo , Transformación Celular Neoplásica/patología , Dermis/patología , Modelos Animales de Enfermedad , Epidermis/patología , Homeostasis , Humanos , Melanocitos/metabolismo , Ratones , Mutación/genética , Células Madre Neoplásicas/metabolismo , Fenotipo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Microambiente Tumoral , Vía de Señalización WntRESUMEN
Death receptors (DRs) and their ligands are expressed in developing nervous system. However, neurons are generally resistant to death induction through DRs and rather their activation promotes neuronal outgrowth and branching. These results suppose the existence of DRs antagonists expressed in the nervous system. Fas apoptosis inhibitory molecule (FAIM(S)) was first identified as a Fas antagonist in B-cells. Soon after, a longer alternative spliced isoform with unknown function was identified and named FAIM(L). FAIM(S) is widely expressed, including the nervous system, and we have shown previously that it promotes neuronal differentiation but it is not an anti-apoptotic molecule in this system. Here, we demonstrate that FAIM(L) is expressed specifically in neurons, and its expression is regulated during the development. Expression could be induced by NGF through the extracellular regulated kinase pathway in PC12 (pheochromocytoma cell line) cells. Contrary to FAIM(S), FAIM(L) does not increase the neurite outgrowth induced by neurotrophins and does not interfere with nuclear factor kappaB pathway activation as FAIM(S) does. Cells overexpressing FAIM(L) are resistant to apoptotic cell death induced by DRs such as Fas or tumor necrosis factor R1. Reduction of endogenous expression by small interfering RNA shows that endogenous FAIM(L) protects primary neurons from DR-induced cell death. The detailed analysis of this antagonism shows that FAIM(L) can bind to Fas receptor and prevent the activation of the initiator caspase-8 induced by Fas. In conclusion, our results indicate that FAIM(L) could be responsible for maintaining initiator caspases inactive after receptor engagement protecting neurons from the cytotoxic action of death ligands.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/fisiología , Proteínas Inhibidoras de la Apoptosis/fisiología , Neuronas/metabolismo , Receptores de Muerte Celular/antagonistas & inhibidores , Receptores de Muerte Celular/fisiología , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/fisiología , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Variación Genética/fisiología , Humanos , Ratones , Neuronas/patología , Células PC12 , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Ratas , Receptores de Muerte Celular/genéticaRESUMEN
Fas Apoptosis Inhibitory Molecule (FAIM) is an evolutionarily highly conserved death receptor antagonist, widely expressed and known to participate in physiological and pathological processes. Two FAIM transcript variants have been characterized to date, namely FAIM short (FAIM-S) and FAIM long (FAIM-L). FAIM-S is ubiquitously expressed and serves as an anti-apoptotic protein in the immune system. Furthermore, in neurons, this isoform promotes NGF-induced neurite outgrowth through NF-кB and ERK signaling. In contrast FAIM-L is found only in neurons, where it exerts anti-apoptotic activity against several stimuli. In addition to these two variants, in silico studies point to the existence of two additional isoforms, neither of which have been characterized to date. In this regard, here we confirm the presence of these two additional FAIM isoforms in human fetal brain, fetal and adult testes, and placenta tissues. We named them FAIM-S_2a and FAIM-L_2a since they have the same sequence as FAIM-S and FAIM-L, but include exon 2a. PCR and western blot revealed that FAIM-S_2a shows ubiquitous expression in all the tissues and cellular models tested, while FAIM-L_2a is expressed exclusively in tissues of the nervous system. In addition, we found that, when overexpressed in non-neuronal cells, the splicing factor nSR100 induces the expression of the neuronal isoforms, thus identifying it as responsible for the generation of FAIM-L and FAIM-L_2a. Functionally, FAIM-S_2a and FAIM-L_2a increased neurite outgrowth in response to NGF stimulation in a neuronal model. This observation thus, supports the notion that these two isoforms are involved in neuronal differentiation. Furthermore, subcellular fractionation experiments revealed that, in contrast to FAIM-S and FAIM-L, FAIM-S_2a and FAIM-L_2a are able to localize to the nucleus, where they may have additional functions. In summary, here we report on two novel FAIM isoforms that may have relevant roles in the physiology and pathology of the nervous system.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Isoformas de Proteínas/metabolismo , Empalme Alternativo , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Exones , Humanos , Conformación de Ácido Nucleico , Células PC12 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estabilidad Proteica , ARN Mensajero/química , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , TermodinámicaRESUMEN
Caspases have recently emerged as key regulators of axonal pruning and degeneration and of long-term depression (LTD), a long-lasting form of synaptic plasticity. However, the mechanism underlying these functions remains unclear. In this context, XIAP has been shown to modulate these processes. The neuron-specific form of FAIM protein (FAIM-L) is a death receptor antagonist that stabilizes XIAP protein levels, thus preventing death receptor-induced neuronal apoptosis. Here we show that FAIM-L modulates synaptic transmission, prevents chemical-LTD induction in hippocampal neurons, and thwarts axon degeneration after nerve growth factor (NGF) withdrawal. Additionally, we demonstrate that the participation of FAIM-L in these two processes is dependent on its capacity to stabilize XIAP protein levels. Our data reveal FAIM-L as a regulator of axonal degeneration and synaptic plasticity.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Degeneración Nerviosa/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Axones/metabolismo , Células Cultivadas , Ganglios Espinales/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Ratones , N-Metilaspartato/metabolismo , Plasticidad Neuronal , Neuronas/metabolismo , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/genética , Receptores AMPA/metabolismo , Regulación hacia ArribaRESUMEN
âHeat-shock factor 1 (âHSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate âHSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase âFBXW7α interacts with âHSF1 through a conserved motif phosphorylated by âGSK3ß and âERK1. âFBXW7α ubiquitylates âHSF1 and loss of âFBXW7α results in impaired degradation of nuclear âHSF1 and defective heat-shock response attenuation. âFBXW7α is either mutated or transcriptionally downregulated in melanoma and âHSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. âFBXW7α deficiency and subsequent âHSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the âHSF1 transcriptional program both in the presence of exogenous stress and in cancer.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas F-Box/genética , Glucógeno Sintasa Quinasa 3/genética , Melanoma/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Procesamiento Proteico-Postraduccional , Neoplasias Cutáneas/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Factores de Transcripción del Choque Térmico , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Trasplante de Neoplasias , Alineación de Secuencia , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Apoptosis is a major mechanism regulating immune tolerance by the elimination of autoreactive T lymphocytes. A failure of activation induced cell-death (AICD) has been described in T lymphocytes from patients with multiple sclerosis (MS). The aims of this study were to evaluate AICD in T lymphocytes from patients with MS and healthy controls, and to explore the molecular mechanisms underlying the deregulation observed in apoptosis induction. PHA-induced AICD was reduced in T lymphocytes from patients with relapsing-remitting MS compared with controls. This finding was associated with a diminished expression of Fas and a failure in caspase 3 activation.
Asunto(s)
Apoptosis/inmunología , Activación de Linfocitos/inmunología , Esclerosis Múltiple/patología , Linfocitos T/patología , Adulto , Antígenos CD/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Células Cultivadas , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Fitohemaglutininas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Receptor fas/metabolismoRESUMEN
X-linked Inhibitor of apoptosis protein (XIAP) has been classically identified as a cell death regulator. Here, we demonstrate a novel function of XIAP as a regulator of neurite outgrowth in neuronal cells. In PC12 cells, XIAP overexpression prevents NGF-induced neuronal differentiation, whereas NGF treatment induces a reduction of endogenous XIAP levels concomitant with the induction of neuronal differentiation. Accordingly, downregulation of endogenous XIAP protein levels strongly increases neurite outgrowth in PC12 cells as well as axonal and dendritic length in primary cortical neurons. The effects of XIAP are mediated by the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERKs) pathway since blocking this pathway completely prevents the neuritogenesis mediated by XIAP downregulation. In addition, we found that XIAP binds to cRaf and Trk receptors. Our results demonstrate that XIAP plays a new role as a negative regulator of neurotrophin-induced neurite outgrowth and neuronal differentiation in developing neurons.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Neuronas/citología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptor trkA/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Diferenciación Celular , Unión Proteica , RatasRESUMEN
Upon activation, tumor necrosis factor alpha (TNF-alpha) receptor can engage apoptotic or survival pathways. Inhibition of macromolecular synthesis is known to sensitize cells to TNF-alpha-induced cell death. It is believed that this sensitization is due to the transcriptional blockade of genes regulated by NF-kappaB. Nevertheless, such evidence has remained elusive in the nervous system. Here, we show that TNF-alpha cannot normally induce apoptosis in PC12 cells or cortical neurons. However, cells treated with Actinomycin D (ActD) become susceptible to TNF-alpha-induced cell death through the activation of caspase-8, generation of tBid and activation of caspase-9 and -3. Analysis of several proteins involved in TNF-alpha receptor signaling showed no significant downregulation of NF-kappaB target genes, such as IAPs or FLIP, under such conditions. However, Bcl-x(L) protein levels, but not those of Bcl-2, Bax and Bak, are reduced by ActD or TNF-alpha/ActD treatments. Moreover, Bcl-x(L) overexpression fully protects cells against TNF-alpha/ActD-induced cell death. When endogenous levels of Bcl-x(L) are specifically downregulated by lentiviral-based RNAi, cells no longer require ActD to be sensitive to TNF-alpha-triggered apoptosis. Furthermore, Bcl-x(L) downregulation does not affect TNF-alpha-mediated NF-kappaB activation. Altogether, our results demonstrate that Bcl-x(L), and not Bcl-2, FLIP or IAPs, acts as the endogenous regulator of neuronal resistance/sensitivity to TNF-alpha-induced apoptosis in an NF-kappaB-independent manner.